A Francisella tularensis Live Vaccine Strain That Improves Stimulation of Antigen-Presenting Cells Does Not Enhance Vaccine Efficacy

Vaccination is a proven strategy to mitigate morbidity and mortality of infectious diseases. The methodology of identifying and testing new vaccine candidates could be improved with rational design and in vitro testing prior to animal experimentation. The tularemia vaccine, Francisella tularensis live vaccine strain (LVS), does not elicit complete protection against lethal challenge with a virulent type A Francisella strain. One factor that may contribute to this poor performance is limited stimulation of antigen-presenting cells. In this study, we examined whether the interaction of genetically modified LVS strains with human antigen-presenting cells correlated with effectiveness as tularemia vaccine candidates. Human dendritic cells infected with wild-type LVS secrete low levels of proinflammatory cytokines, fail to upregulate costimulatory molecules, and activate human T cells poorly in vitro. One LVS mutant, strain 13B47, stimulated higher levels of proinflammatory cytokines from dendritic cells and macrophages and increased costimulatory molecule expression on dendritic cells compared to wild type. Additionally, 13B47-infected dendritic cells activated T cells more efficiently than LVS-infected cells. A deletion allele of the same gene in LVS displayed similar in vitro characteristics, but vaccination with this strain did not improve survival after challenge with a virulent Francisella strain. In vivo, this mutant was attenuated for growth and did not stimulate T cell responses in the lung comparable to wild type. Therefore, stimulation of antigen-presenting cells in vitro was improved by genetic modification of LVS, but did not correlate with efficacy against challenge in vivo within this model system

Genetic and environmental variation in continuous phenotypes in the ABCD Study®

Twin studies yield valuable insights into the sources of variation, covariation and causation in human traits. The ABCD Study® (abcdstudy.org) was designed to take advantage of four universities known for their twin research, neuroimaging, population-based sampling, and expertise in genetic epidemiology so that representative twin studies could be performed. In this paper we use the twin data to: (i) provide initial estimates of heritability for the wide range of phenotypes assessed in the ABCD Study using a consistent direct variance estimation approach, assuring that both data and methodology are sound; and (ii) provide an online resource for researchers that can serve as a reference point for future behavior genetic studies of this publicly available dataset. Data were analyzed from 772 pairs of twins aged 9-10 years at study inception, with zygosity determined using genotypic data, recruited and assessed at four twin hub sites. The online tool provides twin correlations and both standardized and unstandardized estimates of additive genetic, and environmental variation for 14,500 continuously distributed phenotypic features, including: structural and functional neuroimaging, neurocognition, personality, psychopathology, substance use propensity, physical, and environmental trait variables. The estimates were obtained using an unconstrained variance approach, so they can be incorporated directly into meta-analyses without upwardly biasing aggregate estimates. The results indicated broad consistency with prior literature where available and provided novel estimates for phenotypes without prior twin studies or those assessed at different ages. Effects of site, self-identified race/ethnicity, age and sex were statistically controlled. Results from genetic modeling of all 53,172 continuous variables, including 38,672 functional MRI variables, will be accessible via the user-friendly open-access web interface we have established, and will be updated as new data are released from the ABCD Study. This paper provides an overview of the initial results from the twin study embedded within the ABCD Study, an introduction to the primary research domains in the ABCD study and twin methodology, and an evaluation of the initial findings with a focus on data quality and suitability for future behavior genetic studies using the ABCD dataset. The broad introductory material is provided in recognition of the multidisciplinary appeal of the ABCD Study. While this paper focuses on univariate analyses, we emphasize the opportunities for multivariate, developmental and causal analyses, as well as those evaluating heterogeneity by key moderators such as sex, demographic factors and genetic background

The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis

Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensisinvades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks

The use of resazurin as a novel antimicrobial agent against Francisella tularensis

