Genomic attributes of airway commensal bacteria and mucosa.

Microbial communities at the airway mucosal barrier are conserved and highly ordered, in likelihood reflecting co-evolution with human host factors. Freed of selection to digest nutrients, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the initial results of systematic culture and whole-genome sequencing of the thoracic airway bacteria, identifying 52 novel species amongst 126 organisms that constitute 75% of commensals typically present in heathy individuals. Clinically relevant genes encode antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. Using whole-genome content we identify dysbiotic features that may influence asthma and chronic obstructive pulmonary disease. We match isolate gene content to transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways to sustain host interactions with microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosa in lung diseases of global significance.

Vitamin D and COVID-19-Revisited.

Vitamin D, when activated to 1,25-dihydroxyvitamin D, is a steroid hormone that induces responses in several hundred genes, including many involved in immune responses to infection. Without supplementation, people living in temperate zones commonly become deficient in the precursor form of vitamin D, 25-hydroxyvitamin D, during winter, as do people who receive less sunlight exposure or those with darker skin pigmentation. Studies performed pre-COVID-19 have shown significant but modest reduction in upper respiratory infections in people receiving regular daily vitamin D supplementation. Vitamin D deficiency, like the risk of severe COVID-19, is linked with darker skin colour and also with obesity. Greater risk from COVID-19 has been associated with reduced ultraviolet exposure. Various studies have examined serum 25-hydroxyvitamin D levels, either historical or current, in patients with COVID-19. The results of these studies have varied but the majority have shown an association between vitamin D deficiency and increased risk of COVID-19 illness or severity. Interventional studies of vitamin D supplementation have so far been inconclusive. Trial protocols commonly allow control groups to receive low-dose supplementation that may be adequate for many. The effects of vitamin D supplementation on disease severity in patients with existing COVID-19 are further complicated by the frequent use of large bolus dose vitamin D to achieve rapid effects, even though this approach has been shown to be ineffective in other settings. As the pandemic passes into its third year, a substantial role of vitamin D deficiency in determining the risk from COVID-19 remains possible but unproven.

Perspective: Vitamin D supplementation prevents rickets and acute respiratory infections when given as daily maintenance but not as intermittent bolus: implications for COVID-19.

The value of vitamin D supplementation in the treatment or prevention of various conditions is often viewed with scepticism as a result of contradictory results of randomised trials. It is now becoming apparent that there is a pattern to these inconsistencies. A recent large trial has shown that high-dose intermittent bolus vitamin D therapy is ineffective at preventing rickets - the condition that is most unequivocally caused by vitamin D deficiency. There is a plausible biological explanation since high-dose bolus replacement induces long-term expression of the catabolic enzyme 24-hydroxylase and fibroblast growth factor 23, both of which have vitamin D inactivating effects. Meta-analyses of vitamin D supplementation in prevention of acute respiratory infection and trials in tuberculosis and other conditions also support efficacy of low dose daily maintenance rather than intermittent bolus dosing. This is particularly relevant during the current COVID-19 pandemic given the well-documented associations between COVID-19 risk and vitamin D deficiency. We would urge that clinicians take note of these findings and give strong support to widespread use of daily vitamin D supplementation.

Influence of Aerosol Delivered BCG Vaccination on Immunological and Disease Parameters Following SARS-CoV-2 Challenge in Rhesus Macaques.

