Description: Pertussis toxin (PTX) is an AB5-type exotoxin produced by the bacterium Bordetella pertussis , the causative agent of whooping cough. In vivo intoxication with PTX elicits a variety of immunologic and inflammatory responses, including vasoactive amine sensitization (VAAS) to histamine (HA), serotonin (5-HT), and bradykinin (BDK). Previously, by using a forward genetic approach, we identified the HA H 1 receptor ( Hrh1 /H 1 R) as the gene in mice that controls differential susceptibility to B. pertussis PTX-induced HA sensitization (Bphs). Here we show, by using inbred strains of mice, F 1 hybrids, and segregating populations, that, unlike Bphs, PTX-induced 5-HT sensitivity (Bpss) and BDK sensitivity (Bpbs) are recessive traits and are separately controlled by multiple loci unlinked to 5-HT and BDK receptors, respectively. Furthermore, we found that PTX sensitizes mice to HA independently of Toll-like receptor 4, a purported receptor for PTX, and that the VAAS properties of PTX are not dependent upon endothelial caveolae or endothelial nitric oxide synthase. Finally, by using mice deficient in individual Gα i/o G-protein subunits, we demonstrate that Gα i1 and Gα i3 are the critical in vivo targets of ADP-ribosylation underlying VAAS elicited by PTX exposure.

Description: The incidence of infection with any of the four dengue virus serotypes (DENV1 to -4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an human lymphocyte antigen (HLA)-linked protective role for CD8 + T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. Peripheral blood mononuclear cells from these donors were screened in gamma interferon enzyme-linked immunosorbent spot assays with pools of predicted, HLA-matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8 + T cell responses after live attenuated dengue vaccination and show that CD8 + T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, whereas broad responses to structural and nonstructural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused toward the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV. IMPORTANCE The development of effective vaccination strategies against dengue virus (DENV) infection and clinically significant disease is a task of high global public health value and significance, while also being a challenge of significant complexity. A recent efficacy trial of the most advanced dengue vaccine candidate, demonstrated only partial protection against all four DENV serotypes, despite three subsequent immunizations and detection of measurable neutralizing antibodies to each serotype in most subjects. These results challenge the hypothesis that seroconversion is the only reliable correlate of protection. Here, we show that CD8 + T cell responses in vaccinees were readily detectable and comparable to natural dengue virus infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection in natural immunity and vaccination against DENV.

Description: High-throughput transcriptomics was used to identify Fibroporia radiculosa genes that were differentially regulated during colonization of wood treated with a copper-based preservative. The transcriptome was profiled at two time points while the fungus was growing on wood treated with micronized copper quat (MCQ). A total of 917 transcripts were differentially expressed. Fifty-eight of these genes were more highly expressed when the MCQ was protecting the wood from strength loss and had putative functions related to oxalate production/degradation, laccase activity, quinone biosynthesis, pectin degradation, ATP production, cytochrome P450 activity, signal transduction, and transcriptional regulation. Sixty-one genes were more highly expressed when the MCQ lost its effectiveness (〉50% strength loss) and had functions related to oxalate degradation; cytochrome P450 activity; H 2 O 2 production and degradation; degradation of cellulose, hemicellulose, and pectin; hexose transport; membrane glycerophospholipid metabolism; and cell wall chemistry. Ten of these differentially regulated genes were quantified by reverse transcriptase PCR for a more in-depth study (4 time points on wood with or without MCQ treatment). Our results showed that MCQ induced higher than normal levels of expression for four genes (putative annotations for isocitrate lyase, glyoxylate dehydrogenase, laccase, and oxalate decarboxylase 1), while four other genes (putative annotations for oxalate decarboxylase 2, aryl alcohol oxidase, glycoside hydrolase 5, and glycoside hydrolase 10) were repressed. The significance of these results is that we have identified several genes that appear to be coregulated, with putative functions related to copper tolerance and/or wood decay.

Description: A deletion variant of the dengue virus (DENV) serotype 2 (DENV2) Tonga/74 strain lacking 30 nucleotides from its 3' untranslated region (rDEN230) has previously been established for use in a controlled human DENV challenge model. To evaluate if this model is appropriate for the derivation of correlates of protection for DENV vaccines on the basis of cellular immunity, we wanted to compare the cellular immune response to this challenge strain to the response induced by natural infection. To achieve this, we predicted HLA class I- and class II-restricted peptides from rDEN230 and used them in a gamma interferon enzyme-linked immunosorbent spot assay to interrogate CD8 + and CD4 + T cell responses in healthy volunteers infected with rDEN230. At the level of CD8 responses, vigorous ex vivo responses were detected in approximately 80% of donors. These responses were similar in terms of the magnitude and the numbers of epitopes recognized to the responses previously observed in peripheral blood mononuclear cells from donors from regions where DENV is hyperendemic. The similarity extended to the immunodominance hierarchy of the DENV nonstructural proteins, with NS3, NS5, and NS1 being dominant in both donor cohorts. At the CD4 level, the responses to rDEN230 vaccination were less vigorous than those to natural DENV infection and were more focused on nonstructural proteins. The epitopes recognized following rDEN230 infection and natural infection were largely overlapping for both the CD8 (100%) and CD4 (85%) responses. Finally, rDEN230 induced stronger CD8 responses than other, more attenuated DENV isolates. IMPORTANCE The lack of a known correlate of protection and the failure of a neutralizing antibody to correlate with protection against dengue virus have highlighted the need for a human DENV challenge model to better evaluate the candidate live attenuated dengue vaccines. In this study, we sought to characterize the immune profiles of rDEN230-infected subjects and to compare the profiles with those for subjects from areas where DENV is hyperendemic. Our data demonstrate that T cell responses to rDENV230 are largely similar to those to natural infection in terms of specificity, highlighting that the response to this virus in humans is appropriate as a model for the T cell response to primary DENV2 infection.