Identification of vaccine candidate antigens from a genomic analysis of Porphyomonas gingivalis

Porphyromonas gingivalis is a key periodontal pathogen which has been implicated in the etiology of chronic adult periodontitis. Our aim was to develop a protein based vaccine for the prevention and or treatment of this disease. We used a whole genome sequencing approach to identify potential vaccine candidates. From a genomic sequence, we selected 120 genes using a series of bioinformatics methods. The selected genes were cloned for expression in Escherichia coli and screened with P. gingivalis antisera before purification and testing in an animal model. Two of these recombinant proteins (PG32 and PG33) demonstrated significant protection in the animal model, while a number were reactive with various antisera. This process allows the rapid identification of vaccine candidates from genomic data. (C) 2001 Elsevier Science Ltd. All rights reserved.

Genetic dependence of the specific T-cell cytokine response to Porphyromonas gingivalis in mice

Background: Susceptibility to periodontal infections may, in part, be genetically determined. Porphyromonas gingivalis is a major periodontopathogen, and the immune response to this organism requires T-cell help. The aim of the present study was to examine the specific T-cell cytokine responses to P gingivalis outer membrane antigens in a mouse model and their relationship with H-2 haplotype. Methods: BALB/c and DBA/2J (H-2(d)), CBACaH (H-2(k)), and C57BL6 (H-2(b)) mice were immunized with P gingivalis outer membrane antigens weekly for 3 weeks. One week after the final injection, the spleens were removed, and 6 T-cell lines specific for P gingivalis were established for each mouse strain. The percentage of CD4 and CD8 cells in the P gingivalis-specific T-cell lines staining positive for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma, and IL-10 was determined by 2-color flow cytometry. Results: The cytokine profiles of T-cell lines from BALB/c and DBA/2J mice showed no significant differences. Significantly fewer IL4+, IFN-gamma+, and IL-10+ CD4 cells than IL-4+, IFN-gamma+, and IL-10+ CD8 cells, respectively, were demonstrated for both strains. P gingivalis-specific T-cell lines generated from CBACaH mice were similar to those generated from BALB/c and DBA/2J mice; however, the mean percentage of IL4+ CD4 cells in CBACaH mice was lower than the percentage of IFN-gamma+ CD4 cells. Also, the mean percentage of IFN-gamma+ CD4 cells in CBACaH mice was significantly increased compared to DBA/2J mice. Unlike the other 3 strains, T-cell lines established from C57BL6 mice contained similar percentages of cytokine-positive cells, although the percentage of IL-4+ CD4 cells was reduced in comparison to the percentage of CD8 cells. However, comparisons with the other 3 strains demonstrated a higher percentage of IL-4+ CD4 cells than in lines established from the spleens of DBA/2J mice, IFN-gamma+ CD4 cells than in lines established from BALB/c and CBACaH mice, and IL-10+ CD4 cells than in lines established from all 3 other strains. No significant differences in the percentage of positive CD8 cells were demonstrated between lines in the 4 strains of mice. Conclusion: The specific T-cell response to P gingivalis in mice may, in the case of the CD4 response, depend on MHC genes. These findings are consistent with the concept that patient susceptibility is important to the outcome of periodontal infection and may, in part, be genetically determined.

The role of CD4(+) cells in vivo on the induction of the immune response to Porphyromonas gingivalis in mice

Background: It has previously been suggested that CD4(+) T cells play a pivotal role in regulating the immune response to periodontal pathogens. The aim of the present study therefore was to determine delayed type hypersensitivity (DTH), spleen cell proliferation, serum and splenic anti-Porphyromonas gingivalis antibody levels, and lesion sizes following challenge with viable P. gingiualis in CD4-depleted BALB/c mice immunized with P. gingiualis outer membrane proteins (OMP). Methods: Four groups of BALB/c mice were used. Groups 1 and 2 were injected intraperitoneally (ip) with saline for 3 consecutive days and then weekly throughout the experiment. Groups 3 and 4 were injected ip with rat immunoglobulin and a monoclonal rat anti-mouse CD4 antibody, respectively. Two days later, group 1 mice were injected ip with saline only, while all the other groups were immunized ip with P. gingiualis OMP weekly for 3 weeks. One week later following the last immunization of OMP, 3 separate experiments were conducted to determine: 1) the DTH response to P. gingiualis OMP by measuring footpad swelling; 2) the levels of antibodies to P. gingiualis in serum samples and spleen cell cultures using an enzyme-linked immunosorbent assay, as well as spleen cell proliferation after stimulation with OMP; and 3) the lesion sizes after a subcutaneous challenge with viable P. gingiualis cells. Results: In CD4(+) T-cell-depleted mice (group 4), the DTH response and antigen-stimulated cell proliferation were significantly suppressed when compared to groups 2 and 3. Similarly, the levels of serum and splenic IgM, IgG, and all IgG subclass antibodies to P. gingiualis OMP were depressed. Delayed healing of P. gingivalis-induced lesions was also observed in the CD4(+) T-cell-depleted group. Conclusions: This study has shown that depletion of CD4(+) T cells prior to immunization with P. gingiualis OMP led to the suppression of both the humoral and cell-mediated immune response to this microorganism and that this was associated with delayed healing. These results suggest that the induction of the immune response to P. gingiualis is a CD4(+) T-cell-dependent mechanism and that CD4(+) T cells are important in the healing process.