The tuberculosis vaccine, Bacille Calmette-Guerin (BCG), also affords protection against non-tuberculous diseases attributable to heterologous immune mechanisms such as trained innate immunity, activation of non-conventional T-cells, and cross-reactive adaptive immunity. Aerosol vaccine delivery can target immune responses toward the primary site of infection for a respiratory pathogen. Therefore, we hypothesised that aerosol delivery of BCG would enhance cross-protective action against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and be a deployable intervention against coronavirus disease 2019 (COVID-19). Immune parameters were monitored in vaccinated and unvaccinated rhesus macaques for 28 days following aerosol BCG vaccination. High-dose SARS-CoV-2 challenge was applied by intranasal and intrabronchial instillation and animals culled 6-8 days later for assessment of viral, disease, and immunological parameters. Mycobacteria-specific cell-mediated immune responses were detected following aerosol BCG vaccination, but SARS-CoV-2-specific cellular- and antibody-mediated immunity was only measured following challenge. Early secretion of cytokine and chemokine markers associated with the innate cellular and adaptive antiviral immune response was detected following SARS-CoV-2 challenge in vaccinated animals, at concentrations that exceeded titres measured in unvaccinated macaques. Classical CD14+ monocytes and Vδ2 γδ T-cells quantified by whole-blood immunophenotyping increased rapidly in vaccinated animals following SARS-CoV-2 challenge, indicating a priming of innate immune cells and non-conventional T-cell populations. However, viral RNA quantified in nasal and pharyngeal swabs, bronchoalveolar lavage (BAL), and tissue samples collected at necropsy was equivalent in vaccinated and unvaccinated animals, and in-life CT imaging and histopathology scoring applied to pulmonary tissue sections indicated that the disease induced by SARS-CoV-2 challenge was comparable between vaccinated and unvaccinated groups. Hence, aerosol BCG vaccination did not induce, or enhance the induction of, SARS-CoV-2 cross-reactive adaptive cellular or humoral immunity, although an influence of BCG vaccination on the subsequent immune response to SARS-CoV-2 challenge was apparent in immune signatures indicative of trained innate immune mechanisms and primed unconventional T-cell populations. Nevertheless, aerosol BCG vaccination did not enhance the initial clearance of virus, nor reduce the occurrence of early disease pathology after high dose SARS-CoV-2 challenge. However, the heterologous immune mechanisms primed by BCG vaccination could contribute to the moderation of COVID-19 disease severity in more susceptible species following natural infection.

Preventing vitamin D deficiency during the COVID-19 pandemic: UK definitions of vitamin D sufficiency and recommended supplement dose are set too low.

There is growing evidence linking vitamin D deficiency with risk of COVID-19. It is therefore distressing that there is major disagreement about the optimal serum level for 25-hydroxyvitamin D (25(OH)D) and appropriate supplement dose. The UK Scientific Advisory Committee for Nutrition has set the lowest level for defining sufficiency (10 ng/ml or 25 nmol/L) of any national advisory body or scientific society and consequently recommends supplementation with 10 micrograms (400 IU) per day. We have searched for published evidence to support this but not found it. There is considerable evidence to support the higher level for sufficiency (20 ng/ml or 50 nmol/L) recommended by the European Food Safety Authority and the American Institute of Medicine and hence greater supplementation (20 micrograms or 800 IU per day). Serum 25(OH)D concentrations in the UK typically fall by around 50% through winter. We believe that governments should urgently recommend supplementation with 20-25 micrograms (800-1,000 IU) per day.

Vitamin D and COVID-19: evidence and recommendations for supplementation.

Vitamin D is a hormone that acts on many genes expressed by immune cells. Evidence linking vitamin D deficiency with COVID-19 severity is circumstantial but considerable-links with ethnicity, obesity, institutionalization; latitude and ultraviolet exposure; increased lung damage in experimental models; associations with COVID-19 severity in hospitalized patients. Vitamin D deficiency is common but readily preventable by supplementation that is very safe and cheap. A target blood level of at least 50 nmol l-1, as indicated by the US National Academy of Medicine and by the European Food Safety Authority, is supported by evidence. This would require supplementation with 800 IU/day (not 400 IU/day as currently recommended in UK) to bring most people up to target. Randomized placebo-controlled trials of vitamin D in the community are unlikely to complete until spring 2021-although we note the positive results from Spain of a randomized trial of 25-hydroxyvitamin D3 (25(OH)D3 or calcifediol) in hospitalized patients. We urge UK and other governments to recommend vitamin D supplementation at 800-1000 IU/day for all, making it clear that this is to help optimize immune health and not solely for bone and muscle health. This should be mandated for prescription in care homes, prisons and other institutions where people are likely to have been indoors for much of the summer. Adults likely to be deficient should consider taking a higher dose, e.g. 4000 IU/day for the first four weeks before reducing to 800 IU-1000 IU/day. People admitted to the hospital with COVID-19 should have their vitamin D status checked and/or supplemented and consideration should be given to testing high-dose calcifediol in the RECOVERY trial. We feel this should be pursued with great urgency. Vitamin D levels in the UK will be falling from October onwards as we head into winter. There seems nothing to lose and potentially much to gain.

