Breast carcinoma cell lysate-pulsed dendritic cells cross-prime MUC1-specific CD8+ T cells identified by peptide-MHC-class-I tetramers

For cancer immunotherapy the loading of dendritic cells (DCs) with whole tumor cell lysate preparations represents a simple and promising approach for presentation of tumor-associated antigens (TAAs), avoiding the disadvantages of HLA-matching and definition of TAAs. The aim of this study was to investigate whether lysate-pulsed DCs efficiently cross-prime CD8+ T cells and induce a strong T(H)1 cell response, as compared to DCs pulsed with specific peptides (FLU M1 and Melan-A/Mart-1). As a model system breast carcinoma cell lysate from either MCF-7 or MDA-MB-231 cell lines (both HLA-A*0201+) expressing the TAA MUC1 were selected. Both cell lines expressed MUC1, the epithelial mucin, which is a large molecular weight O-glycosylated protein expressed in the majority of breast, ovarian, and other epithelial malignancies and is under evaluation as a target antigen in cancer immunotherapy. We developed a simple lysate preparation method to solubilize all cell proteins without degradation. For loading of monocyte-derived dendritic cells, 100 microgmL(-1) of breast carcinoma cell lysate was used, accompanied by an adjuvant consisting of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin-E2. T cells were co-cultivated with lysate or peptide pulsed DCs and were restimulated weekly. Before cultivation, and after the 3rd stimulation, tetramer frequencies for the MUC1 epitopes M1.2 and F7 as well as for the FLU M1 and Melan-A/Mart-1 epitopes were determined. After stimulation with lysate, higher frequencies for M1.2-specific T cells were observed compared with the F7 epitope. Furthermore, we found expansion factors for M1.2-specific T cells that had been stimulated with MCF-7 lysate-pulsed DCs of up to 43-fold. The analysis of typical T(H)1/T(H)2 cytokines (IFN-gamma, TNF-alpha, IL-12p70, IL-2, IL-4, IL-5, and IL-10) revealed a strong T(H)1 response. These results provide evidence for a strong T(H)1 polarization and cross-priming of MUC1-specific CD8+ T cells and demonstrate the feasibility of using lysate-pulsed dendritic cells in breast cancer immunotherapy.     

Individual differences in trait anxiety predict the response of the basolateral amygdala to unconsciously processed fearful faces

Responses to threat-related stimuli are influenced by conscious and unconscious processes, but the neural systems underlying these processes and their relationship to anxiety have not been clearly delineated. Using fMRI, we investigated the neural responses associated with the conscious and unconscious (backwardly masked) perception of fearful faces in healthy volunteers who varied in threat sensitivity (Spielberger trait anxiety scale). Unconscious processing modulated activity only in the basolateral subregion of the amygdala, while conscious processing modulated activity only in the dorsal amygdala (containing the central nucleus). Whereas activation of the dorsal amygdala by conscious stimuli was consistent across subjects and independent of trait anxiety, activity in the basolateral amygdala to unconscious stimuli, and subjects' reaction times, were predicted by individual differences in trait anxiety. These findings provide a biological basis for the unconscious emotional vigilance characteristic of anxiety and a means for investigating the mechanisms and efficacy of treatments for anxiety.     

A molecular beacon-based real-time NASBA assay for detection of Mycobacterium avium subsp. paratuberculosis in water and milk

A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp. paratuberculosis has been developed. It targets and amplifies sequences from the dnaA gene which are specific for this bacterium. The assay includes an internal amplification control, to allow identification of inhibited reactions. The assay was tested against 18 isolates of M. avium subsp. paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets. The lowest number of cells which the assay can detect with 99% probability is 150-200 cells per reaction (as determined using pure culture suspensions). Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 10(3) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(4) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium subsp. paratuberculosis.     