Embryogenesis and metamorphosis in a haplosclerid demosponge: gastrulation and transdifferentiation of larval ciliated cells to choanocytes

Early development and metamorphosis of Reniera sp., a haplosclerid demosponge, have been examined to determine how gastrulation occurs in this species, and whether there is an inversion of the primary germ layers at metamorphosis. Embryogenesis occurs by unequal cleavage of blastomeres to form a solid blastula consisting micro- and macromeres; multipolar migration of the micromeres to the surface of the embryo results in a bi-layered embryo and is interpreted as gastrulation. Polarity of the embryo is determined by the movement of pigment-containing micromeres to one pole of the embryo; this pole later becomes the posterior pole of the swimming larva. The bi-layered larva has a fully differentiated monociliated outer cell layer, and a solid interior of various cell types surrounded by dense collagen. The pigmented cells at the posterior pole give rise to long cilia that are capable of responding to environmental stimuli. Larvae settle on their anterior pole. Fluorescent labeling of the monociliated outer cell layer with a cell-lineage marker (CMFDA) demonstrates that the monociliated cells resorb their cilia, migrate inwards, and transdifferentiate into the choanocytes of the juvenile sponge, and into other amoeboid cells. The development of the flagellated choanocytes and other cells in the juvenile from the monociliated outer layer of this sponge's larva is interpreted as the dedifferentiation of fully differentiated larval cells-a process seen during the metamorphosis of other ciliated invertebrate larvae-not as inversion of the primary germ layers. These results suggest that the sequences of development in this haplosclerid demosponge are not very different than those observed in many cnidarians.

Factors affecting the establishment and dispersal of nymphs of Pristhesancus plagipennis Walker (Hemiptera : Reduviidae) when released onto soybean, cotton and sunflower crops

A problem with augmenting predatory bugs through mass release is the logistical difficulty of delivering nymphs onto the foliage of field crops. In this paper we examine postrelease establishment and dispersal of the nymphs of the predatory bug Pristhesancus plagipennis on soybean, cotton and sunflower in an effort to devise an appropriate strategy for field release. The effects of predator stadia and release rates on field establishment and within-crop-canopy dispersal after hand release were recorded in soybean, cotton and sunflower. Field establishment improved with the release of more-developed nymphs, with third instars providing the most appropriate compromise between field hardiness and rearing cost. Increased nymphal density at the point of release had little effect on nymphal dispersal throughout the crop canopy. The patterns of nymphal dispersal observed on the three crops suggest that crop-canopy architecture may have an effect on the ability of nymphs to spread out postrelease, as nymphs dispersed poorly in cotton and sunflower compared to soybean. To overcome poor dispersal of nymphs after release, a mechanical release method, where nymphs were mixed with vermiculite and delivered onto a target crop through a spinning disk fertiliser spreader, was tested, and provided similar nymph establishment rates and dispersal patterns as releasing nymphs individually by hand. The implications of nymph dispersal and field hardiness in regard to inundative field release techniques are discussed.

