Herbert F. Sewell

Migration of human intestinal lamina propria lymphocytes, macrophages and eosinophils following the loss of surface epithelial cells

Lymphocytes and macrophages are present in the normal intestinal lamina propria, separated from the epithelial monolayer by the basement membrane. There is evidence for movement of mononuclear cells through the lamina propria, entering from the systemic circulation and exiting via lymphatic channels. The goal of our studies was to investigate the capacity of cells to migrate out from the lamina propria into the lumen following the loss of surface epithelial cells. An in vitro model was therefore established in which normal human intestinal mucosal samples, denuded of the surface epithelium, were maintained in culture. Electron microscopy showed that during culture, large numbers (> 2 × 106/g tissue per 24 h) of cells migrated out of the lamina propria via discrete ‘tunnels’ which were in continuity with pores (diameter < 4 μm) in the basement membrane. The emigrating cells were T cells (68.5 ± 5.1%), macrophages (10.5 ± 1.3%) and eosinophils (7.1 ± 1.3%). Our studies have therefore demonstrated, for the first time, the capacity for large numbers of lymphocytes, macrophages and eosinophils to migrate out of the lamina propria, via basement membrane pores. We postulate that such emigration of cells occurs in vivo following the loss of surface epithelial cells due to injury, and could represent an important form of host defence against luminal microorganisms and also facilitate wound repair by enhancing restitution by neighbouring epithelial cells, via peptide factors.

The protease allergen Der p 1 cleaves cell surface DC‐SIGN and DC‐SIGNR: experimental analysis of in silico substrate identification and implications in allergic responses

Background The cysteine protease Der p 1 from the house dust mite Dermatophagoides pteronyssinus is one of the most potent allergens known. An attractive mechanism for a component of Der p 1 allergenicity lies in its ability to cleave key regulatory molecules from leucocyte surfaces, subverting cellular function and driving abnormal immunoglobulin E (IgE) responses.

Comparative molecular modelling identifies a common putative IgE epitope on cysteine protease allergens of diverse sources

Previous approaches for studying common allergenic epitopes have mainly focused on sequence comparisons, which unfortunately yield little or no information on the shape of the epitope which is the most important determinant of cross‐reactivity.

The interaction between the dust mite antigen Der p 1 and cell-signalling molecules in amplifying allergic disease.

It has recently been suggested that nonimmunological properties of some allergens, such as enzymatic activity, might contribute to their allergenicity [1]. Within this context, a number of studies have focused on the proteolytic activity of the dust mite cysteine protease Der p 1, the most significant domestic allergen. In this article, we review the proteolytic activity of Der p 1 and its effects on cells of the innate (mast cells) and adaptive (lymphocytes) immune systems, with a view to demonstrating a link between enzymatic activity and allergenicity. Collectively, these findings reveal previously unrecognized mechanisms by which the proteolytic activity of Der p 1 may perturb immunological signalling in favour of an allergic outcome [2].

Differential lamina propria cell migration via basement membrane pores of inflammatory bowel disease mucosa

BACKGROUND & AIMS In active inflammatory bowel disease (IBD), the intestinal mucosa is infiltrated by polymorphonuclear cells (PMNs), lymphocytes, and monocytes from the systemic circulation. Using an ex vivo model, we have investigated luminally directed migration of cells out of the lamina propria. METHODS Fresh untreated and deepithelialized mucosal samples were studied by electron microscopy. Cells migrating out of the lamina propria were investigated by immunohistochemistry and fluorescence-activated cell sorter analysis. RESULTS In intact IBD mucosal samples, tunnels containing cells were prominent in the lamina propria matrix, and PMNs, but not other cell types, were prominent in the epithelium. In deepithelialized mucosal samples, the basement membrane was either destroyed or contained numerous large pores. During culture of deepithelialized mucosal samples, many cells (3.3 [+/-0.8] x 10(5) . g tissue-1 . h-1) migrated out of the lamina propria via basement membrane pores. PMNs and eosinophils were prominent during the first 3 hours of culture, but T cells predominated thereafter. Macrophages also migrated, but B cells were the minority population (<2%) at all times. CONCLUSIONS In active IBD mucosa with an intact epithelium, luminally directed migration of lamina propria cells is restricted mainly to PMNs. After loss of the epithelium, other cell types also migrate into the lumen via numerous, large, basement membrane pores.

Circulating antibody and memory B-cell responses to C. difficile toxins A and B in patients with C. difficile- associated diarrhoea, inflammatory bowel disease and cystic fibrosis