Identification of common subpopulations of non-sorbitol-fermenting, beta-glucuronidase-negative Escherichia coli O157:H7 from bovine production environments and human clinical samples

Non-sorbitol-fermenting, beta-glucuronidase-negative Escherichia coli O157:H7 strains are regarded as a clone complex, and populations from different geographical locations are believed to share a recent common ancestor. Despite their relatedness, high-resolution genotyping methods can detect significant genome variation among different populations. Phylogenetic analysis of high-resolution genotyping data from these strains has shown that subpopulations from geographically unlinked continents can be divided into two primary phylogenetic lineages, termed lineage I and lineage II, and limited studies of the distribution of these lineages suggest there could be differences in their propensity to cause disease in humans or to be transmitted to humans. Because the genotyping methods necessary to discriminate the two lineages are tedious and subjective, these methods are not particularly suited for studying the large sets of strains that are required to systematically evaluate the ecology and transmission characteristics of these lineages. To overcome this limitation, we have developed a lineage-specific polymorphism assay (LSPA) that can readily distinguish between the lineage I and lineage II subpopulations. In the studies reported here, we describe the development of a six-marker test (LSPA-6) and its validation in a side-by-side comparison with octamer-based genome scanning. Analysis of over 1,400 O157:H7 strains with the LSPA-6 demonstrated that five genotypes comprise over 91% of the strains, suggesting that these subpopulations may be widespread.     

Vibrio fischeri sigma54 controls motility, biofilm formation, luminescence, and colonization

In this study, we demonstrated that the putative Vibrio fischeri rpoN gene, which encodes sigma(54), controls flagellar biogenesis, biofilm development, and bioluminescence. We also show that rpoN plays a requisite role initiating the symbiotic association of V. fischeri with juveniles of the squid Euprymna scolopes.     

Functional and regulatory analysis of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> CAX2 cation transporter

The vacuolar sequestration of metals is an important metal tolerance mechanism in plants. The Arabidopsis thaliana vacuolar transporters CAX1 and CAX2 were originally identified in a Saccharomyces cerevisiae suppression screen as Ca²⁺/H⁺ antiporters. CAX2 has a low affinity for Ca²⁺ but can transport other metals including Mn²⁺ and Cd²⁺. Here we demonstrate that unlike cax1 mutants, CAX2 insertional mutants caused no discernable morphological phenotypes or alterations in Ca²⁺/H⁺ antiport activity. However, cax2 lines exhibited a reduction in vacuolar Mn²⁺/H⁺ antiport and, like cax1 mutants, reduced V-type H⁺-ATPase (V-ATPase) activity. Analysis of a CAX2 promoter -glucoronidase (GUS) reporter gene fusion confirmed that CAX2 was expressed throughout the plant and strongly expressed in flower tissue, vascular tissue and in the apical meristem of young plants. Heterologous expression in yeast identified an N-terminal regulatory region in CAX2, suggesting that Arabidopsis contains multiple cation/H⁺ antiporters with shared regulatory features. Furthermore, despite significant variations in morphological and biochemical phenotypes, cax1 and cax2 lines both significantly alter V-ATPase activity, hinting at coordinate regulation among transporters driven by H⁺ gradients and the V-ATPase.     

Characterization of Drosophila nitric oxide synthase: a biochemical study

The heme and flavin-binding domains of Drosophila nitric oxide synthase (DNOS) were expressed in Escherichia coli using the expression vector pCW. The denatured molecular mass of the expressed protein was 152kDa along with a proteolytically cleaved product of 121kDa. The DNOS heme protein exhibited very low Ca(2+)/calmodulin-dependent NO synthase activity. The trypsin digestion patterns were different from nNOS. The full-length DNOS protein had high degree of stability against trypsin. The activity assay of trypsin-digested protein confirmed the same result. Urea dissociation profile of DNOS full-length protein showed that the reductase domain activity was much more susceptible towards urea than the oxygenase domain activity. Urea gradient gel of DNOS full-length protein established distinct transition of dissociation and unfolding in the range 3-4M urea. Reductase domain activity of full-length DNOS protein against external electron acceptors like cytochrome c indicated slow electron transfer from FMN. The bacterial expression of DNOS full-length protein represents an important development in structure-function studies of this enzyme and comparison with other mammalian NOS enzymes which is evolutionary significant.