Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alpha beta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes, Here we describe a DNA plasmid encoding a polyepitope or polytope protein, which contained multiple contiguous minimal murine CTL epitopes, Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models, CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help, The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.

The development of an early warning system to detect trends in illicit drug use in Australia: The illicit drug reporting-system

Appropriate ways to monitor the availability and use of illicit drugs were examined. Four methods were tested concurrently: (1) a quantitative survey of injecting drug users, (2) a qualitative key informant study of illicit drug users and professionals working in the drug field, (3) examination of existing sources of survey, health and law enforcement data and (4) an ethnographic study of a high risk group of illicit drug users. The first three methods were recommended for inclusion in an ongoing national monitoring system, enabling the collection of both quantitative and qualitative data on a range of illicit drugs in a relatively brief, quick and cost-effective manner. A degree of convergent validity was also noted among these methods, improving the degree of confidence in drug trends. The importance of injecting drug users as a sentinel population of illicit drug users was highlighted, along with optimal methods for qualitative research.

Effects of incubation temperature on the energetics of embryonic development and hatchling morphology in the Brisbane river turtle Emydura signata

Incubation temperature and the amount of water taken up by eggs from the substrate during incubation affects hatchling size and morphology in many oviparous reptiles. The Brisbane river turtle Emydura signata lays hard-shelled eggs and hatchling mass was unaffected by the amount of water gained or lost during incubation. Constant temperature incubation of eggs at 24 degrees C, 26 degrees C, 28 degrees C and 31 degrees C had no effect on hatchling mass, yolk-free hatchling mass, residual yolk mass, carapace length, carapace width, plastron length or plastron width. However, hatchlings incubated at 26 degrees C and 28 degrees C had wider heads than hatchlings incubated at 24 degrees C and 31 degrees C. Incubation period varied inversely with incubation temperature, while the rate of increase in oxygen consumption during the first part of incubation and the peak rate of oxygen consumption varied directly with incubation temperature. The total amount of oxygen consumed during development and hatchling production cost was significantly greater at 24 degrees C than at 26 degrees C, 28 degrees C and 31 degrees C. Hatchling mass and dimensions and total embryonic energy expenditure was directly proportional to initial egg mass.

Retinoic acid disrupts anterior ectodermal and endodermal development in ascidian larvae and postlarvae

In vertebrates, excess all-trans retinoic acid (RA) applied during axis formation leads to the apparent truncation of anterior structures. In this study we sought to determine the type of defects caused by ectopic RA on the development of the ascidian Herdmania curvata. We demonstrate that H. curvata embryos cultured in the presence of RA develop into larvae whose trunks are shortened and superficially resemble those of early metamorphosing postlarvae. Despite RA-treated larvae lacking papillar structures they respond normally to natural cues that induce metamorphosis, indicating that chemosensory functionality previously mapped to the most anterior region of normal larvae is unaffected by RA. Excess RA applied during postlarval development leads to a graded loss of the juvenile pharynx, apparently by respecifying anterior endoderm to a more posterior fate. This structure is considered homologous to the gill slits of amphioxus. which are also lost upon RA treatment. This suggests that RA may have had a role in the development of the pharynx of the ancestral chordate and that this function has been maintained in ascidians and cephalochordates and lost in vertebrates.

Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes

When expressed as a transgene from the keratin 14 (K14) promoter in an MHC class II-deficient mouse, I-Ab expressed in thymic cortical epithelium promotes positive but not negative selection of I-Ab-restricted CD4(+) T cells (Laufer, T. M. et al., Nature 1996. 383:81-85). Transgenic mice expressing the E7 protein of human papilloma virus 16 from the K14 promoter were studied to determine the consequence of expression of a cytoplasmic/nuclear protein from the K14 promoter. K14E7-transgenic mice express E7 in the thymus and skin without evidence for autoimmunity to E7. Repeated immunization of FVB(H-2(q)) or F1(C57BV6JxFVB) mice with E7 elicited similar antibody responses to the defined B cell epitopes of E7 in K14E7-transgenic and non-transgenic animals. In contrast, for each genetic background, a single immunization with E7 elicited demonstrable T cell proliferative responses to the major promiscuous T helper epitope of E7 in the transgenic but not the non-transgenic animals. Further,E7-immunized non-transgenic F1 (FVBxC57BL/6J) animals developed strong E7-specific cytotoxic T lymphocyte (CTL) responses and were protected against challenge with E7(+) tumors, whereas similarly immunized K14E7-transgenic animals had a markedly reduced CTL response to E7 and no E7-specific tumor protection was observed, although the antibody and CTL response to ovalbumin was normal. Expression of E7 protein as a transgene from the K14 promoter in the skin and thymus thus induces E7-specific tolerance in the cytotoxic T effector repertoire, together with expansion of the E7-specific T helper repertoire. These findings demonstrate that limited tissue distribution of an autoantigen may result in split tolerance to that autoantigen.

