Wet chemical silver treatment of endotracheal tubes to produce antibacterial surfaces.

Mechanically ventilated patients in hospitals are subjected to an increased risk of acquiring nosocomial pneumonia that sometimes has a lethal outcome. One way to minimize the risk could be to make the surfaces on endotracheal tubes antibacterial. In this study, bacterial growth was inhibited or completely prevented by silver ions wet chemically and deposited onto the tube surface. Through the wet chemical treatment developed here, a surface precipitate was formed containing silver chloride and a silver stearate salt. The identity and morphology of the surface precipitate was studied using x-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, and x-ray powder diffraction. Leaching of silver ions into solution was examined, and bacterial growth on the treated surfaces was assayed using Pseudomonas aeruginosa wild type (PAO1) bacteria. Furthermore, the minimum inhibitory concentration of silver ions was determined in liquid- and solid-rich growth medium as 23 and 18 microM, respectively, for P. aeruginosa.

Neocucullanus neocucullanus Travassos, Artigas et Pereira, 1928 (Nematoda: Cucullanidae) from the Characidae fish, Brycon hilarii Valenciennes, 1850, from Brazil

During investigation on the helminth parasites from Brycon hilarii Valenciennes, 1850 (Characiformes, Characidae), from River Juba, Tangará da Serra, state of Mato Grosso, Brazil, several specimens of the nematode Neocucullanus Travassos, Artigas et Pereira, 1928 were detected. A detailed study of this material, including scanning electron microscopy, allowed to identify these nematodes as N. neocucullanus Travassos, Artigas et Pereira, 1928 and to confirm N. multipapillatus Petter, 1989 as a junior synonym of N. neocucullanus.

Cross-immunoreactivity between anti-potato apyrase antibodies and mammalian ATP diphosphohydrolases: potential use of the vegetal protein in experimental schistosomiasis

We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models.

Estudo de ovos de quatro espécies do gênero Meccus (Hemiptera, Reduviidae, Triatominae), vetores da doença de Chagas

The objective of this work was to analyze the morphological, morphometrical, and histological characteristics of eggs of four triatomine bugs species still not studied, in order to understand phylogenetic aspects and to facilitate parameters used in taxonomy, with the purpose of a specific and generic characterization of these vectors in public health. The eggs of each species had come from the collections of the Laboratory of Triatomine bugs and Culicid mosquitoes of the Faculdade de Saúde Pública - USP (Faculty of Public Health/USP). The morphologic studies were carried out through optic microscopy (OM) and scanning electronic microscopy (SEM). The eggs were measured with the help of the digital paquimeter Starrett® 727. In relation to histological measures, Microtome Leica RM 2145® was used. Similarities were evidenced in the exocorial architecture of the eggs when they were studied by OM and SEM. This similarity among the eggs suggests a recent speciation, probably derived from common ancestry, representing a monophyletic group. Some structures were also detected by the histological cuts. The study has contributed to the magnification and recognition in generic and specific terms of the Triatominae subfamily. These new data will be able to subsidize a better understanding to determine roles for each vector species and to facilitate parameters to be used in taxonomy.

Functionalization of microstructured open-porous bioceramic scaffolds with human fetal bone cells.

Bone substitute materials allowing trans-scaffold migration and in-scaffold survival of human bone-derived cells are mandatory for development of cell-engineered permanent implants to repair bone defects. In this study, we evaluated the influence on human bone-derived cells of the material composition and microstructure of foam scaffolds of calcium aluminate. The scaffolds were prepared using a direct foaming method allowing wide-range tailoring of the microstructure for pore size and pore openings. Human fetal osteoblasts (osteo-progenitors) attached to the scaffolds, migrated across the entire bioceramic depending on the scaffold pore size, colonized, and survived in the porous material for at least 6 weeks. The long-term biocompatibility of the scaffold material for human bone-derived cells was evidenced by in-scaffold determination of cell metabolic activity using a modified MTT assay, a repeated WST-1 assay, and scanning electron microscopy. Finally, we demonstrated that the osteo-progenitors can be covalently bound to the scaffolds using biocompatible click chemistry, thus enhancing the rapid adhesion of the cells to the scaffolds. Therefore, the different microstructures of the foams influenced the migratory potential of the cells, but not cell viability. Scaffolds allow covalent biocompatible chemical binding of the cells to the materials, either localized or widespread integration of the scaffolds for cell-engineered implants.