An epigenome-wide association study of total serum IgE in Hispanic children.

BACKGROUND: Total IgE is a therapeutic target in patients with allergic diseases. DNA methylation in white blood cells (WBCs) was associated with total IgE levels in an epigenome-wide association study of white subjects. Whether DNA methylation of eosinophils explains these findings is insufficiently understood. METHODS: We tested for association between genome-wide DNA methylation in WBCs and total IgE levels in 2 studies of Hispanic children: the Puerto Rico Genetics of Asthma and Lifestyle Study (PR-GOAL; n = 306) and the Genes-environments and Admixture in Latino Americans (GALA II) study (n = 573). Whole-genome methylation of DNA from WBCs was measured by using the Illumina Infinium HumanMethylation450 BeadChip. Total IgE levels were measured by using the UniCAP 100 system. In PR-GOAL WBC types (ie, neutrophils, eosinophils, basophils, lymphocytes, and monocytes) in peripheral blood were measured by using Coulter Counter techniques. In the GALA II study WBC types were imputed. Multivariable linear regression was used for the analysis of DNA methylation and total IgE levels, which was first conducted separately for each cohort, and then results from the 2 cohorts were combined in a meta-analysis. RESULTS: CpG sites in multiple genes, including novel findings and results previously reported in white subjects, were significantly associated with total IgE levels. However, adjustment for WBC types resulted in markedly fewer significant sites. Top findings from this adjusted meta-analysis were in the genes ZFPM1 (P = 1.5 × 10-12), ACOT7 (P = 2.5 × 10-11), and MND1 (P = 1.4 × 10-9). CONCLUSIONS: In an epigenome-wide association study adjusted for WBC types (including eosinophils), methylation changes in genes enriched in pathways relevant to asthma and immune responses were associated with total IgE levels among Hispanic children.

An epigenome-wide association study of total serum immunoglobulin E concentration.

Immunoglobulin E (IgE) is a central mediator of allergic (atopic) inflammation. Therapies directed against IgE can alleviate hay fever and allergic asthma. Genetic association studies have not yet identified novel therapeutic targets or pathways underlying IgE regulation. We therefore surveyed epigenetic associations between serum IgE concentrations and methylation at loci concentrated in CpG islands genome wide in 95 nuclear pedigrees, using DNA from peripheral blood leukocytes. We validated positive results in additional families and in subjects from the general population. Here we show replicated associations--with a meta-analysis false discovery rate less than 10(-4)--between IgE and low methylation at 36 loci. Genes annotated to these loci encode known eosinophil products, and also implicate phospholipid inflammatory mediators, specific transcription factors and mitochondrial proteins. We confirmed that methylation at these loci differed significantly in isolated eosinophils from subjects with and without asthma and high IgE levels. The top three loci accounted for 13% of IgE variation in the primary subject panel, explaining the tenfold higher variance found compared with that derived from large single-nucleotide polymorphism genome-wide association studies. This study identifies novel therapeutic targets and biomarkers for patient stratification for allergic diseases.

Development of pre-clinical models for evaluating the therapeutic potential of candidate siRNA targeting STAT6.