The highly infectious and deadly pathogen, Francisella tularensis, is classified by the CDC as a Category A bioterrorism agent. Inhalation of a single bacterium results in an acute pneumonia with a 30-60% mortality rate without treatment. Due to the prevalence of antibiotic resistance, there is a strong need for new types of antibacterial drugs. Resazurin is commonly used to measure bacterial and eukaryotic cell viability through its reduction to the fluorescent product resorufin. When tested on various bacterial taxa at the recommended concentration of 44 µM, a potent bactericidal effect was observed against various Francisella and Neisseria species, including the human pathogens type A F. tularensis (Schu S4) and N. gonorrhoeae. As low as 4.4 µM resazurin was sufficient for a 10-fold reduction in F. tularensis growth. In broth culture, resazurin was reduced to resorufin by F. tularensis. However, resorufin also suppressed the growth of F. tularensis suggesting that the process of reducing resazurin was not responsible for the observed antimicrobial effect. Replication of F. tularensis in primary human macrophages and non-phagocytic cells was abolished following treatment with 44 μM resazurin indicating this compound could be an effective therapy for tularemia in vivo

Human monocyte-derived DCs mature following exposure to LVS strain 13B47.

<p>DCs were stimulated with either LVS, 13B47, ΔcapC, 1664d, or <i>E. coli</i> for 24 hours (MOI = 10). Cells were harvested and analyzed for changes in surface expression of CD86, CD80, and HLA-DR. Cells were gated on CD1b-positive population. (A) Representative histograms for CD86, CD80, and HLA-DR expression on LVS-, 13B47-, and <i>E. coli</i>-treated DCs from one experiment. Histograms for ΔcapC- and 1664d-infected DCs were similar to LVS (data not shown). (B) Mean percentages of DCs with high CD86, CD80, and HLA-DR expression (± SEM) from three individual experiments with different donors. (C) Geometric mean fluorescence intensities (GMFI) of CD86, CD80, and HLA-DR (± SEM) on DCs from three individual experiments with different donors. Statistically significant differences in CD86, CD80, and HLA-DR expression by infected DCs were determined by one-way ANOVA, followed by Bonferroni comparison of means (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Bacterial strains, plasmids, and primers used in this study.

<p>Bacterial strains, plasmids, and primers used in this study.</p

Enhanced proliferation and IFN-γ production by CD4⁺ T cells stimulated with LVS strain 13B47-infected DCs.

<p>Purified CFSE-labeled CD4⁺ T cells from a single donor were co-cultured with either <i>E. coli</i>-infected, <i>F. tularensis</i> LVS-infected, or 13B47-infected DCs from a different donor at a ratio of 10∶1 (2×10⁵ T cells/2×10⁴ DCs/well) for 5 days. (A) Representative dot plots showing loss of CFSE fluorescence versus CD4 staining on day 5 for each group from one experiment. (B) The mean percentages of proliferating CD4⁺ T cells were calculated (± SEM) from five individual experiments with different donors. (C) IFN-γ levels were measured in day 5 supernatants by ELISA. Data are presented as the mean ± SEM from four individual experiments with different donors that were represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031172#pone-0031172-g004" target="_blank">Figure 4B</a>. BLD = below limits of detection of the ELISA. Statistically significant differences in mean percentages and GMFI for all groups were determined by one-way ANOVA, followed by Bonferroni comparison of means (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Systemic Immunomodulatory Treatments for Atopic Dermatitis:Update of a Living Systematic Review and Network Meta-analysis

