Previous publications from our and other groups identified E2F1 as a transcription factor involved in the regulation of inflammatory response to Toll-like receptor ligands including LPS. In this ...study, we challenged E2F1-deficient mice with LPS systemically and demonstrated decreased survival despite attenuated inflammatory response compared with controls. Gene expression profiling of liver tissue identified a dampened transcriptional response in the coagulation cascade in B6;129(E2F1-/-) compared with B6x129 F2 mice. These data were further corroborated by increased prothrombin time, activated partial thromboplastin time, and fibrin split products in the blood of E2F1-deficient mice, suggesting disseminated intravascular coagulation as a consequence of uncontrolled sepsis providing at least a partial explanation for their decreased survival despite attenuated inflammatory response. To identify novel miRNAs involved in the innate immune response to LPS, we also performed miRNA profiling of liver tissue from B6;129(E2F1-/-) and B6x129 F2 mice treated with LPS systemically. Our analysis identified a set of miRNAs and their mRNA targets that are significantly differentially regulated in E2F1-deficient but not control mice including let-7g, miR-101b, miR-181b, and miR-455. These miRNAs represent novel regulators of the innate immune response. In summary, we used transcriptional and miRNA profiling to characterize the response of E2F1-deficient mice to systemic LPS.
Previous work demonstrated that pre-exposure to ozone primes innate immunity and increases Toll-like receptor-4 (TLR4)-mediated responses to subsequent stimulation with LPS. To explore the pulmonary ...innate immune response to ozone exposure further, we investigated the effects of ozone in combination with Pam3CYS, a synthetic TLR2/TLR1 agonist. Whole-lung lavage (WLL) and lung tissue were harvested from C57BL/6 mice after exposure to ozone or filtered air, followed by saline or Pam3CYS 24 hours later. Cells and cytokines in the WLL, the surface expression of TLRs on macrophages, and lung RNA genomic expression profiles were examined. We demonstrated an increased WLL cell influx, increased IL-6 and chemokine KC (Cxcl1), and decreased macrophage inflammatory protein (MIP)-1α and TNF-α in response to Pam3CYS as a result of ozone pre-exposure. We also observed the increased cell surface expression of TLR4, TLR2, and TLR1 on macrophages as a result of ozone alone or in combination with Pam3CYS. Gene expression analysis of lung tissue revealed a significant increase in the expression of genes related to injury repair and the cell cycle as a result of ozone alone or in combination with Pam3CYS. Our results extend previous findings with ozone/LPS to other TLR ligands, and suggest that the ozone priming of innate immunity is a general mechanism. Gene expression profiling of lung tissue identified transcriptional networks and genes that contribute to the priming of innate immunity at the molecular level.
BACKGROUND: Soybean seeds are rich in natural protein and are favorable environments for targeted protein expression. Soybeans represent an ideal platform for the production of novel vaccines that, ...in theory, do not require a cold chain. This study investigated the stability of a soybean-derived antigen following long-term storage, formulation, and shipment overseas in the absence of refrigeration.RESULTS: Transgenic seeds can be stored for more than 4 years at ambient temperature with no detectable loss of FanC antigen stability. Conventional processing methods utilizing heat, mechanical extraction and solvent extraction resulted in practical formulations that could be lyophilized, stored as dried milk powder and rehydrated in water without loss of FanC antigen stability. Overseas shipment of transgenic seed powder and soymilk formulations in the absence of a cold chain had no adverse effects on formulations or the FanC antigen.CONCLUSION: The feasibility of long-term storage, processing and shipment of transgenic soy products in the absence of a cold chain was demonstrated. Soybeans represent a practical platform for development of novel vaccines to potentially address the worldwide need for vaccines that are cost-effective, easy to formulate and can be manufactured, stored and transported without refrigeration.
