Andrew J. Wardlaw's research while affiliated with University of Leicester and other places

Background Component‐resolved diagnosis allows detection of IgE sensitization having the advantage of reproducibility and standardization compared to crude extracts. The main disadvantage of the traditional allergen identification methods, 1‐ or 2‐dimensional western blotting and screening of expression cDNA libraries with patients' IgEs, is that the native structure of the protein is not necessarily maintained. Methods We used a novel immunoprecipitation technique in combination with mass spectrometry to identify new allergens of Aspergillus fumigatus. Magnetic Dynabeads coupled with anti‐human IgE antibodies were used to purify human serum IgE and subsequently allergens from A. fumigatus protein extract. Results Of the 184 proteins detected by subsequent mass peptide fingerprinting, a subset of 13 were recombinantly expressed and purified. In a panel of 52 A. fumigatus‐sensitized people with asthma, 23 non‐fungal‐sensitized asthmatics and 18 healthy individuals, only the former showed an IgE reaction by immunoblotting and/or ELISA. We discovered 11 proteins not yet described as A. fumigatus allergens, with fructose‐bisphosphate aldolase class II (FBA2) (33%), NAD‐dependent malate dehydrogenase (31%) and Cu/Zn superoxide dismutase (27%) being the most prevalent. With respect to these three allergens, native versus denatured protein assays indicated a better recognition of the native proteins. Seven of 11 allergens fulfilled the WHO/IUIS criteria and were accepted as new A. fumigatus allergens. Conclusion In conclusion, we introduce a straightforward method of allergen identification from complex allergenic sources such as A. fumigatus by immunoprecipitation combined with mass spectrometry, which has the advantage over traditional methods of identifying allergens by maintaining the structure of the proteins.

Background: Fungal respiratory allergy is believed to affect up to 30% of hayfever sufferers and up to 70% of severe asthmatics in the UK, however trends in fungal spore seasonality are not well described. Information about seasonal trends would help allergists determine sources of fungal sensitisation and aid disease management. Method: Daily monitoring was carried out at Leicester from 2007 to 2020 using a Burkard volumetric spore trap. Fungal spore concentrations were analysed by microscopy, identifying 23 morphologically distinct taxa. Daily average concentrations were calculated as spores/m3 of air sampled and a 90% method used to determine the spore seasons. Results: Thirteen years of data were used to develop a fungal spore calendar for the nine most abundant spore types identified; Alternaria, Cladosporium, Didymella, Leptosphaeria, Sporobolomyces, Tilletiopsis and Ustilago plus the wider groupings of Aspergillus/Penicillium type and coloured basidiospores. All have been implicated in fungal allergy. We observed long seasons for, Cladosporium, Sporobolomyces and Tilletiopsis, beginning in late spring and ending in late autumn. In contrast Ustilago and the highly allergenic Alternaria showed relatively short seasons, spanning summer and early autumn. Temperature and precipitation were the main meteorological factors related to spore concentration with wind speed appearing to have little influence. Over the study period, there was a reducing trend for total spore concentrations, driven by a reduction in wet weather spores, in line with a reduction in precipitation. Conversely, the dry weather spores of Alternaria and Cladosporium demonstrated an increasing trend. Conclusion: We present an aeroallergen calendar to provide readily accessible information to patients, healthcare professionals and pharmaceutical companies on exposure concentrations over the year in central England and potentially more widely across the UK. More research on allergenic thresholds would enhance the clinical usefulness of aeroallergen calendars.

