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.

Data analysis in multiple-frequency bioelectrical impedance analysis

The performance of three analytical methods for multiple-frequency bioelectrical impedance analysis (MFBIA) data was assessed. The methods were the established method of Cole and Cole, the newly proposed method of Siconolfi and co-workers and a modification of this procedure. Method performance was assessed from the adequacy of the curve fitting techniques, as judged by the correlation coefficient and standard error of the estimate, and the accuracy of the different methods in determining the theoretical values of impedance parameters describing a set of model electrical circuits. The experimental data were well fitted by all curve-fitting procedures (r = 0.9 with SEE 0.3 to 3.5% or better for most circuit-procedure combinations). Cole-Cole modelling provided the most accurate estimates of circuit impedance values, generally within 1-2% of the theoretical values, followed by the Siconolfi procedure using a sixth-order polynomial regression (1-6% variation). None of the methods, however, accurately estimated circuit parameters when the measured impedances were low (<20 Omega) reflecting the electronic limits of the impedance meter used. These data suggest that Cole-Cole modelling remains the preferred method for the analysis of MFBIA data.

Type 1 and Type 2 cytokines: from basic science to fungal infections

At the present time, it is clear that Th1 responses afford protection against the fungi; however, the development, maintenance and function of the protective immune responses are complex mechanisms and are influenced by multiple factors. The route of infection has been shown to affect initial cytokine production and, consequently, the induction of protective Th1 responses. The ability of different isolates of the same fungal agent to induce and sustain a protective response has also been emphasized. Protective immune responses have been shown to vary in genetically different mouse strains after infection. In addition, these protective responses, such as cellular influx and cytokine production, also vary within the same animal depending on the tissue infected. The functional dominance of certain cytokines over others in influencing development and maintenance of protective responses has been discussed. Certain cytokines may act differently in hosts lacking important components of their innate or immune repertoire. It is evident from these presentations that a more comprehensive understanding of the protective mechanisms against different fungal agents is emerging. However, there is still much to learn before cytokine modulatory therapy can be used effectively without risk in the human host.

Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: characterisation and role in organophosphate resistance

Acetylcholinesterase is the target of organophosphate and carbamate pesticides. Organophosphate resistance is widespread in the cattle tick, Boophilus microplus, in Australia. We have isolated a cDNA of acetylcholinesterase from B. microplus and show that it would encode a protein 62 kDa in size. The predicted amino acid sequence contains all the residues characteristic of an acetylcholinesterase. Alternative splicing of the transcript was detected at both the 5' and 3' ends. Alternative splicing at the 5' end would result in two proteins differing by six amino acids. This is the first report of alternative splicing of the N-terminal coding region in a cholinesterase. No point mutations were detected in the acetylcholinesterase gene from organophosphate resistant strains of B. microplus. Alternative explanations for resistance to organophosphates in B. microplus are discussed. (C) 1998 Elsevier Science Ltd. All rights reserved.

Simultaneous detection of multiple antigens with triple immunolabeling

Immunolabeling is commonly used to localize antigens within frozen or paraffin tissue sections. We modified existing immunolabeling techniques to allow the detection of three antigens simultaneously within the one tissue section. The approach relies on the use of three monoclonal antibodies in sequential immunoperoxidase staining steps, each with colored substrates, resulting in the deposition of black, brown, and rose stains. The method is rapid and does not require novel techniques or materials. In this report, we demonstrate the colocalization of mast cell tryptase, neurofilament protein, and CD31 (platelet-endothelial cell adhesion molecule) or laminin in normal human skin and normal buccal mucosa, as an illustration of the power and simplicity of the multiple antigen localization technique.

Bioelectrical impedance analysis predicts outcome in patients with suspected bacteremia

Fluid shifts from intracellular to extracellular water (ICW to ECW) are a feature of sepsis, caused by increased vascular permeability and cell catabolism. Changes in ECW and total body water (TBW) were assessed in a prospective observational study of patients with bacteremia by a bedside technique, and its prognostic impact determined; In 78 hospital patients with fever, the resistance ratio (Rinf/RO) and estimated ECW/TBW ratio from multifrequency bioelectrical impedance analysis, and serum albumin concentration were measured. Rinf/RO and ECW/TBW ratios decreased from day 0 to 2 in patients with significant bacteremia (n = 31), but not in patients with doubtful or negative blood cultures (n = 22 and 25), Increased Rinf/RO at baseline, and further increase of ECW/TBW from day 0 to 2, were associated with lower rate of recovery after 1 week and with higher mortality. Baseline Rinf/RO above the median (0.75) had positive and negative predictive values of 0.31 and 0.95 for death. This prognostic effect was independent of underlying disease and blood culture result in a multivariate model. Hypoalbuminemia at baseline was predictive of outcome, but changes in albumin from day 0 to 2 were unrelated to blood culture results or outcome. In patients with bacteremia,fluid shifts from intracellular to extracellular,vater occur early are rapidly reversible by antibiotic treatment but are associated with adverse prognosis. Bioelectrical impedance deserves further study as a tool for bedside monitoring of patients with bacteremia.

