Characterization of human immunodeficiency virus Gag-specific gamma interferon-expressing cells following protective mucosal immunization with alphavirus replicon particles

A safe, replication-defective viral vector that can induce mucosal and systemic immune responses and confer protection against many infectious pathogens, such as human immunodeficiency virus type 1 (HIV-1), may be an ideal vaccine platform. Accordingly, we have generated and tested alphavirus replicon particles encoding HIV-1 Gag from Sindbis virus (SIN-Gag) and Venezuelan equine encephalitis virus (VEE-Gag), as well as chimeras between the two (VEE/SIN-Gag). Following intramuscular (i.m.), intranasal (i.n.), or intravaginal (IVAG) immunization with VEE/SIN-Gag and an IVAG challenge with vaccinia virus encoding HIV Gag (VV-Gag), a larger number of Gag-specific CD8+ intracellular gamma interferon-expressing cells (iIFNEC) were detected in iliac lymph nodes (ILN), which drain the vaginal/uterine mucosa (VUM), than were observed after immunizations with SIN-Gag. Moreover, a single i.n. or IVAG immunization with VEE/SIN-Gag induced a larger number of cells expressing HIV Gag in ILN, and immunizations with VEE/SIN-Gag through any route induced better protective responses than immunizations with SIN-Gag. In VUM, a larger percentage of iIFNEC expressed alpha4beta7 or alpha(Ebeta)7 integrin than expressed CD62L integrin. However, in spleens (SP), a larger percentage of iIFNEC expressed alpha4beta7 or CD62L than expressed alpha(Ebeta)7. Moreover, a larger percentage of iIFNEC expressed the chemokine receptor CCR5 in VUM and ILN than in SP. These results demonstrate a better induction of cellular and protective responses following immunizations with VEE/SIN-Gag than that following immunizations with SIN-Gag and also indicate a differential expression of homing and chemokine receptors on iIFNEC in mucosal effector and inductive sites versus systemic lymphoid tissues.     

Species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae) from Epinephelus fasciatus and E. merra (Perciformes: Serranidae) off New Caledonia and other parts of the Indo-Pacific Ocean, with a comparison of measurements of specimens prepared using different methods, and a description of P. caledonicus n. sp.

Species of Pseudorhabdosynochus were studied from fresh specimens collected from Epinephelus fasciatus and E. merra off New Caledonia, South Pacific, and specimens deposited in Museums. Experiments on two species demonstrated that the sclerotised hollow organs, such as the quadriloculate male copulatory organ and the vagina, may show differences in measurements of up to 50% when flattened. P. caledonicus n. sp. is described from E. fasciatus in New Caledonia, on which it is relatively rare; it is distinguished on the basis of the quadriloculate organ, which has a very thin anterior wall, the sclerotised parts of the vagina in form of a straight tube with a star-shaped lateral structure, and the squamodiscs composed of 11 open rows of rodlets. P. cupatus (Young, 1969) is redescribed from abundant material from E. fasciatus off New Caledonia (new geographical record) and compared with paratype specimens from Australia (from E. fasciatus and E. merra) and specimens from E. fasciatus in the Red Sea (both herein redescribed and figured); a specimen was also found on a slide from E. merra off Vanuatu. P. melanesiensis (Laird, 1958) is redescribed from material from E. merra off New Caledonia (new geographical record) and compared with type-specimens (herein redescribed and figured) from the same host off Vanuatu. The structure of the sclerotised vagina in P. cupatus and P. melanesiensis is very similar, with a thin-walled tube and a heavily sclerotised structure with three loculi. P. vagampullum (Young, 1969) is redescribed from the paratypes from E. merra from Australia, but was not found in New Caledonia; specimens included among its paratypes (from E. merra in Australia), but different, are herein attributed to Pseudorhabdosynochus sp. 3. P. lantauensis (Beverley-Burton & Suriano, 1981) is redescribed from the paratype specimens from E. longispinis off Hong-Kong. A specimen found among the paratypes of P. cupatus belongs to a different species, herein designated as Pseudorhabdosynochus sp. 1. Specimens from E. longispinis off Hong-Kong, previously attributed to P. cupatus, are attributed to another species, Pseudorhabdosynochus sp. 2. The three species P. cupatus, Pseudorhabdosynochus sp. 1 and Pseudorhabdosynochus sp. 2 have in common a 'lamellosquamodisc' composed of central telescopic lamellae and peripheral rows of rodlets; they can be distinguished by the shape of the sclerotised vagina and measurements of the haptoral hard-parts. Specimens from E. longispinis off Hong-Kong, previously attributed to P. vagampullum, probably belong to a different species. Consequently, after these modified determinations, P. cupatus parasitises only E. fasciatus and E. merra, and P. melanesiensis and P. vagampullum parasitise only E. merra. With their wide geographical distribution and different species of Pseudorhabdosynochus in different localities, E. fasciatus and E. merra appear to represent excellent models for investigating monogenean biogeography in the Indo-Pacific Ocean.     