Eosinophils are a characteristic feature of a number of pulmonary pathologies that are characterized by elevated numbers of eosinophils in the blood and the airway or lung parenchyma. They are stained by acidophilic dyes such as eosin, hence their name [1]. They have segmented nuclei, and their nucleo-cytoplasmic ratio is considered to be 30% [2]. Having large specific granules is a characteristic feature of eosinophils and these granules contain variable mediators such as eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), major basic protein (MBP), and eosinophil-derived neurotoxin (EDN). These molecules each play a part in eosinophilic inflammation and/or tissue damage [2]. Mature eosinophils are nondividing and develop in the bone marrow differentiating from myeloid precursors. Their maturation, activation, migration, and survival are mainly regulated by cytokines such as interleukin (IL)-5, IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF) [2, 3]. Mature eosinophils are released into the circulation and are recruited into tissues [4]. Chemokines such as CCL11, CCL24, and CCL26 are thought to be involved in the recruitment of eosinophils towards certain tissues from the bone marrow by virtue of expression of CCR3 [5]. VLA-4/VCAM-1 and PSGLI-P-selectin adhesion interactions favor eosinophil over neutrophil recruitment [6]. IL-5 plays a crucial role in the survival of blood and tissue eosinophils [3]. IL-5 is produced predominantly by T helper (TH)2 and group 2 innate lymphoid cell subset (ILC2s), although mast cells and basophils express low levels. Targeting IL-5 provides a novel therapeutic option for patients with eosinophilic pulmonary disease.

The term allergic fungal airways disease has a liberal definition based on IgE sensitisation to thermotolerant fungi and evidence of fungal-related lung damage. It arose from a body of work looking into the role of fungi in asthma. Historically fungi were considered a rare complication of asthma, exemplified by allergic bronchopulmonary aspergillosis; however, there is a significant proportion of individuals with Aspergillus fumigatus sensitisation who do not meet these criteria, who are at high risk for the development of lung damage. The fungi that play a role in asthma can be divided into two groups; those that can grow at body temperature referred to as thermotolerant, which are capable of both infection and allergy, and those that cannot but can still act as allergens in IgE sensitised individuals. Sensitisation to thermotolerant filamentous fungi (Aspergillus and Penicillium), and not non-thermotolerant fungi (Alternaria and Cladosporium) is associated with lower lung function and radiological abnormalities (bronchiectasis, tree-in-bud, fleeting shadows, collapse/consolidation and fibrosis). For antifungals to play a role in treatment, the focus should be on fungi capable of growing in the airways thereby causing a persistent chronic allergenic stimulus and releasing tissue damaging proteases and other enzymes which may disrupt the airway epithelial barrier and cause mucosal damage and airway remodelling. All patients with IgE sensitisation to thermotolerant fungi in the context of asthma and other airway disease are at risk of progressive lung damage, and as such should be monitored closely.

Eosinophils play a homeostatic role in the body's immune responses. These cells are involved in combating some parasitic, bacterial, and viral infections and certain cancers and have pathologic roles in diseases including asthma, chronic rhinosinusitis with nasal polyps, eosinophilic gastrointestinal disorders, and hypereosinophilic syndromes. Treatment of eosinophilic diseases has traditionally been through nonspecific eosinophil attenuation by use of glucocorticoids. However, several novel biologic therapies targeting eosinophil maturation factors, such as interleukin (IL)-5 and the IL-5 receptor or IL-4/IL-13, have recently been approved for clinical use. Despite the success of biologic therapies, some patients with eosinophilic inflammatory disease may not achieve adequate symptom control, underlining the need to further investigate the contribution of patient characteristics, such as comorbidities and other processes, in driving ongoing disease activity. New research has shown that eosinophils are also involved in several homeostatic processes, including metabolism, tissue remodeling and development, neuronal regulation, epithelial and microbiome regulation, and immunoregulation, indicating that these cells may play a crucial role in metabolic regulation and organ function in healthy humans. Consequently, further investigation is needed into the homeostatic roles of eosinophils and eosinophil-mediated processes across different tissues and their varied microenvironments. Such work may provide important insights into the role of eosinophils not only under disease conditions but also in health. This narrative review synthesizes relevant publications retrieved from PubMed informed by author expertise to provide new insights into the diverse roles of eosinophils in health and disease, with particular emphasis on the implications for current and future development of eosinophil-targeted therapies.