Developing siRNA therapeutics poses technical challenges including appropriate molecular design and testing in suitable pre-clinical models. We previously detailed sequence-selection and modification strategies for siRNA candidates targeting STAT6. Here, we describe methodology that evaluates the suitability of candidate siRNA for respiratory administration. Chemically-modified siRNA exhibited similar inhibitory activity (IC50) against STAT6 in vitro compared to unmodified siRNA and apical exposure testing with Caco-2 cell monolayers showed modification was not associated with cellular toxicity. Use of a modified RNA extraction protocol improved the sensitivity of a PCR-based bio-analytical assay (lower limit of siRNA strand quantification  =  0.01 pg/µl) which was used to demonstrate that lung distribution profiles for both siRNAs were similar following intra-tracheal administration. However, after 6 hours, modified siRNA was detected in lung tissue at concentrations >1000-fold higher than unmodified siRNA. Evaluation in a rat model of allergic inflammation confirmed the persistence of modified siRNA in vivo, which was detectable in broncho-alveolar lavage (BAL) fluid, BAL cells and lung tissue samples, 72 hours after dosing. Based upon the concept of respiratory allergy as a single airway disease, we considered nasal delivery as a route for respiratory targeting, evaluating an intra-nasal exposure model that involved simple dosing followed by fine dissection of the nasal cavity. Notably, endogenous STAT6 expression was invariant throughout the nasal cavities and modified siRNA persisted for at least 3 days after administration. Coupled with our previous findings showing upregulated expression of inflammatory markers in nasal samples from asthmatics, these findings support the potential of intranasal siRNA delivery. In summary, we demonstrate the successful chemical modification of STAT6 targeting siRNA, which enhanced bio-availability without cellular toxicity or reduced efficacy. We have established a robust, sensitive method for determining siRNA bio-distribution in vivo, and developed a nasal model to aid evaluation. Further work is warranted.

Interactive effect of STAT6 and IL13 gene polymorphisms on eczema status: results from a longitudinal and a cross-sectional study.

BACKGROUND: Eczema is a prevalent skin disease that is mainly characterized by systemic deviation of immune response and defective epidermal barrier. Th2 cytokines, such as IL-13 and transcription factor STAT6 are key elements in the inflammatory response that characterize allergic disorders, including eczema. Previous genetic association studies showed inconsistent results for the association of single nucleotide polymorphisms (SNPs) with eczema. Our aim was to investigate whether SNPs in IL13 and STAT6 genes, which share a biological pathway, have an interactive effect on eczema risk. METHODS: Data from two independent population-based studies were analyzed, namely the Isle of Wight birth cohort study (IOW; n = 1,456) and for the purpose of replication the Swansea PAPA (Poblogaeth Asthma Prifysgol Abertawe; n = 1,445) cross-sectional study. Log-binomial regressions were applied to (i) account for the interaction between IL13 (rs20541) and STAT6 (rs1059513) polymorphisms and (ii) estimate the combined effect, in terms of risk ratios (RRs), of both risk factors on the risk of eczema. RESULTS: Under a dominant genetic model, the interaction term [IL13 (rs20541) × STAT6 (rs1059513)] was statistically significant in both studies (IOW: adjusted P(interaction) = 0.046; PAPA: P(interaction) = 0.037). The assessment of the combined effect associated with having risk genotypes in both SNPs yielded a 1.52-fold increased risk of eczema in the IOW study (95% confidence interval (CI): 1.05 - 2.20; P = 0.028) and a 2.01-fold higher risk of eczema (95% CI: 1.29 - 3.12; P = 0.002) in the PAPA study population. CONCLUSIONS: Our study adds to the current knowledge of genetic susceptibility by demonstrating for the first time an interactive effect between SNPs in IL13 (rs20541) and STAT6 (rs1059513) on the occurrence of eczema in two independent samples. Findings of this report further support the emerging evidence that points toward the existence of genetic effects that occur via complex networks involving gene-gene interactions (epistasis).

Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA-based therapeutics for asthma.

The development of siRNA-based asthma therapeutics is currently hampered by a paucity of relevant biomarkers and the need to ascertain tissue-specific gene targeting in the context of active disease. Epithelial STAT6 expression is fundamental to asthma pathogenesis in which inflammatory changes are found throughout the respiratory tract. Therefore, to improve preclinical evaluation, we tested the efficacy of STAT6-targeting siRNA within nasal epithelial cells (NEC's) obtained from asthmatic and non-asthmatic donors. STAT6 expression was invariant in both donor groups and amenable to suppression by siRNA treatment. In addition, STAT6 mRNA was also suppressible by apically delivered siRNA treatment in comparative differentiated nasal epithelial cell-line monolayer cultures. Analysis of donor NEC's showed consistent elevation in CCL26 (eotaxin-3) mRNA within the asthmatic group suggesting potential as a relevant biomarker. Furthermore, targeting of STAT6 with siRNA attenuated IL-13-driven CCL26 expression in these cells, pointing to the utility of this approach in preclinical testing. Finally, siRNA-mediated suppression of STAT6 was independent of donor disease phenotype or epithelial cell differentiation status, signifying therapeutic potential.