Collagen (NeuraGen®) nerve conduits and stem cells for peripheral nerve gap repair.

Collagen nerve guides are used clinically for peripheral nerve defects, but their use is generally limited to lesions up to 3 cm. In this study we combined collagen conduits with cells as an alternative strategy to support nerve regeneration over longer gaps. In vitro cell adherence to collagen conduits (NeuraGen(®) nerve guides) was assessed by scanning electron microscopy. For in vivo experiments, conduits were seeded with either Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC) or left empty (control group), conduits were used to bridge a 1cm gap in the rat sciatic nerve and after 2-weeks immunohistochemical analysis was performed to assess axonal regeneration and SC infiltration. The regenerative cells showed good adherence to the collagen walls. Primary SC showed significant improvement in distal stump sprouting. No significant differences in proximal regeneration distances were noticed among experimental groups. dMSC and dASC-loaded conduits showed a diffuse sprouting pattern, while SC-loaded showed an enhanced cone pattern and a typical sprouting along the conduits walls, suggesting an increased affinity for the collagen type I fibrillar structure. NeuraGen(®) guides showed high affinity of regenerative cells and could be used as efficient vehicle for cell delivery. However, surface modifications (e.g. with extracellular matrix molecule peptides) of NeuraGen(®) guides could be used in future tissue-engineering applications to better exploit the cell potential.

SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: a comparative analysis.

Several evidences suggest that astrocytes release small transmitter molecules, peptides, and protein factors via regulated exocytosis, implying that they function as specialized neurosecretory cells. However, very little is known about the molecular and functional properties of regulated secretion in astrocytes in the adult brain. Establishing these properties is central to the understanding of the communication mode(s) of these cells and their role(s) in the control of synaptic functions and of cerebral blood flow. In this study, we have set-up a high-resolution confocal microscopy approach to distinguish protein expression in astrocytic structures and neighboring synaptic terminals in adult brain tissue. This approach was applied to investigate the expression pattern of core SNARE proteins for vesicle fusion in the dentate gyrus and CA1 regions of the mouse hippocampus. Our comparative analysis shows that astrocytes abundantly express, in their cell body and main processes, all three protein partners necessary to form an operational SNARE complex but not in the same isoforms expressed in neighbouring synaptic terminals. Thus, SNAP25 and VAMP2 are absent from astrocytic processes and typically concentrated in terminals, while SNAP23 and VAMP3 have the opposite expression pattern. Syntaxin 1 is present in both synaptic terminals and astrocytes. These data support the view that astrocytes in the adult hippocampus can communicate via regulated exocytosis and also indicates that astrocytic exocytosis may differ in its properties from action potential-dependent exocytosis at neuronal synapses, as it relies on a distinctive set of SNARE proteins.

Spontaneous stage differentiation of mouse-virulent Toxoplasma gondii RH parasites in skeletal muscle cells: an ultrastructural evaluation

Although the predilection for Toxoplasma gondii to form cysts in the nervous system and skeletal and heart muscles has been described for more than fifty years, skeletal muscle cells (SkMCs) have not been explored as a host cell type to study the Toxoplasma-host cell interaction and investigate the intracellular development of the parasite. Morphological aspects of the initial events in the Toxoplasma-SkMC interaction were analysed and suggest that there are different processes of protozoan adhesion and invasion and of the subsequent fate of the parasite inside the parasitophorous vacuole (PV). Using scanning electron microscopy,Toxoplasma tachyzoites from the mouse-virulent RH strain were found to be attached to SkMCs by the anterior or posterior region of the body, with or without expansion of the SkMC membrane. This suggests that different types of parasite internalization occurred. Asynchronous multiplication and differentiation of T. gondii were observed. Importantly, intracellular parasites were seen to display high amounts of amylopectin granules in their cytoplasm, indicating that tachyzoites of the RH strain were able to differentiate spontaneously into bradyzoites in SkMCs. This stage conversion occurred in approximately 3% of the PVs. This is particularly intriguing as tachyzoites of virulent Toxoplasma strains are not thought to be prone to cyst formation. We discuss whether biological differences in host cells are crucial to Toxoplasma stage conversion and suggest that important questions concerning the host cell type and its relevance in Toxoplasma differentiation are still unanswered.