The upper 50-kDa region of myosin may be critical for coupling between the nucleotide- and actin-binding regions. We introduced a tetracysteine motif in the upper 50-kDa domain (residues 292-297) of ...myosin V containing a single IQ domain (MV 1IQ), allowing us to label this site with the fluorescein biarscenical hairpin-binding dye (FlAsH) (MV 1IQ FlAsH). The enzymatic properties of MV 1IQ FlAsH were similar to those of unlabeled MV 1IQ except for a 3-fold reduced ADP-release rate. MV 1IQ FlAsH was also capable of moving actin filaments in the in vitro motility assay. To examine rotation of the upper 50-kDa region, we determined the difference in the degree of energy transfer from N-methylanthraniloyl (mant)-labeled nucleotides to FlAsH in both steady-state and transient kinetic experiments. The energy transfer efficiency was higher with mant-ATP (0.65 ± 0.02) compared with mant-ADP (0.55 ± 0.02) in the absence of actin. Stopped-flow measurements suggested that the energy transfer efficiency decreased with phosphate release (0.04 s-1) in the absence of actin. In contrast, upon mixing MV 1IQ FlAsH in the ADP·Pi state with actin, a decrease in the energy transfer signal was observed at a rate of 13 s-1, similar to the ADP release rate. Our results demonstrate there was no change in the energy transfer signal upon actin-activated phosphate release and suggest that actin binding alters the dynamics of the upper 50-kDa region, which may be critical for the ability of myosin to bind tightly to both ADP and actin.
Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of enteric diseases affecting livestock and humans. Edible transgenic plants producing E. coli fimbrial subunit proteins have the ...potential to vaccinate against these diseases, but have not reached their full potential as a renewable source of oral vaccines due in part to insufficient levels of recombinant protein accumulation. Previously, we reported that cytosol targeting of the E. coli K99 fimbrial subunit antigen resulted in FanC accumulation to ~0.4% of total soluble protein in soybean leaves (Piller et al. in Planta 222:6-18, 2005). In this study, we report on the subcellular targeting of FanC to chloroplasts. Twenty-two transgenic T₁ progeny derived from seven individual T₀ transformation events were characterized, and 17 accumulated transgenic FanC. All of the characterized events displayed relatively low T-DNA complexity, and all exhibited proper targeting of FanC to the chloroplast. Accumulation of chloroplast-targeted FanC was ~0.08% of total soluble leaf protein, or ~5-fold less than cytosol-targeted FanC. Protein analysis of leaves at various stages of maturity suggested stability of chloroplast-targeted FanC throughout leaf maturation. Furthermore, mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing chloroplast-targeted FanC developed significant antibody titers against FanC. This is the first report of subcellular targeting of a vaccine subunit antigen in soybean.
IMPORTANCE: Preclinical models suggest dysregulation of the renin-angiotensin system (RAS) caused by SARS-CoV-2 infection may increase the relative activity of angiotensin II compared with ...angiotensin (1-7) and may be an important contributor to COVID-19 pathophysiology. OBJECTIVE: To evaluate the efficacy and safety of RAS modulation using 2 investigational RAS agents, TXA-127 (synthetic angiotensin 1-7) and TRV-027 (an angiotensin II type 1 receptor–biased ligand), that are hypothesized to potentiate the action of angiotensin (1-7) and mitigate the action of the angiotensin II. DESIGN, SETTING, AND PARTICIPANTS: Two randomized clinical trials including adults hospitalized with acute COVID-19 and new-onset hypoxemia were conducted at 35 sites in the US between July 22, 2021, and April 20, 2022; last follow-up visit: July 26, 2022. INTERVENTIONS: A 0.5-mg/kg intravenous infusion of TXA-127 once daily for 5 days or placebo. A 12-mg/h continuous intravenous infusion of TRV-027 for 5 days or placebo. MAIN OUTCOMES AND MEASURES: The primary outcome was oxygen-free days, an ordinal outcome that classifies a patient’s status at day 28 based on mortality and duration of supplemental oxygen use; an adjusted odds ratio (OR) greater than 1.0 indicated superiority of the RAS agent vs placebo. A key secondary outcome was 28-day all-cause mortality. Safety outcomes included allergic reaction, new kidney replacement therapy, and hypotension. RESULTS: Both trials met prespecified early stopping criteria for a low probability of efficacy. Of 343 patients in the TXA-127 trial (226 65.9% aged 31-64 years, 200 58.3% men, 225 65.6% White, and 274 79.9% not Hispanic), 170 received TXA-127 and 173 received placebo. Of 290 patients in the TRV-027 trial (199 68.6% aged 31-64 years, 168 57.9% men, 195 67.2% White, and 225 77.6% not Hispanic), 145 received TRV-027 and 145 received placebo. Compared with placebo, both TXA-127 (unadjusted mean difference, −2.3 95% CrI, −4.8 to 