The diagnosis of asthma, a clinical syndrome.

Clinical experience and now genetic data indicate that asthma is a heterogeneous clinical syndrome--clinical cases emerge, proceed and respond to treatments in different ways. Currently the diagnosis of asthma (as enunciated in national guidelines) is based on incisive clinical methods, supported by lung function testing that substantiates labile or reversible bronchial airflow obstruction. But this approach alone is insufficient to address the diagnostic and therapeutic challenges presented by asthma's heterogeneity. This article contends that bronchial pathology (with molecular and morphologic analysis) should be adopted into the mainstream clinical practice of asthma so as to clarify the nature of the bronchial disorder in compliant patients not settling securely on moderate-dose inhaled corticosteroid. This would allow a differentiated approach to appropriate therapeutics--those already available and those yet to be developed.

The diagnosis of asthma / O diagnóstico da asma

O diagnóstico de asma — como exposto em diversas diretrizes nacionais — é fundamentado na história clínica e corroborado pelo exame clínico e pela função pulmonar, que demonstra obstrução ao fluxo aéreo, reversível espontaneamente ou após o uso de broncodilatador ou corticosteroide. Diversos diagnósticos diferenciais devem ser cuidadosamente excluídos na avaliação clínica — incluindo bronquiolite viral na infância e DPOC nos adultos. Neste artigo, consideramos que o diagnóstico de asma deve agora avançar com o reconhecimento de que a asma é uma síndrome clínica heterogênea (casos individuais têm evolução e resposta ao tratamento diversos).Recomendamos que a broncoscopia e a biópsia brônquica devam participar do processo diagnóstico nos casos de pacientes que seguem o tratamento e, mesmo assim, não obtêm o controle da asma com doses moderadas de corticosteroides inalatórios. Desse modo, uma melhor caracterização da alteração clínica do paciente será obtida, visando o uso de terapias alternativas (disponíveis ou ainda a serem desenvolvidas)

Hyperexpression of the high-affinity IgE receptor-beta chain in chronic allergic keratoconjunctivitis.

PURPOSE: Although the existence of Fc(epsilon)RI-alphabetagamma(2) and Fc(epsilon)RI-alphagamma(2) receptor subtypes was reported, there has been no direct evidence of these two subtypes of Fc(epsilon)RI in vivo. To investigate the existence of these two subtypes of Fc(epsilon)RI in vivo, the authors evaluated the expression of Fc(epsilon)RI-beta in the giant papillae of chronic allergic conjunctivitis and compared the expression level of Fc(epsilon)RI-beta with control conjunctivae using the anti-human Fc(epsilon)RI-beta antibody. METHODS: Fc(epsilon)RI-beta expression in giant papillae obtained from patients with atopic keratoconjunctivitis and vernal keratoconjunctivitis in control conjunctivae was evaluated by immunohistochemistry using anti-Fc(epsilon)RI-beta, -alpha, -gamma, and anti-human mast cell tryptase, anti-chymase, anti-basophil, and anti-CD1a antibodies. RESULTS: Statistical analyses revealed that the densities of Fc(epsilon)RI-beta(+) cells, Fc(epsilon)RI-alpha(+) cells, tryptase(+) cells, and Fc(epsilon)RI-beta(+)/tryptase(+) cells were significantly increased in giant papillae compared with controls. There were two types of Fc(epsilon)RI (alphabetagamma(2) and alphagamma(2)) on the mast cells of the giant papillae. The ratio of the Fc(epsilon)RI-beta(+) cell number/Fc(epsilon)RI-alpha(+) cell number in the giant papillae (0.69 +/- 0.08 [mean +/- SD]) was significantly higher than that of the controls (0.07 +/- 0.16). Fc(epsilon)RI-beta/tryptase double immunostaining revealed that 81% +/- 13% of tryptase(+) cells expressed Fc(epsilon)RI-beta. Fc(epsilon)RI-beta(+) cells were preferentially localized within and around epithelial tissue. The authors also found that Fc(epsilon)RI-beta was expressed by basophils but not by Fc(epsilon)RI-alphagamma(2)-positive Langerhans cells in the giant papillae samples. CONCLUSIONS: Preferential Fc(epsilon)RI-beta expression observed in the mast cells and basophils of giant papillae suggests important roles of Fc(epsilon)RI-beta in the pathophysiology of atopic keratoconjunctivitis and vernal keratoconjunctivitis.