Pigments and plasters discovered in the House of Diana (Cosa, Grosseto, Italy): An integrated study between art history, archaeology and scientific analyses

The pigments and the plasters of the Roman frescoes discovered at the House of Diana (Cosa, Grosseto, Italy) were analysed using non-destructive and destructive mineralogical and chemical techniques. The characterization of both pigments and plasters was performed through optical microscopy, scanning electron microscopy and electron microprobe analysis. The pigments were identified by Raman spectroscopy and submitted to stable isotope analysis. The results were integrated with the archaeological data in order to determine and reconstruct the provenance, trade patterns and the employment of the raw materials used for the elaboration of the frescoes.

Infection kinetics of human adenovirus serotype 41 in HEK 293 cells

The purpose of this work was to acquire an overview of the infectious cycle of HAdV-41 in permissive HEK 293 cells and compare it to that observed with the prototype of the genus, Human adenovirus C HAdV-2. HEK 293 cells were infected with each virus separately and were harvested every 12 h for seven days. Infection kinetics were analysed using confocal and electronic microscopy. The results show that, when properly cultivated, HAdV-41 was not fastidious. It had a longer multiplication cycle, which resulted in the release of complete viral particles and viral stocks reached high titres. After 60 h of infection, the export of viral proteins from the infected cell to the extracellular milieu was observed, with a pattern similar to that previously described for HAdV-2 penton-base trafficking after 30 h of infection. HAdV-41 had a non-lytic cycle and the infection spread from the first infected cell to its neighbours. The release process of the viral particles is unknown. The results observed for HAdV-41 infection in HEK 293 cells show how different this virus is from the prototype HAdV-2 and provides information for the development of this vector for use in gene therapy.

Comparative descriptions of eggs from three species of Rhodnius (Hemiptera: Reduviidae: Triatominae)

The authors describe and compare the morphological and ultrastructural characteristics of eggs from the three most recent described species of the genus Rhodnius Stål, 1859, which have not previously been studied. These species are Rhodnius colombiensis (Mejia, Galvão & Jurberg 1999), Rhodnius milesi (Carcavallo, Rocha, Galvão & Jurberg 2001) and Rhodnius stali (Lent, Jurberg & Galvão 1993). The results revealed that there are similarities in the exochorial architecture of optical microscopy and scanning electron microscopy; these include the predominance of hexagonal cells that are common to all Rhodnius species and variable degrees of lateral flattening, which is common not only to species of this genus, but also to the Rhodniini tribe. Differences in overall colour, the presence of a collar in R. milesi, a longitudinal bevel in R. stali and the precise length of R. colombiensis can be useful distinguishing features. As a result of this study, the key for egg identification proposed by Barata in 1981 can be updated.

Dengue-2 infection and the induction of apoptosis in human primary monocytes

Monocytes/macrophages are important targets for dengue virus (DENV) replication; they induce inflammatory mediators and are sources of viral dissemination in the initial phase of the disease. Apoptosis is an active process of cellular destruction genetically regulated, in which a complex enzymatic pathway is activated and may be trigged by many viral infections. Since the mechanisms of apoptotic induction in DENV-infected target cells are not yet defined, we investigated the virus-cell interaction using a model of primary human monocyte infection with DENV-2 with the aim of identifying apoptotic markers. Cultures analyzed by flow cytometry and confocal microscopy yielded DENV antigen positive cells with rates that peaked at the second day post infection (p.i.), decayed afterwards and produced the apoptosis-related cytokines TNF-α and IL-10. Phosphatidylserine, an early marker for apoptosis, was increased at the cell surface and the Fas death receptor was upregulated at the second day p.i. at significantly higher rates in DENV infected cell cultures than controls. However, no detectable changes were observed in the expression of the anti-apoptotic protein Bcl-2 in infected cultures. Our data support virus modulation of extrinsic apoptotic factors in the in vitro model of human monocyte DENV-2 infection. DENV may be interfering in activation and death mechanisms by inducing apoptosis in target cells.