The ultrastructure of Macropodinium moiri and revised diagnosis of the Macropodiniidae (Litostomatea : Trichostomatia)

The ultrastructural features of Macropodinium moiri were investigated. The somatic cortex is composed of two lateral non-ciliated zones covered with trapezoidal plates and separated by a trough-like dorsoventral groove (DVG) which divides the cell into left and right halves. The somatic kineties occupy the margins of the DVG and are composed of monokinetids whose infraciliature shows a typical litostome pattern. The pellicular plates are lamellate, and separated by V-shaped grooves which are lined by thick-walled vacuoles. The DVG cortex is composed of electron-opaque U-shaped ribs which alternate with electron-lucent saccular structures. The DVG surface is composed of small regular pellicular sacs built up to form the ridges of the dorsal DVG. The vestibulum forms a laterally compressed cone with left/right differentiation. The basal section of its non-ciliated right side is internally lined (outer to innermost) by longitudinal fibres, nematodesmata and transverse microtubular ribbons. The left side bears the vestibular kineties and in its basal section is lined (outer to innermost) by small nematodesmata and transverse tubules. Cytoplasmic organelles include endoplasmic reticulum, starch granules and a single contactile vacuole surrounded by patches of nephridioplasm. Hydrogenosomes are absent and coccoid Gram-positive bacteria lie under the ciliated portions of the cell. This set of characteristics differs significantly from those of the all other trichostomes; Macropodiniidae is therefore designated Trichostomatia incertae sedis. A revised familial diagnosis of the Macropodiniidae is proposed.

Welding and rheomorphism of phonolitic fallout deposits from the Las Canadas caldera, Tenerife, Canary Islands

The Las Canadas caldera is a nested collapse caldera formed by the successive migration and collapse of shallow magmatic chambers. Among the pyroclastic products of this caldera are phonolitic fallout deposits that crop out in the caldera wall and on the extracaldera slopes. These deposits exhibit an uninterrupted facies gradation from nonwelded to lava-like and record continuous volcanic deposition. Densely welded and lava-like facies result from the extreme attenuation and complete homogenization of juvenile clasts that destroy original clast outlines and any evidence of fallout deposition. Agglutination contributes significantly to the final degree of flattening observed in the welded facies. After deposition, rheomorphic flowage occurs. Emplacement temperatures for one of the welding sequences are calculated from magmatic temperatures and a model of tephra cooling during fallout. Results are 486 degreesC for the nonwelded facies and 740 degreesC for the moderately welded facies. For the same welding sequence, a cooling time between 25 and 54 days is estimated from published experimental and computational data as the possible duration of welding and rheomorphism. Following deposition and agglutination, the lava-like pyroclastic facies had the rheological properties of viscous lavas and flowed down the outer slopes away from the caldera. Some lava-like masses detached from proximal areas to more distal regions. During deposition, the eruptive style evolved from Plinian fallout to fountain-fed spatter deposition. This evolution was accompanied by a decrease in explosive power and a lower height of the eruptive column, which produce higher emplacement temperatures and more effective heat retention of pyroclasts.

Phase variation in meningococcal lipooligosaccharide biosynthesis genes

Neisseria meningitidis expresses a range of lipooligosaccharide (LOS) structures, comprising of at least 13 immunotypes (ITs). Meningococcal LOS is subject to phase variation of its terminal structures allowing switching between ITs, which is proposed to have functional significance in disease. The objectives of this study were to investigate the repertoire of structures that can be expressed in clinical isolates, and to examine the role of phase-variable expression of LOS genes during invasive disease. Southern blotting was used to detect the presence of LOS biosynthetic genes in two collections of meningococci, a global set of strains previously assigned to lineages of greater or lesser virulence, and a collection of local clinical isolates which included paired throat and blood isolates from individual patients. Where the phase-variable genes lgtA, lgtC or IgtG were identified, they were amplified by PCR and the homopolymeric tracts, responsible for their phase-variable expression, were sequenced. The results revealed great potential for variation between alternate LOS structures in the isolates studied, with most strains capable of expressing several alternative terminal structures. The structures predicted to be currently expressed by the genotype of the strains agreed well with conventional immunotyping. No correlation was observed between the structural repertoire and virulence of the isolate. Based on the potential for LOS phase variation in the clinical collection and observations with the paired patient isolates, our data suggest that phase variation of LOS structures is not required for translocation between distinct compartments in the host. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Powder forging of a sintered Al-3.8Cu-1Mg-0.8Si-O.1Sn alloy