Two anisakid nematodes from marine fishes off New Caledonia, including Raphidascaris (Ichthyascaris) nemipteri n. sp. from Nemipterus furcosus

One new and one known species of the ascaridoid family Anisakidae are reported from marine fishes off the southwestern coast of New Caledonia: Raphidascaris (Ichthyascaris) nemipteri n. sp. from the intestine of the forked-tailed threadfin bream Nemipterus furcosus (Nemipteridae, Perciformes) and Hysterothylacium cenaticum (Bruce & Cannon, 1989) from the intestine of the striped marlin Tetrapturus audax (Istiophoridae, Perciformes). R. nemipteri is characterised mainly by the shape (wider than long) of the lips, the length of the spicules (225–399 μm, which represent 2.7–4.2% of the body length), the number (22–33) of caudal pre-anal papillae, the position of the vulva (at 16–20% of the body length), and the presence of cuticular spines on the tip of the female tail. Specimens of H. cenaticum from New Caledonia generally exhibited smaller body measurements than those originally described from Australian waters; the deirids and eggs are described for the first time. Maricostula Bruce & Cannon, 1989 is considered a junior synonym of Hysterothylacium, to which three species are transferred as H. cenaticum (Bruce & Cannon, 1989) n. comb., H. makairi (Bruce & Cannon, 1989) n. comb. and H. tetrapteri (Bruce & Cannon, 1989) n. comb.     

Vav family GEFs link activated Ephs to endocytosis and axon guidance

Ephrin signaling through Eph receptor tyrosine kinases can promote attraction or repulsion of axonal growth cones during development. However, the mechanisms that determine whether Eph signaling promotes attraction or repulsion are not known. We show here that the Rho family GEF Vav2 plays a key role in this process. We find that, during axon guidance, ephrin binding to Ephs triggers Vav-dependent endocytosis of the ligand-receptor complex, thus converting an initially adhesive interaction into a repulsive event. In the absence of Vav proteins, ephrin-Eph endocytosis is blocked, leading to defects in growth cone collapse in vitro and significant defects in the ipsilateral retinogeniculate projections in vivo. These findings suggest an important role for Vav family GEFs as regulators of ligand-receptor endocytosis and determinants of repulsive signaling during axon guidance.     

Antidiabetic potential of two novel fatty acid derivatised, N-terminally modified analogues of glucose-dependent insulinotropic polypeptide (GIP): N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37)

Fatty acid derivatisation was used to develop two novel, long-acting, N-terminally modified, glucose-dependent insulinotropic polypeptide (GIP) analogues, N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37). In contrast to GIP, which was rapidly degraded by in vitro incubation with dipeptidylpeptidase IV (DPP IV) (52% intact after 2 h), the analogues remained fully intact for up to 24 h. Both fatty acid-derivatised analogues stimulated cAMP production in GIP receptor Chinese hamster lung (CHL) fibroblasts (EC50 12.1-13.0 nM) and significantly improved in vitro insulin secretion from BRIN-BD11 cells (1.1- to 2.4-fold; p < 0.05 to p < 0.001) compared to control (5.6 mM glucose). Administration of N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) together with glucose in obese diabetic (ob/ob) mice significantly reduced the glycaemic excursion (1.4- and 1.5-fold, respectively; p < 0.05 to p < 0.01) and improved the insulinotropic response (1.5- and 2.3-fold, respectively; p < 0.01 to p < 0.001) compared to native peptide. Dose-response studies with N-AcGIP(LysPAL37) revealed that even the lowest concentration (6.25 nmol/kg) induced a highly significant decrease (1.4-fold; p < 0.001) in the overall glycaemic excursion, coupled with a significant increase (2.0-fold; p < 0.01) in circulating insulin. Furthermore, basal glucose values remained significantly reduced (p < 0.05) and insulin values increased 24 h following a single injection of N-AcGIP(LysPAL37). The glucose-lowering action of the fatty acid-derivatised peptide was greater than that of N-AcGIP. These data demonstrate that novel fatty acid-derivatised analogues of N-terminally modified AcGIP function as long-acting GIP-receptor agonists, with significant antidiabetic potential.     