Spinitectus osorioi (Nematoda: Cystidicolidae) in the Mexican endemic fish Atherinella alvarezi (Atherinopsidae) from the Atlantic River drainage system in Chiapas, Southern Mexico

Specimens of Spinitectus osorioi Choudhury and Pérez-Ponce de León, an intestinal nematode species previously considered to be specific to Chirostoma spp and endemic to some lakes in the Pacific drainage in Michoacán, were collected from the freshwater fish Atherinella alvarezi (Díaz-Pardo) (Atherinopsidae) of the Michol River near Palenque, Chiapas, Southern Mexico, which belongs to the Atlantic drainage system. Studies using light and scanning electron microscopy revealed some taxonomically important, previously unreported or erroneously reported features of S. osorioi, such as the location of the vulva, the actual number and distribution of postanal papillae and phasmids and the presence of a short median cuticular ridge anterior to the cloacal opening (in addition to two long subventral ridges). The recorded somewhat shorter spicules (420-465 and 105-111 μm) and mostly smaller eggs (33-36 × 18-20 μm) as compared to the original species description may be due to a different type of host, geographical region or generally smaller body measurements of these specimens. These biometrical differences are considered to be within the limits of the intraspecific variability of S. osorioi. A key to species of Spinitectus parasitizing freshwater fishes in Mexico is provided.

Rhabdias paraensis sp. nov.: a parasite of the lungs of Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

The nematode parasites of Rhinella marina include species of the genus Rhabdias (Rhabdiasidae: Rhabditoidea). The present study describes Rhabdias paraensis sp. nov., which parasitizes the lungs of R. marina in Brazilian Amazonia. Of the more than 70 known species of this genus, 18 are parasites of bufonids, of which, eight are Neotropical. The new species described here is similar to Rhabdias alabialis in the absence of lips is different by the presence of conspicuous cephalic papillae. We describe details of the four rows of pores, which are distributed equally along the whole of the length of the body and connected with hypodermal cells, using histology and scanning electron microscopy. Other histological aspects of the internal structure of this nematode are also described.

Lanfrediella amphicirrus gen. nov. sp. nov. Nematotaeniidae (Cestoda: Cyclophyllidea), a tapeworm parasite of Rhinella marina (Linnaeus, 1758) (Amphibia: Bufonidae)

The family Nematotaeniidae, tapeworms commonly found in the small intestines of amphibians and reptiles, includes 27 recognised species distributed among four genera: Bitegmen Jones, Cylindrotaenia Jewell, Distoichometra Dickey and Nematotaenia Lühe. The taxonomy of these cestodes is poorly defined, due in part to the difficulties of observing many anatomical traits. This study presents and describes a new genus and species of nematotaeniid parasite found in cane toads (Rhinella marina) from eastern Brazilian Amazonia. The cestodes were collected during the necropsy of 20 hosts captured in the urban area of Belém, Pará. The specimens were fixed and processed for light microscopy, scanning electron microscopy (SEM) and three-dimensional (3D) reconstruction. Samples were also collected for molecular analyses. The specimens presented a cylindrical body, two testes and paruterine organs. However, they could not be allocated to any of the four existing nematotaeniid genera due to the presence of two each of dorsal compact medullary testes, cirri, cirrus pouches, genital pores, ovaries and vitelline glands per mature segment. Lanfrediella amphicirrus gen. nov. sp. nov. is the first nematotaeniid studied using Historesin analysis, SEM and 3D reconstruction, and it is the second taxon for which molecular data have been deposited in GenBank.