Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness

<div><p>Background</p><p><b>P</b>ractical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF).</p><p>Methods</p><p>We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 <i>per protocol</i>). Doses of ultrapure LPS (1, 10, 30 or 100μg/100μl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge.</p><p>Results</p><p>Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1β, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100μg LPS). At 100μg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10μg and 30μg LPS).</p><p>Conclusions</p><p>Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa.</p><p>Key Messages</p><p>Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1β, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS.</p><p>Trial Registration</p><p>Clinical Trials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT02284074?term=nasal+lipopolysaccharide&rank=1" target="_blank">NCT02284074</a></p></div

Genetic dissection of maturity using RFLPs.

Several genetic regions having major effects on maturity have been identified using RFLP analysis. One such region on chromosomes 8 is important across several diverse genotypes and ccounts for up to 50% of the variation for maturity in a cross involving an inbred line (N28) and a 20-backcross generation derivative (N28E). In addition to the chromsome 8 QTL for maturity, we have evidence using the backcross-derived line approach for regions controlling maturity on chromosomes 1, 2, 3, 5, 7, and 9. Chromosome 5 appears to be especially important in both magnitude of effect and across several genetic hackground. Maturity as measured by days to pollen shed or silking may be controlled by additive or nearly dominant gene action depending on the chromosome region. The marker-trait linkages were consistent across environnments based on A662 X B73 F3 per se and testcross evaluations from three locations in one year. UMC12 (chromosome 8) and UMC54 (chromosome 5) also marked important regions for maturity in these materials

Quantifying the impact of the modifiable areal unit problem when estimating the health effects of air pollution

Air pollution is a major public health concern, and large numbers of epidemiological studies have been conducted to quantify its impacts. One study design used to quantify these impacts is a spatial areal unit design, which estimates a population level association using data on air pollution concentrations and disease incidence that have been spatially aggregated to a set of non‐overlapping areal units. A major criticism of this study design is that the specification of these areal units is arbitrary, and if one changed their boundaries then the aggregated datawould change despite the locations of the disease cases and the air pollution surface remaining the same. This is known as the modifiable areal unit problem (MAUP), and this is the first paper to quantify its likely effects in air pollution and health studies. Additionally, we derive an aggregate model for these data directly from an idealised individual level risk model, and show that it provides better estimation than the commonly used ecological model. Our work is motivated by a new study of air pollution and health in Scotland, and we find consistent significant associations between air pollution and respiratory disease but not for circulatory disease

Patterns of systemic and local inflammation in patients with asthma hospitalised with influenza.

BACKGROUND: Patients with asthma are at risk of hospitalisation with influenza, but the reasons for this predisposition are unknown. STUDY SETTING: A prospective observational study of adults with PCR-confirmed influenza in 11 UK hospitals, measuring nasal, nasopharyngeal and systemic immune mediators and whole-blood gene expression. RESULTS: Of 133 admissions, 40 (30%) had previous asthma; these were more often female (70% versus 38.7%, OR 3.69, 95% CI 1.67 to 8.18, p=0.0012), required less mechanical ventilation (15% versus 37.6%, χ2 6.78, p=0.0338) and had shorter hospital stays (mean 8.3 versus 15.3 d, p=0.0333) than those without. In patients without asthma, severe outcomes were more frequent in those given corticosteroids (OR=2.63, 95% CI=1.02-6.96, p=0.0466) or presenting >4 days after disease onset (OR 5.49, 95% CI 2.28-14.03, p=0.0002). Influenza vaccination in at-risk groups (including asthma) were lower than intended by national policy and the early use of antiviral medications were less than optimal. Mucosal immune responses were equivalent between groups. Those with asthma had higher serum IFN-α but lower serum TNF, IL-5, IL-6, CXCL8, CXCL9, IL-10, IL-17 and CCL2 levels (all p<0.05); both groups had similar serum IL-13, total IgE, periostin and blood eosinophil gene expression levels. Asthma diagnosis was unrelated to viral load, IFN-α, IFN-γ, IL-5 or IL-13 levels. CONCLUSIONS: Asthma is common in those hospitalised with influenza, but may not represent classical Type 2-driven disease. Those admitted with influenza tend to be female with mild serum inflammatory responses, increased serum IFN-α levels and good clinical outcomes.MOSAIC (Mechanisms of Severe Influenza Consortium) was supported by the MRC (UK) and Wellcome Trust (090382/Z/09/Z). The study was also supported by the National Institute of Healthcare Research (NIHR) Biomedical Research Centres (BRCs) in London and Liverpool and by the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Respiratory Infections at Imperial College London in partnership with Public Health England (PHE). P.J.O. was supported by EU FP7 PREPARE project 602525

Regulatory T cells expressing granzyme B play a critical role in controlling lung inflammation during acute viral infection