The forging characteristics of an Al-Cu-Mg-Si-Sn alloy are examined using it new testing strategy which incorporates a double truncated cone specimen and finite element modelling. This sample geometry produces controlled strain distributions within a single specimen and can readily identify the specific strain required to achieve a specific microstructural event by matching the metallographic data with the strain profiles calculated from finite element software, The friction conditions were determined using the conventional friction ring test, which was evaluated using finite element software. The rheological properties of the alloy, evaluated from compression testing of right cylinders, are similar to the properties of conventional aluminium forgings. A hoop strain develops at the outer diameter of the truncated cones and this leads to pore opening at the outer few millimetres. The porosity is effectively removed when the total strain equals the net compressive strain. The strain profiles that develop in the truncated cones are largely independent of the processing temperature and the strain rate although the strain required for pore closure increases as the forging temperature is reduced. This suggests that the microstructure and the strain rate sensitivity may also be important factors controlling pore behaviour. (C) 2002 Elsevier Science B.V. All rights reserved.

The prediction of sharkskin instability observed during film blowing

The characteristics of sharkskin surface instability for linear low density polyethylene are studied as a function of film blowing processing conditions. By means of scanning electron microscopy and surface profilometry, is it found that for the standard industrial die geometry studied, sharkskin only occurs on the inside of the film bubble. Previous work suggests that this instability may be due to critical extensional stress levels at the exit of the die. Isothermal integral viscoelastic simulations of the annular extrusion process are reported, and confirm that the extensional stress at the die exit is large enough to cause local melt rupture. However the extensional stress level at the outer die wall predicts melt rupture of the outside bubble surface also, which contradicts the experimental findings. A significant temperature gradient is expected to exist across the die gap at the exit of the die, due to the external heating of the die and the low conductivity, of the polymer melt. It is shown that a gradient of 20 degreesC is required to cause sharkskin to only appear on the inner bubble surface.

Progressive Age-Related Changes Similar to Age-Related Macular Degeneration in a Transgenic Mouse Model

Age-related macular degeneration (AMD) is the major cause of blindness in the developed world. its pathomechanism is unknown and its late onset, complex genetics and strong environmental components have all hampered investigations. Here we demonstrate the development of an animal model for AMD that reproduces features associated with geographic atrophy, a transgenic mouse line (mcd/mcd) expressing a mutated form of cathepsin D that is enzymatically inactive thus impairing processing of phagocytosed photoreceptor outer segments in the retinal pigment epithelial (RPE) cells. Pigmentary changes indicating RPE cell atrophy and a decreased response to flash electroretinograms were observed in 11- to 12-month-old mcd/mcd mice. Histological studies showed RPE cell proliferation, photoreceptor degeneration, shortening of photoreceptor outer segments, and accumulation of immunoreactive photoreceptor breakdown products in the RPE cells. An accelerated photoreceptor cell death was detected in 12-month-old mcd/mcd mice. Transmission electron microscopy demonstrated presence of basal laminar and linear deposits that are considered to be the hallmarks of AMD. Small hard drusen associated with human age-related maculopathy were absent in the mcd/mcd mouse model at the ages analyzed. in summary, this model presents several features of AMD, thus providing a valuable tool for investigating the underlying biological processes and pathomechanism of AMD.

Winter intervention against Aedes aegypti (Diptera : Culicidae) larvae in subterranean habitats slows surface recolonization in summer

At semiarid Charters Towers, north Queensland, Australia, the importance of Aedes aegypti (L.) in wells was assessed in relation to the colonization of surface habitats during the wet season. From April to July 1999, 10 wells (five positive for Ae. aegypti) were monitored to assess their status and larvae population numbers therein. All surface containers located within a 100 m radius of each well were removed, treated with s-methoprene or sealed to prevent the utilization of these containers by mosquitoes. These inner cores were surrounded by outer zones for a further 100 m in which surface containers were left untreated but all subterranean habitats were treated. Ovitraps were monitored monthly in the inner cores for 36 wk from August 1999 to April 2000 and differences in the proportions of ovitraps positive for Ae. aegypti and Ochlerotatus notoscriptus (Skuse) were analyzed by logistic regression. Analysis of the proportions of ovitraps positive for Ae. aegypti near positive wells indicated significantly greater colonization from November to March (the wet season), compared with those situated near Ae. aegypti negative wells. As Oc. notoscriptus were not produced from subterranean sites, comparisons of the proportions of ovitraps positive for Oc. notoscriptus in positive and negative inner cores provided an indication of the relative productivity of the uncontrolled surface containers in the outer zones. Differences in the utililization of ovitraps by Oc. notoscriptus among positive and negative cores were observed during only one month (March), when oviposition was greater in ovitraps in the negative cores, compared with the positive cores. Best subsets linear regression analysis of the proportion of ovitraps positive for Ae. aegypti against meteorological variables (rainfall, mean wind speed, mean relative humidity, mean minimum, and maximum temperature) during the week of ovitrapping indicated that minimum temperature and wind speed accounted for 63.4% of the variability. This study confirms that for semiarid towns such as Charters Towers, the practice of treating a relatively small number of key subterranean habitats during winter will significantly affect Ae. aegypti recolonization of surface container habitats during summer, the period of greatest risk for dengue.