Background: A peripheral blood eosinophilia of greater than 1.0 × 109 /L is relatively unusual and offers a clue to the underlying diagnosis. In 2003, we established a specialist service to diagnose unexplained eosinophilia. Objective: To describe the causes of an eosinophilia in our service and the diagnostic algorithm we developed. Methods: Subjects were referred by physician colleagues across a range of specialties and undertook standard investigations following a semi-structured protocol. Data were extracted from a bespoke database. Results: Three hundred and eighty two subjects were referred over a 17-year period. Average age was 54 years and 183 (48%) of subjects were female, with 21 of 25 (84%) females in the idiopathic eosinophilic pneumonia group (p < 0001), 22 of 30 (73%) females in the gastrointestinal disease group (p < .008), but 11 of 37 (30%) females in the eosinophilic granulomatosis with polyangiitis group (p < .04). A diagnosis was assigned after systematic evaluation using a pre-defined algorithm in 361 (94.5%) of cases. Fungal allergy (82 subjects: 21%), parasitic infection (61 subjects: 16%) and severe eosinophilic asthma (50 subjects: 13%) were the three commonest individual diagnoses. Hypereosinophilic syndrome (HES) disease including eosinophilic granulomatosis with polyangiitis (EGPA) accounted for 85 subjects (20%) of which seven subjects (2%) had myeloproliferative disease (M-HES). A high IgE was common, and 79 (91%) of subjects with complete data who had an IgE of ≥1000 IU/L had fungal allergy or parasite infection. The serum tryptase was raised in 44 of 302 (14.5%) of individuals across all diagnostic groups, though none had mastocytosis. Conclusion: A diagnosis of an unexplained eosinophilia can usually be determined using as semi-structured algorithm. Parasitic infection and fungal allergy often with severe eosinophilic asthma were common causes, whereas HES, particularly myeloproliferative, disease was relatively rare.

Allergy to airway-colonising, thermotolerant, filamentous fungi represents a distinct eosinophilic endotype of often severe lung disease. This endotype, which particularly affects adult asthma, but also complicates other airway diseases and sometimes occurs de novo, has a heterogeneous presentation ranging from severe eosinophilic asthma to lobar collapse. Its hallmark is lung damage, characterised by fixed airflow obstruction (FAO), bronchiectasis and lung fibrosis. It has a number of monikers including severe asthma with fungal sensitisation (SAFS) and allergic bronchopulmonary aspergillosis/mycosis (ABPA/M), but these exclusive terms constitute only sub-sets of the condition. In order to capture the full extent of the syndrome we prefer the inclusive term allergic fungal airway disease (AFAD), the criteria for which are IgE sensitisation to relevant fungi in association with airway disease. The primary fungus involved is Aspergillus fumigatus, but a number of other thermotolerant species from several genera have been implicated. The unifying mechanism involves germination of inhaled fungal spores in the lung in the context of IgE sensitisation, leading to a persistent and vigorous eosinophilic inflammatory response in association with release of fungal proteases. Most allergenic fungi, including Alternaria and Cladosporium species, are not thermotolerant and cannot germinate in the airways so only act as aeroallergens and do not cause AFAD. Studies of the airway mycobiome have shown that A. fumigatus colonises the normal as much as the asthmatic airway, suggesting it is the tendency to become IgE-sensitised that is the critical triggering factor for AFAD rather than colonisation per se. Treatment is aimed at preventing exacerbations with glucocorticoids and increasingly by the use of anti-T2 biological therapies. Anti-fungal therapy has a limited place in management, but is an effective treatment for fungal bronchitis which complicates AFAD in about 10% of cases.

A persistent unexplained eosinophilia is relatively unusual and has a number of causes of which allergy and infection with helminthic parasites are the most common diagnoses. Less common causes include idiopathic hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA) and single organ conditions such as eosinophilic gastrointestinal disease (EGID) (1). Although occasionally caused by myeloproliferative neoplasm in most cases eosinophilia is due to excess production of growth factors, particularly interleukin‐5, by bystander cells which are often lymphoid in nature. Sometimes an environmental trigger can be identified, but the underlying cause of the eosinophilia often remains obscure.