The inflammatory response to lung infections must be tightly regulated, enabling pathogen elimination while maintaining crucial gas exchange. Using recently described “depletion of regulatory T cell” (DEREG) mice, we found that selective depletion of regulatory T cells (Tregs) during acute respiratory syncytial virus (RSV) infection enhanced viral clearance but increased weight loss, local cytokine and chemokine release, and T-cell activation and cellular influx into the lungs. Conversely, inflammation was decreased when Treg numbers and activity were boosted using interleukin-2 immune complexes. Unexpectedly, lung (but not draining lymph node) Tregs from RSV-infected mice expressed granzyme B (GzmB), and bone marrow chimeric mice with selective loss of GzmB in the Treg compartment displayed markedly enhanced cellular infiltration into the lung after infection. A crucial role for GzmB-expressing Tregs has not hitherto been described in the lung or during acute infections, but may explain the inability of children with perforin/GzmB defects to regulate immune responses to infection. The effects of RSV infection in mice with defective immune regulation closely parallel the observed effects of RSV in children with bronchiolitis, suggesting that the pathogenesis of bronchiolitis may involve an inability to regulate virus-induced inflammation

Measurement of two-halo neutron transfer reaction p(11^{11}Li,9^{9}Li)t at 3AA MeV

The p(\nuc{11}{Li},\nuc{9}{Li})t reaction has been studied for the first time at an incident energy of 3$A$ MeV delivered by the new ISAC-2 facility at TRIUMF. An active target detector MAYA, build at GANIL, was used for the measurement. The differential cross sectionshave been determined for transitions to the \nuc{9}{Li} ground andthe first excited states in a wide range of scattering angles. Multistep transfer calculations using different \nuc{11}{Li} model wave functions, shows that wave functions with strong correlations between the halo neutrons are the most successful in reproducing the observation.Comment: 6 pages, 3 figures, submitted to Physical Review Letter

Eliminating a Region of Respiratory Syncytial Virus Attachment Protein Allows Induction of Protective Immunity without Vaccine-enhanced Lung Eosinophilia

In a murine model of respiratory syncytial virus disease, prior sensitization to the attachment glycoprotein (G) leads to pulmonary eosinophilia and enhanced illness. Three different approaches were taken to dissect the region of G responsible for enhanced disease and protection against challenge. First, mutant viruses, containing frameshifts that altered the COOH terminus of the G protein, were used to challenge mice sensitized by scarification with recombinant vaccinia virus (rVV) expressing wild-type G. Second, cDNA expressing these mutated G proteins were expressed by rVV and used to vaccinate mice before challenge with wild-type respiratory syncytial virus (RSV). These studies identified residues 193–205 to be responsible for G-induced weight loss and lung eosinophilia and showed that this region was not was not necessary for induction of protective immunity. Third, mice were sensitized using an rVV that expressed only amino acids 124–203 of the G protein. Upon RSV challenge, mice sensitized with this rVV developed enhanced weight loss and eosinophilia. This is the first time that a region within RSV (amino acids 193–203) has been shown to be responsible for induction of lung eosinophilia and disease enhancement. Moreover, we now show that it is possible to induce protective immunity with an altered G protein without inducing a pathological response

Potential therapeutic implications of new insights into respiratory syncytial virus disease

Viral bronchiolitis is the most common cause of hospitalization in infants under 6 months of age, and 70% of all cases of bronchiolitis are caused by respiratory syncytial virus (RSV). Early RSV infection is associated with respiratory problems such as asthma and wheezing later in life. RSV infection is usually spread by contaminated secretions and infects the upper then lower respiratory tracts. Infected cells release proinflammatory cytokines and chemokines, including IL-1, tumor necrosis factor-α, IL-6, and IL-8. These activate other cells and recruit inflammatory cells, including macrophages, neutrophils, eosinophils, and T lymphocytes, into the airway wall and surrounding tissues. The pattern of cytokine production by T lymphocytes can be biased toward 'T-helper-1' or 'T-helper-2' cytokines, depending on the local immunologic environment, infection history, and host genetics. T-helper-1 responses are generally efficient in antiviral defense, but young infants have an inherent bias toward T-helper-2 responses. The ideal intervention for RSV infection would be preventive, but the options are currently limited. Vaccines based on protein subunits, live attenuated strains of RSV, DNA vaccines, and synthetic peptides are being developed; passive antibody therapy is at present impractical in otherwise healthy children. Effective vaccines for use in neonates continue to be elusive but simply delaying infection beyond the first 6 months of life might reduce the delayed morbidity associated with infantile disease

Biphasic activation of complement and fibrinolysis during the human nasal allergic response

Complement, coagulation and fibrinolysis contribute to the pathology of many respiratory diseases. Here we detail the biphasic activation of these pathways following nasal allergen challenge. Understanding these mechanisms may lead to therapeutic insight in common respiratory diseases

Early mucosal events drive distinct mucosal and systemic antibody responses to live attenuated influenza vaccine

Compared to intramuscular vaccines, nasally administered vaccines have the advantage of inducing local mucosal immune responses that may block infection and interrupt transmission of respiratory pathogens. Live attenuated influenza vaccine (LAIV) is effective in preventing influenza in children, but a correlate of protection for LAIV remains unclear. Studying young adult volunteers, we observe that LAIV induces distinct, compartmentalized, antibody responses in the mucosa and blood. Seeking immunologic correlates of these distinct antibody responses we find associations with mucosal IL-33 release in the first 8 hours post-inoculation and divergent CD8+ and circulating T follicular helper (cTfh) T cell responses 7 days post-inoculation. Mucosal antibodies are induced separately from blood antibodies, are associated with distinct immune responses early post-inoculation, and may provide a correlate of protection for mucosal vaccination. This study was registered as NCT04110366 and reports primary (mucosal antibody) and secondary (blood antibody, and nasal viral load and cytokine) endpoint data