Epidermal iontophoresis: I. Development of the ionic mobility-pore model

Purpose, An integrated ionic mobility-pore model for epidermal iontophoresis is developed from theoretical considerations using both the free volume and pore restriction forms of the model for a range of solute radii (r(j)) approaching the pore radii (r(p)) as well as approximation of the pore restriction form for r(j)/r(p) < 0.4. In this model, we defined the determinants for iontophoresis as solute size (defined by MV, MW or radius), solute mobility, solute shape, solute charge, the Debye layer thickness, total current applied, solute concentration, fraction ionized, presence of extraneous ions (defined by solvent conductivity), epidermal permselectivity, partitioning rates to account for interaction of unionized and ionized lipophilic solutes with the wall of the pore and electroosmosis. Methods, The ionic mobility-pore model was developed from theoretical considerations to include each of the determinants of iontophoretic transport. The model was then used to reexamine iontophoretic flux conductivity and iontophoretic flux-fraction ionized literature data on the determinants of iontophoretic flux. Results. The ionic mobility-pore model was found to be consistent with existing experimental data and determinants defining iontophoretic transport. However, the predicted effects of solute size on iontophoresis are more consistent with the pore-restriction than free volume form of the model. A reanalysis of iontophoretic flux-conductivity data confirmed the model's prediction that, in the absence of significant electroosmosis, the reciprocal of flux is linearly related to either donor or receptor solution conductivity. Significant interaction with the pore walls, as described by the model, accounted for the reported pH dependence of the iontophoretic transport for a range of ionizable solutes. Conclusions. The ionic mobility-pore iontophoretic model developed enables a range of determinants of iontophoresis to be described in a single unifying equation which recognises a range of determinants of iontophoretic flux.

Molecular development of the olfactory nerve pathway

There are, at least, two major questions concerning the molecular development of the olfactory nerve pathway. First, what are the molecular cues responsible for guiding axons from the nasal cavity to the olfactory bulb? Second, what is the molecular basis of axon targeting to specific glomeruli once axons reach the olfactory bulb? Studies in the primary olfactory pathway have focused on the role of the extracellular matrix and ensheathing cells in establishing an initial substrate for growth of pioneer axons between the periphery and brain. The primary axons also express a multitude of cell adhesion molecules that regulate fasciculation of axons and hence may play a role in fascicle formation in the olfactory nerve. Although the olfactory neuroepithelium principally consists of a morphologically homogeneous class of primary olfactory neurons, there are numerous subpopulations of olfactory neurons expressing chemically distinct phenotypes. In particular, numerous subpopulations have been characterized by expression of unique carbohydrate residues and olfactory receptor proteins. Some of these molecules have recently been implicated in axon guidance and targeting to specific glomeruli.

Development of composite adsorbents of carbon and intercalated clay for N2 and O2 adsorption: A preliminary study

Composite adsorbents of carbon and alumina intercalated montmorillonite were prepared and characterized by adsorption of N-2 and O-2 at various temperatures. The effects of pyrolysis, temperature, heating rate, subsequent degassing, and doping of cations and anions were investigated. The adsorption capacities of the composite adsorbents developed at higher temperatures (0 and -79 degrees C) are found to be larger than those of normal alumina pillared clays. The experimental results showed that the framework of these adsorbents is made of alumina particles and clay sheets while the pyrolyzed carbon distributes in the space of interlayers and interpillars. The pores between the carbon particles, clay sheets, and alumina pillars are very narrow with very strong adsorption forces, leading to enhanced adsorption capacities at 0 and -79 degrees C. The composite adsorbents exhibit features similar to those of carbonaceous adsorbents. Their pore structures, adsorption capacities, and selectivities to oxygen can be tailored by a controlled degassing procedure. Meanwhile, ions can be doped into the adsorbents to modify their adsorption properties, as usually observed for oxide adsorbents like zeolite and pillared clays. Such flexibility in pore structure tailoring is a potential advantage of the composite adsorbents developed for their adsorption and separation applications. (C) 1999 Academic Press.

Strategies of accommodation: Development of a coding system for conversational interaction

This study describes a coding system developed to operationalize the sociolinguistic strategies proposed by communication accommodation theory (CAT) in an academic context. Fifty interactions between two students (of Australian or Chinese ethnic background) or a student and faculty member were videotaped. A turn- and episode-based coding system was developed, focusing on verbal and nonverbal behavior. The development of this system is described in detail, before results are presented. Results indicated that status was the main influence on choice of strategies, particularly the extent and type of discourse management and interpersonal control. Participants' sew and ethnicity also played a role: Male participants made more use of interpretability (largely questions), whereas female participants used discourse management to develop a shared perspective. The results make clear that there is no automatic correspondence between behaviors and the strategies they constitute, and they point to the appropriateness of conceptualizing behavior and strategies separately in CAT.

m144, a murine cytomegalovirus (MCMV)-encoded major histocompatibility complex class I homologue, confers tumor resistance to natural killer cell-mediated rejection

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2-deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell-mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell-mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2-activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.