Effects of polyunsaturated fatty acids on voltage-gated K+ and Na+ channels in rat olfactory receptor neurons

Although the polyunsaturated fatty acids arachidonic acid (AA) and docosahexaenoic acid (DHA) are enriched in the olfactory mucosa, their possible contribution to olfactory transduction has not been investigated. This study characterized their effects on voltage-gated K+ and Na+ channels of rat olfactory receptor neurons. Physiological (3-10 mum) concentrations of AA and DHA potently and irreversibly inhibited the voltage-gated K+ current in a voltage-independent manner. In addition, both compounds significantly reduced the inhibitory potency of the odorants acetophenone and amyl acetate at these channels. By comparison, the steady-state effects of both AA and DHA on the voltage-gated Na+ channel were relatively weak, with half-maximal inhibition requiring approximate to 35 mum of either compound. However, a surprising finding was that the initial application of 3 mum AA to a naive neuron caused a strong but transient inhibition of the Na+ current. The channels became almost completely resistant to this inhibition within 1 min, and a 2-min wash in control solution was insufficient to restore the strong inhibitory effect. These observations suggest that polyunsaturated fatty acids have the potential to strongly influence the coding of odorant information by olfactory receptor neurons.

Distribution of two splice variants of the glutamate transporter GLT-1 in the developing rat retina

The distributions of a carboxyl terminal splice variant of the glutamate transporter GLT-1, referred to as GLT-1B, and the carboxyl terminus of the originally described variant of GLT-1, referred to hereafter as GLT-1alpha, were examined using specific antisera. GLT-1B was present in the retina at very early developmental stages. Labelling was demonstrable at embryonic day 14, and strong labelling was evident by embryonic day 18. Such labelling was initially restricted to populations of cone photoreceptors, the processes of which extended through the entire thickness of the retina and appeared to make contact with the retinal ganglion cells. During postnatal development the GLT-1B-positive photoreceptor processes retracted to form the outer plexiform layer, and around postnatal day 7, GLT-1B-immunoreactive bipolar cells appeared. The pattern of labelling of bipolar cell processes within the inner plexiform layer changed during postnatal development. Two strata of strongly immunoreactive terminals were initially evident in the inner plexiform layer, but by adulthood these two bands were no longer evident and labelling was restricted to the somata and processes (but not synaptic terminals) of the bipolar cells, as well as the somata, processes, and terminals of cone photoreceptors. By contrast, GLT-1alpha appeared late in postnatal development and was restricted mainly to a population of amacrine cells, although transient labelling was also associated with punctate elements in the outer plexiform layer, which may represent photoreceptor terminals, (C) 2002 Wiley-Liss, Inc.

Cauda epididymal spermatozoa of the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia) imaged by electron and confocal microscopy

The ultrastructure of mature Lagorchestes hirsutus spermatozoa is described for the first time, revealing unusual aspects of sperm structure in macropodid species. The sperm head is ovoid rather than cuneiform, lacks a ventral nuclear groove and has an acrosomal distribution over approximately 85-90% of its dorsal surface. Immediately adjacent to the nuclear membrane the peripheral nucleoplasm in most spermatozoa form an irregular series of distinctive evaginations previously not described in the spermatozoa of any other marsupial. The midpiece is extremely thickened and short, containing no helical network or peripheral plasma membrane specializations. Axonemal structure is unspecialized with no connecting lamellae; dense outer fibres are closely adherent to axonemal doublets. The sperm morphology of this species is highly aberrant in comparison to other macropod taxa and supports the retention of Lagorchestes as a distinctive genus. In light of this new information, skeletal and serological data should be re-evaluated to determine the true taxonomic and phylogenetic position of this species.