RNA interference of STAT6 rapidly attenuates ongoing interleukin-13-mediated events in lung epithelial cells.

Signal transducer and activator of transcription 6 (STAT6) expression in lung epithelial cells plays a central role in asthma pathogenesis, with its activation driving the development of airway hyper-reactivity and local inflammation. Therefore, inhibition of local STAT6 expression provides a rationale for therapeutic intervention in bronchial asthma. Given the absence of specific inhibitory drugs, we tested the ability of small interfering RNAs (siRNAs) to target STAT6 gene expression through the molecular process of RNA interference (RNAi). At pico-molar concentrations, STAT6-specific siRNAs potently inhibited STAT6 mRNA expression in lung epithelial cells (50% inhibitory concentration range = 134-861 pm) without inducing cellular interferon responses. Detectable STAT6 protein expression was rapidly abolished within 48 hr of treatment (t(1/2) range = or < 12-37 hr) and this was unaffected by pretreatment with STAT6-activating cytokines. Furthermore, STAT6 suppression by RNAi produced downstream functional inhibitory effects in that interleukin (IL)-13- or IL-4-driven eotaxin chemokine family [chemokine (C-C motif) ligand 11 (CCL11), CCL24 and CCL26] mRNA expression was markedly inhibited. Induction of detectable CCL26 protein synthesis was completely ablated by pretreating cells with STAT6-specific siRNA. The therapeutic potential of this approach is further demonstrated by novel findings that cells pre-exposed to IL-13 or IL-4 and subsequently treated with STAT6-targeting siRNA exhibited a rapid and significant attenuation of ongoing CCL26 protein expression, suggesting that chronic asthma-associated lung inflammation will be responsive to this approach.

High-affinity IgE receptor-beta chain expression in human mast cells.

The high-affinity IgE receptor (FcepsilonRI)-beta gene is one of the atopy-associated genes, but its biological significance is largely unknown. In this study, we generated the anti-FcepsilonRI-beta chain antibody to clarify beta-chain protein expression in human mast cells. The FcepsilonRI-beta antibody showed specific binding to a 27 kDa protein with Western blotting and membrane bound immunostaining using cultured mast cells. Monomeric IgE sensitization increased beta-chain expression as well as mature alpha-chain expression in mast cells. Upregulation of beta-chain expression with monomeric IgE treatment suggests possible roles of FcepsilonRI-beta protein as an atopy-related molecule.

Genetic haplotypes of Th-2 immune signalling link allergy to enhanced protection to parasitic worms.

Parasitic worm infection, allergy and asthma involve increased IgE production, eosinophil activity, mucus secretion and smooth muscle reactivity, effected through Th-2 immune signalling. These pathological features of allergic disorder, common in developed countries, appear to be protective features in resistance to parasitic worm infections prevalent in many developing countries. We investigated how genetic variation in the Th-2 signalling transduction molecule STAT6 relates to these clinical disorders, using immune phenotyping by serum IgE levels and haplotyping nine STAT6 genetic variants in a rural Chinese population, where Ascaris infection is prevalent, and an urban UK population where Ascaris is largely unknown but asthma and allergy are prevalent. We show for the first time that STAT6 haplotypes relate clearly to IgE levels, allergy and worm burden. The haplotypes segregated into two groups: those with raised IgE/low worm burden tended to have increased risk of allergic disorder, whereas low IgE/high worm burden tended to have a reduced risk of allergies. By estimating the mean worm burden for each haplotype in China and the relative risk of asthma for the matching haplotype in the UK, we draw a cross-population comparison and show a negative correlation between worm burden and expected risk of asthma. These data imply that the origin of common up-regulating variants of Th-2 signalling, involving STAT6, promotes asthma and allergy in developed countries, whereas in developing countries it protects against parasitic worm infections. Selective evolutionary mechanisms, driven by parasitic worm infection, may underlie the genetic contribution to risk of allergy and asthma in humans.