A survey of the methods for the characterization of microbial consortia and communities

A survey of the available literature on methods most frequently used for the identification and characterization of microbial strains, communities, or consortia is presented. The advantages and disadvantages of the various methodologies were examined from several perspectives including technical, economic (time and cost), and regulatory. The methods fall into 3 broad categories: molecular biological, biochemical, and microbiological. Molecular biological methods comprise a broad range of techniques that are based on the analysis and differentiation of microbial DNA. This class of methods possesses several distinct advantages. Unlike most other commonly used methods, which require the production of secondary materials via the manipulation of microbial growth, molecular biological methods recover and test their source materials (DNA) directly from the microbial cells themselves, without the requirement for culturing. This eliminates both the time required for growth and the biases associated with cultured growth, which is unavoidably and artificially selective. The recovered nucleic acid can be cloned and sequenced directly or subpopulations can be specifically amplified using polymerase chain reaction (PCR), and subsequently cloned and sequenced. PCR technology, used extensively in forensic science, provides researchers with the unique ability to detect nucleic acids (DNA and RNA) in minute amounts, by amplifying a single target molecule by more than a million-fold. Molecular methods are highly sensitive and allow for a high degree of specificity, which, coupled with the ability to separate similar but distinct DNA molecules, means that a great deal of information can be gleaned from even very complex microbial communities. Biochemical methods are composed of a more varied set of methodologies. These techniques share a reliance on gas chromatography and mass spectrometry to separate and precisely identify a range of biomolecules, or else investigate biochemical properties of key cellular biomolecules. Like the molecular biological methods, some biochemical methods such as lipid analyses are also independent of cultured growth. However, many of these techniques are only capable of producing a profile that is characteristic of the microbial community as a whole, providing no information about individual members of the community. A subset of these methodologies are used to derive taxonomic information from a community sample; these rely on the identification of key subspecies of biomolecules that differ slightly but characteristically between species, genera, and higher biological groupings. However, when the consortium is already growing in chemically defined media (as is often the case with commercial products), the rapidity and relatively low costs of these procedures can mitigate concerns related to culturing biases. Microbiological methods are the most varied and the least useful for characterizing microbial consortia. These methods rely on traditional tools (cell counting, selective growth, and microscopic examination) to provide more general characteristics of the community as a whole, or else to narrow down and identify only a small subset of the members of that community. As with many of the biochemical methods, some of the microbiological methods can fairly rapidly and inexpensively create a community profile, which can be used to compare 2 or more entire consortia. However, for taxonomic identification of individual members, microbiological methods are useful only to screen for the presence of a few key predetermined species, whose preferred growth conditions and morphological characteristics are well defined and reproducible.     

Potential activity of methane production in soil, peat, and lacustrine sediments and in the Robert Bourassa hydro-electric reservoir in northern Canada

Flooding of land associated with the creation of reservoirs may increase, at least in the short term, methane flux to the atmosphere. To evaluate the potential contribution of such land use on methane production, field samples were studied in vitro for the potential activity of methanogenic bacteria in unflooded or flooded boreal forest soils, together with lacustrine sediments. From this comparative study, periodically flooded or flooded peats contribute more to methane production than do unflooded peats, soils, and natural lake sediment. The intensity and temporal changes in the activity of methanogenic archaea in the different systems depended on a combination of environmental factors, such as the amount and quality of organic carbon, the water level, and the concentration of oxidizing ions (SO42-, Fe3+).     

CD8-mediated protection against Ebola virus infection is perforin dependent

CD8 T cells have been shown to play an important role in the clearance and protection against fatal Ebola virus infection. In this study, we examined the mechanisms by which CD8 T cells mediate this protection. Our data demonstrate that all normal mice infected s.c. with a mouse-adapted Ebola virus survived the infection, as did 100% of mice deficient in Fas and 90% of those deficient in IFN-gamma. In contrast, perforin-deficient mice uniformly died after s.c. challenge. Perforin-deficient mice failed to clear viral infection even though they developed normal levels of neutralizing anti-Ebola Abs and 5- to 10-fold higher levels of IFN-gamma than control mice. Using MHC class I tetramers, we have also shown that perforin-deficient mice have 2- to 4-fold higher numbers of Ebola-specific CD8s than control mice. These findings suggest that the clearance of Ebola virus is perforin-dependent and provide an additional example showing that this basic immunologic mechanism is not limited to the clearance of noncytopathic viruses.     

Serine residues 286, 288, and 293 within the CIITA: a mechanism for down-regulating CIITA activity through phosphorylation

CIITA is the primary factor activating the expression of the class II MHC genes necessary for the exogenous pathway of Ag processing and presentation. Strict control of CIITA is necessary to regulate MHC class II gene expression and induction of an immune response. We show in this study that the nuclear localized form of CIITA is a predominantly phosphorylated form of the protein, whereas cytoplasmic CIITA is predominantly unphosphorylated. Novel phosphorylation sites were determined to be located within a region that contains serine residues 286, 288, and 293. Double mutations of these residues increased nuclear CIITA, indicating that these sites are not required for nuclear import. CIITA-bearing mutations of these serine residues significantly increased endogenous MHC class II expression, but did not significantly enhance trans-activation from a MHC class II promoter, indicating that these phosphorylation sites may be important for gene activation from intact chromatin rather than artificial plasmid-based promoters. These data suggest a model for CIITA function in which phosphorylation of these specific sites in CIITA in the nucleus serves to down-regulate CIITA activity.