0.2; adjusted OR, 0.88 95% CrI, 0.59 to 1.30) and TRV-027 (unadjusted mean difference, −2.4 95% CrI, −5.1 to 0.3; adjusted OR, 0.74 95% CrI, 0.48 to 1.13) resulted in no difference in oxygen-free days. In the TXA-127 trial, 28-day all-cause mortality occurred in 22 of 163 patients (13.5%) in the TXA-127 group vs 22 of 166 patients (13.3%) in the placebo group (adjusted OR, 0.83 95% CrI, 0.41 to 1.66). In the TRV-027 trial, 28-day all-cause mortality occurred in 29 of 141 patients (20.6%) in the TRV-027 group vs 18 of 140 patients (12.9%) in the placebo group (adjusted OR, 1.52 95% CrI, 0.75 to 3.08). The frequency of the safety outcomes was similar with either TXA-127 or TRV-027 vs placebo. CONCLUSIONS AND RELEVANCE: In adults with severe COVID-19, RAS modulation (TXA-127 or TRV-027) did not improve oxygen-free days vs placebo. These results do not support the hypotheses that pharmacological interventions that selectively block the angiotensin II type 1 receptor or increase angiotensin (1-7) improve outcomes for patients with severe COVID-19. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04924660
In the present study, we questioned whether an oral formulation made from soybean seeds and containing the mucosal adjuvant, cholera toxin (CT), could induce any detectable antibody responses against ...soy proteins. Groups of mice were repeatedly gavaged with formulations containing high (15 mg) and low (1 mg) doses of soybean seed protein extracts, along with CT as an oral adjuvant. While robust mucosal and systemic antibody responses against CT were observed, no detectable IgA, IgM, IgG or IgE anti-soy protein antibodies could be found. Furthermore, mice in all treatment groups remained healthy and continued to gain weight over the course of the study. Collectively, these data demonstrate that oral soybean-based vaccine formulations do not induce detectable antibody responses to soy proteins, even when the formulations contain a potent mucosal adjuvant. These studies underscore the safety of oral vaccine formulations which might contain soy proteins as a component.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Maternal nutrition plays an essential role in offspring health and development. Critical stages include: (1) preconception, affecting oocyte development and uterine environment preparation; (2) ...gestation, affecting uterine environment and placental nutrient transfer and (3) postnatal, through lactation. It remains debatable which stage is most important, but arguably, the most complex cellular events occur during gestation. During this time, embryo development requires a well orchestrated and tightly regulated cascade of genetic, molecular and biochemical events. Among these are epigenetic events necessary for gene expression regulation. These produce heritable yet often reversible states established based on transcriptional needs of the cell. A growing body of research highlights the role of maternal nutrition in determining epigenetic states important for pre- and postnatal development. Here, we discuss recent findings addressing epigenetic response to nutrition with relevance to developmental origins of phenotypic outcome.
Abstract
We are exploring a novel platform technology using soybean seed-based formulations for the development of subunit vaccines that can be delivered via an oral route. One requirement for an ...effective subunit vaccine formulation is the inclusion of an adjuvant in order to elicit a robust immune response within the intestinal mucosa. When considering a soybean seed based formulation it is critical that there is no immune response to native soy proteins when administered orally with an adjuvant. One of the many advantages of a soybean seed-based platform for vaccine development is the fact that virtually every animal and human has been exposed to soy proteins given that soy is a major component of almost every diet. We hypothesize that routine consumption of soy proteins in a diet should effectively tolerize against soybean proteins and therefore abrogate the adjuvant effect of soy proteins. To test this hypothesis, we orally vaccinated groups of mice with formulations containing high (15mg) and low (1mg) doses of soybean seed extracts, along with cholera toxin (CT) as an adjuvant. As expected, there were no detectable sera IgE or IgG response in either group of vaccinated mice. Furthermore, there was no detectable IgA response against soy proteins in fecal extracts of immunized animals. Immune responses to CT in all groups were as predicted with a robust IgG response reflecting a Th2 subtype and an IgA response in fecal extract. Collectively, these data demonstrate that adjuvanted soybean-based formulations do not induce immune responses to soybean seed proteins, and underscore the vast potential for a novel soybean-based platform technology that can provide safe, stable and cost-effective vaccines worldwide.
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