Importance: Systemic treatments for atopic dermatitis are being evaluated primarily in placebo-controlled trials; network meta-analysis can provide relative efficacy and safety estimates for treatments that have not been compared head to head. Objective: To compare reported measures of efficacy and assessments of safety in clinical trials of systemic treatments for atopic dermatitis in a living systematic review and network meta-analysis. Data Sources: The Cochrane Central Register of Controlled Trials, MEDLINE, Embase, Latin American and Caribbean Health Science Information database, Global Resource of EczemA Trials database, and trial registries were searched through June 15, 2021. Study Selection: Randomized clinical trials examining 8 or more weeks of treatment with systemic immunomodulatory medications for moderate-to-severe atopic dermatitis were included after screening titles, abstracts, and papers in duplicate. Data Extraction and Synthesis: Data were abstracted in duplicate. Bayesian network meta-analyses and assessed Grading of Recommendations Assessment, Development and Evaluation certainty of evidence were performed. The updated analysis was completed from June to December 2021. Main Outcomes and Measures: Outcomes include change in Eczema Area and Severity Index (EASI), Patient Oriented Eczema Measure (POEM), Dermatology Life Quality Index (DLQI), and Peak Pruritus Numeric Rating Scales (PP-NRS). Results: Since October 2019, 21 new studies were added, for a total of 60 trials with 16579 patients. Up to 16 weeks of treatment in adults, abrocitinib, 200 mg daily (mean difference [MD], 2.2; 95% credible interval [CrI], 0.2-4.0; high certainty) and upadacitinib, 30 mg daily (MD, 2.7; 95% CrI, 0.6-4.7; high certainty) were associated with reduced EASI slightly more than dupilumab, 600 mg then 300 mg every 2 weeks. Abrocitinib, 100 mg daily (MD,-2.1; 95% CrI,-4.1 to-0.3; high certainty), baricitinib, 4 mg daily (MD,-3.2; 95% CrI,-5.7 to-0.8; high certainty), baricitinib, 2 mg daily (MD,-5.2; 95% CrI,-7.5 to-2.9; high certainty) and tralokinumab, 600 mg then 300 mg every 2 weeks (MD,-3.5; 95% CrI,-5.8 to-1.3; high certainty) were associated with reduced EASI slightly less than dupilumab. There was little or no difference between upadacitinib, 15 mg daily, and dupilumab (MD, 0.2; 95% CrI,-1.9 to 2.2; high certainty). The pattern of results was similar for POEM, DLQI, and PP-NRS. Conclusions and Relevance: In this systematic review and meta-analysis, abrocitinib, 200 mg; and upadacitinib, 30 mg daily, were associated with slightly better scores than dupilumab, and upadacitinib, 15 mg daily, was associated with similar scores to dupilumab. Abrocitinib, 100 mg daily, baricitinib, 4 mg and 2 mg daily, and tralokinumab, 300 mg, every 2 weeks were associated with slightly worse scores

<i>F. tularensis</i> LVS strain 13B47 stimulates human monocyte-derived DCs and macrophages to produce proinflammatory cytokines.

<p>LVS and LVS mutants, 13B47, ΔcapC, and 1664d, were cultured overnight in a chemically defined media (CDM) or Mueller-Hinton (MH) broth. The four bacterial cultures were used to inoculate macrophages (A, 1.5×10⁵ cells/well) and DCs (B and C, 5×10⁵ cells/well) at an MOI of 10. As a positive control, DCs and macrophages were cultured with <i>E. coli</i> strain sd-4 (MOI = 10). Supernatants were harvested after 24 hours (A–B) or at indicated times (C), and TNF-α, IL-6, and IL-12p40 were measured by ELISA. Data are expressed as the mean ± SEM of three individual experiments with different donors. The level of cytokine production from each group was compared by a one (A–B) or two-way ANOVA (C), followed by the Bonferroni comparison of means. ($$$, <i>p</i><0.001 for <i>E. coli</i> vs. all other groups). When comparing only the DCs infected with the <i>F. tularensis</i> strains, 13B47 elicited higher cytokine production than the uninfected group (A–B) or LVS cultured in the same media (C). *, p<0.05; **, p<0.01; ***, p<0.001. BLD = below limits of detection of the ELISA.</p

Survival of immunized mice following intratracheal Schu S4 challenge^a.

a<p>Mice were immunized with either LVS or ΔFTL_0883 at the indicated dose and then challenged with 100 CFU of Schu S4 i.t.</p>b<p>Significant difference p<0.005 by log rank test.</p