Dehydromonocrotaline generates sequence-selective N-7 guanine alkylation and heat and alkali stable multiple fragment DNA crosslinks

Monocrotaline is a pyrrolizidine alkaloid known to cause toxicity in humans and animals. Its mechanism of biological action is still unclear although DNA crosslinking has been suggested to a play a role in its activity. In this study we found that an active metabolite of monocrotaline, dehydromonocrotaline (DHM), alkylates guanines at the N7 position of DNA with a preference for 5'-GG and 5'-GA sequences; In addition, it generates piperidine- and heat-resistant multiple DNA crosslinks, as confirmed by electrophoresis and electron microscopy. On the basis of these findings, we propose that DHM undergoes rapid polymerization to a structure which is able to crosslink several fragments of DNA.

Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H, curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

Towards a validation of multiple features in the assessment of emotions

This study extends previous attempts to assess emotion with single adjective descriptors, by examining semantic as well as cognitive, motivational, and intensity features of emotions. The focus was on seven negative emotions common to several emotion typologies: anger, fear, sadness, shame, pity, jealousy, and contempt. For each of these emotions, seven items were generated corresponding to cognitive appraisal about the self, cognitive appraisal about the environment, action tendency, action fantasy, synonym, antonym, and intensity range of the emotion, respectively. A pilot study established that 48 of the 49 items were linked predominantly to the specific emotions as predicted. The main data set comprising 700 subjects' ratings of relatedness between items and emotions was subjected to a series of factor analyses, which revealed that 44 of the 49 items loaded on the emotion constructs as predicted. A final factor analysis of these items uncovered seven factors accounting for 39% of the variance. These emergent factors corresponded to the hypothesized emotion constructs, with the exception of anger and fear, which were somewhat confounded. These findings lay the groundwork for the construction of an instrument to assess emotions multicomponentially.

A gene (Cargl) that regulates tissue resistance to Candida albicans maps to chromosome 14 of the mouse

Tissue susceptibility and resistance to infection with the yeast Candida albicans is genetically regulated. Analysis of the strain distribution pattern of the C. albicans resistance gene (Carg1) and additional gene and DNA segment markers in the AKXL recombinant inbred (RI) set showed that 13/15 RI strains were concordant for Carg1, Tcra and Rib1. Therefore, Carg1 is probably located within a 17 cM segment of chromosome 14, within approximately 4 cM of the other two genes. (C) 1998 Academic Press.

A second Candida albicans resistance gene (Carg2) regulates tissue damage, but not fungal clearance, in sub-lethal murine systemic infection

The severity of systemic infection with the yeast Candida albicans has been shown to be under complex genetic control. C57/L mice carry an allele that is associated with an increase in tissue destruction when compared with C57BI/6 mice; however, the gene affects only the severity of tissue lesions, and does not influence the magnitude of the fungal burden in either kidney or brain. Studies in [C57/L x C57BI/6]F1 hybrid mice, and [C57/L x C57BI/6]F1 x C57/L backcross mice, demonstrated that the gene behaves as a simple Mendelian co-dominant. (C) 1998 Academic Press.

A mathematical model for estimating the extent of solute- and water-flux heterogeneity in multiple sample percolation experiments

Multiple sampling is widely used in vadose zone percolation experiments to investigate the extent in which soil structure heterogeneities influence the spatial and temporal distributions of water and solutes. In this note, a simple, robust, mathematical model, based on the beta-statistical distribution, is proposed as a method of quantifying the magnitude of heterogeneity in such experiments. The model relies on fitting two parameters, alpha and zeta to the cumulative elution curves generated in multiple-sample percolation experiments. The model does not require knowledge of the soil structure. A homogeneous or uniform distribution of a solute and/or soil-water is indicated by alpha = zeta = 1, Using these parameters, a heterogeneity index (HI) is defined as root 3 times the ratio of the standard deviation and mean. Uniform or homogeneous flow of water or solutes is indicated by HI = 1 and heterogeneity is indicated by HI > 1. A large value for this index may indicate preferential flow. The heterogeneity index relies only on knowledge of the elution curves generated from multiple sample percolation experiments and is, therefore, easily calculated. The index may also be used to describe and compare the differences in solute and soil-water percolation from different experiments. The use of this index is discussed for several different leaching experiments. (C) 1999 Elsevier Science B.V. All rights reserved.

Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds

Peanut, one of the world's most important oilseed crops, has a narrow germplasm base and lacks sources of resistance to several major diseases. The species is considered recalcitrant to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants, or availability of explants. Here we present a method to efficiently transform cultivars in both botanical types of peanut, by (1) particle bombardment into embryogenic callus derived from mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media; resulting in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bombardment of 10 cm(2) embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment. Transgene copy number ranged from one to 20 with multiple integration sites. There was ca. 50% coexpression of hph and luc or uidA genes coprecipitated on separate plasmids. Reporter gene (luc) expression was confirmed in T-1 progeny from each of six tested independent transformants. Insufficient seeds were produced under containment conditions to determine segregation ratios. The practicality of the technique for efficient cotransformation with selected and unselected genes is demonstrated using major commercial peanut varieties in Australia (cv. NC-7, a virginia market type) and Indonesia (cv. Gajah, a spanish market type).

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.