Background: Grass pollen subcutaneous immunotherapy (SCIT) is associated with induction of serum IgG 4-associated inhibitory antibodies that prevent IgE-facilitated allergen binding to B cells. Objective: We sought to determine whether SCIT induces nasal allergen-specific IgG 4 antibodies with inhibitory activity that correlates closely with clinical response. Methods: In a cross-sectional controlled study, nasal fluid and sera were collected during the grass pollen season from 10 SCIT-treated patients, 13 untreated allergic patients (with seasonal allergic rhinitis [SAR]), and 12 nonatopic control subjects. Nasal and serum IgE and IgG 4 levels to Phleum pratense components were measured by using the Immuno Solid Allergen Chip microarray. Inhibitory activity was measured by IgE-facilitated allergen binding assay. IL-10 1 regulatory B cells were quantified in peripheral blood by using flow cytometry. Results: Nasal and serum Phl p 1-and Phl p 5-specific IgE levels were increased in patients with SAR compared to nonatopic control subjects (all, P < .001) and SCIT-treated patients (nasal, P < .001; serum Phl p 5, P 5 .073). Nasal IgG 4 levels were increased in the SCIT group compared to those in the SAR group (P < .001) during the pollen season compared to out of season. IgG-associated inhibitory activity in nasal fluid and serum was significantly increased in the SCIT group compared to that in the SAR (both, P < .01). The magnitude of the inhibitory activity was 93% (P < .
The initial adaptive responses to nutrient depletion in bacteria often occur at the level of gene expression. Hfq is an RNA-binding protein present in diverse bacterial lineages and contributes to many different aspects of RNA metabolism during gene expression. Using photoactivated localization microscopy (PALM) and single molecule tracking, we demonstrate that Hfq forms a distinct and reversible focus-like structure in Escherichia coli specifically experiencing long-term nitrogen (N) starvation. Using the ability of T7 phage to replicate in N-starved bacteria as a biological probe of E. coli cell function during N starvation, we demonstrate that Hfq foci have a role in the adaptive response of E. coli to long-term N starvation. We further show that Hfq foci formation does not depend on gene expression once N starvation has set in and occurs independently of the transcription factor N-regulatory protein C (NtrC), that activates the initial adaptive response to N starvation in E. coli. These results serve as a paradigm to demonstrate that bacterial adaptation to long-term nutrient starvation can be spatiotemporally coordinated and can occur independently of de novo gene expression during starvation.
RationaleNasal allergen provocations may be useful in investigating the pathophysiology of allergic rhinitis and effects of treatments.ObjectiveTo use grass pollen nasal allergen challenge (NAC) to investigate the effects of allergen immunotherapy in a cross‐sectional study.MethodsWe studied nasal and cutaneous responses in untreated subjects with seasonal grass‐pollen allergic rhinitis (n = 14) compared with immunotherapy‐treated allergics (n = 14), plus a nonatopic control group (n = 14). Volunteers underwent a standardized NAC with 2000 biological units of timothy grass allergen (equivalent to 1.3 μg major allergen, Phl p5). Nasal fluid was collected and analysed by ImmunoCAP and multiplex assays. Clinical response was assessed by symptom scores and peak nasal inspiratory flow (PNIF). Cutaneous response was measured by intradermal allergen injection. Retrospective seasonal symptom questionnaires were also completed.ResultsImmunotherapy‐treated patients had lower symptom scores (P = 0.04) and higher PNIF (P = 0.02) after challenge than untreated allergics. They had reduced early (P = 0.0007) and late (P < 0.0001) skin responses, and lower retrospective seasonal symptom scores (P < 0.0001). Compared to untreated allergics, immunotherapy‐treated patients had reduced nasal fluid concentrations of IL‐4, IL‐9 and eotaxin (all P < 0.05, 8 h level and/or area under the curve comparison), and trends for reduced IL‐13 (P = 0.07, area under the curve) and early‐phase tryptase levels (P = 0.06).ConclusionsNasal allergen challenge is sensitive in the detection of clinical and biological effects of allergen immunotherapy and may be a useful surrogate marker of treatment efficacy in future studies.
SummaryBackgroundCat allergen is widely distributed in homes and schools; allergic sensitization is common.ObjectiveTo develop a model of cat allergen nasal challenge to establish dose–response and time–course characteristics and investigate local and systemic biomarkers of allergic inflammation.MethodsNineteen cat‐allergic individuals underwent titrated nasal challenge, range 0.243 to 14.6 μg/mL Fel d1, and matched diluent‐only provocation. Clinical response to 8 h was assessed by symptom scores and peak nasal inspiratory flow (PNIF). Nasal fluid was collected using polyurethane sponges and analysed by ImmunoCAP and multiplex assays. Whole blood flow cytometry for basophil surface CD63, CD107a, and CD203c was carried out at baseline and 6 h post‐challenge.ResultsA dose–response to allergen was seen in symptom scores and PNIF, maximal at 10 000 BU/mL (4.87 μg/mL Fel d1), P < 0.0001 vs. diluent. Nasal fluid tryptase was elevated at 5 min after challenge (P < 0.05 vs. diluent); eotaxin, IL‐4, ‐5, ‐9, and ‐13 were increased at 8 h (P < 0.05 to P < 0.0001 vs. diluent); TSLP was undetectable; IL‐10, IL‐17A, and IL‐33 were unchanged compared to diluent challenge. Nasal fluid IL‐5 and IL‐13 correlated inversely with PNIF after challenge (IL‐5, r = −0.79, P < 0.0001; IL‐13, r = −0.60, P = 0.006). Surface expression of CD63 and CD107a was greater at 6 h than at baseline, both in the presence (both P < 0.05) and absence (CD63, P < 0.01; CD107a, P < 0.05) of in vitro allergen stimulation; no changes were seen on diluent challenge day.ConclusionsCat allergen nasal challenge produces local and systemic Th2‐driven inflammatory responses and has potential as a surrogate outcome measure in clinical trials.
The initial adaptive transcriptional response to nitrogen (N) starvation in Escherichia coli involves large-scale alterations to the transcriptome mediated by the transcriptional activator, NtrC. One of these NtrC-activated genes is yeaG, which encodes a conserved bacterial kinase. Although it is known that YeaG is required for optimal survival under sustained N starvation, the molecular basis by which YeaG benefits N starved E. coli remains elusive. By combining transcriptomics with targeted metabolomics analyses, we demonstrate that the methionine biosynthesis pathway becomes transcriptionally dysregulated in ΔyeaG bacteria experiencing sustained N starvation. It appears the ability of MetJ, the master transcriptional repressor of methionine biosynthesis genes, to effectively repress transcription of genes under its control is compromised in ΔyeaG bacteria under sustained N starvation, resulting in transcriptional derepression of MetJ-regulated genes. Although the aberrant biosynthesis does not appear to be a contributing factor for the compromised viability of ΔyeaG bacteria experiencing sustained N starvation, this study identifies YeaG as a novel regulatory factor in E. coli affecting the transcription of methionine biosynthesis genes under sustained N starvation.
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