C. difficile infection (CDI) is rarely reported in cystic fibrosis (CF) patients despite frequent hospitalisations and antibiotic usage. Conversely, the prevalence of CDI in inflammatory bowel disease (IBD) has received increased attention. We investigated components of the IgG-specific humoral immune response to C. difficile toxins A and B in patients with C. difficile-associated diarrhoea (CDAD), IBD patients with CDI, CF patients and healthy controls. Serum anti-toxin IgG was determined by ELISA. Circulating antigen-activated B-cells were investigated using Alexa Fluor 488-labelled toxin A and assessed by flow cytometry. Following induction of differentiation of memory B-cells, toxin A- and B-specific antibody secreting cells (ASCs) were quantified using ELISpot. We present the first data showing levels of serum anti-toxin A and B antibodies were significantly higher in patients with CF (without a history of CDI) than in CDAD patients and were stably maintained over time. Notably, the CDAD patients were significantly older than the CF patients. We also show that circulating toxin A-specific memory B-cells (IgD-negative) can be detected in CDAD patients [0.92 (0.09–1.78)%], and were prominent (5.64%, 1.14%) in two CF patients who were asymptomatic carriers of C. difficile. There was correlation between toxin A- and B-specific ASCs, with significantly higher proportions of the latter seen. In some with CDAD, high serum antibody levels were seen to only one of the two toxins. Mucosal secretion of toxin-specific IgG was detected in an additional group of IBD patients with no history of CDI. We conclude that enhanced and stable humoral immune responses to toxins A and B may protect CF and some IBD patients against CDI. The impaired ability to generate strong and/or sustained toxin-specific antibody and memory B-cell responses may increase susceptibility of older patients to CDI and highlight the need to investigate the role of immune senescence in future studies.

Application of a High Throughput Method of Biomarker Discovery to Improvement of the EarlyCDT®-Lung Test

Background The National Lung Screening Trial showed that CT screening for lung cancer led to a 20% reduction in mortality. However, CT screening has a number of disadvantages including low specificity. A validated autoantibody assay is available commercially (EarlyCDT®-Lung) to aid in the early detection of lung cancer and risk stratification in patients with pulmonary nodules detected by CT. Recent advances in high throughput (HTP) cloning and expression methods have been developed into a discovery pipeline to identify biomarkers that detect autoantibodies. The aim of this study was to demonstrate the successful clinical application of this strategy to add to the EarlyCDT-Lung panel in order to improve its sensitivity and specificity (and hence positive predictive value, (PPV)). Methods and Findings Serum from two matched independent cohorts of lung cancer patients were used (n = 100 and n = 165). Sixty nine proteins were initially screened on an abridged HTP version of the autoantibody ELISA using protein prepared on small scale by a HTP expression and purification screen. Promising leads were produced in shake flask culture and tested on the full assay. These results were analyzed in combination with those from the EarlyCDT-Lung panel in order to provide a set of re-optimized cut-offs. Five proteins that still displayed cancer/normal differentiation were tested for reproducibility and validation on a second batch of protein and a separate patient cohort. Addition of these proteins resulted in an improvement in the sensitivity and specificity of the test from 38% and 86% to 49% and 93% respectively (PPV improvement from 1 in 16 to 1 in 7). Conclusion This is a practical example of the value of investing resources to develop a HTP technology. Such technology may lead to improvement in the clinical utility of the EarlyCDT-Lung test, and so further aid the early detection of lung cancer.

Human Blood Autoantibodies in the Detection of Colorectal Cancer.

Colorectal cancer (CRC) is the second most common malignancy in the western world. Early detection and diagnosis of all cancer types is vital to improved prognosis by enabling early treatment when tumours should be both resectable and curable. Sera from 3 different cohorts; 42 sera (21 CRC and 21 matched controls) from New York, USA, 200 sera from Pittsburgh, USA (100 CRC and 100 controls) and 20 sera from Dundee, UK (10 CRC and 10 controls) were tested against a panel of multiple tumour-associated antigens (TAAs) using an optimised multiplex microarray system. TAA specific IgG responses were interpolated against the internal IgG standard curve for each sample. Individual TAA specific responses were examined in each cohort to determine cutoffs for a robust initial scoring method to establish sensitivity and specificity. Sensitivity and specificity of combinations of TAAs provided good discrimination between cancer-positive and normal serum. The overall sensitivity and specificity of the sample sets tested against a panel of 32 TAAs were 61.1% and 80.9% respectively for 6 antigens; p53, AFP, K RAS, Annexin, RAF1 and NY-CO16. Furthermore, the observed sensitivity in Pittsburgh sample set in different clinical stages of CRC; stage I (n = 19), stage II (n = 40), stage III (n = 34) and stage IV (n = 6) was similar (73.6%, 75.0%, 73.5% and 83.3%, respectively), with similar levels of sensitivity for right and left sided CRC. We identified an antigen panel of sufficient sensitivity and specificity for early detection of CRC, based upon serum profiling of autoantibody response using a robust multiplex antigen microarray technology. This opens the possibility of a blood test for screening and detection of early colorectal cancer. However this panel will require further validation studies before they can be proposed for clinical practice.

Development and Validation of a High Throughput System for Discovery of Antigens for Autoantibody Detection

An assay employing a panel of tumor-associated antigens has been validated and is available commercially (EarlyCDT®-Lung) to aid the early detection of lung cancer by measurement of serum autoantibodies. The high throughput (HTP) strategy described herein was pursued to identify new antigens to add to the EarlyCDT-Lung panel and to assist in the development of new panels for other cancers. Two ligation-independent cloning vectors were designed and synthesized, producing fusion proteins suitable for the autoantibody ELISA. We developed an abridged HTP version of the validated autoantibody ELISA, determining that results reflected the performance of the EarlyCDT assay, by comparing results on both formats. Once validated this HTP ELISA was utilized to screen multiple fusion proteins prepared on small-scale, by a HTP expression screen. We determined whether the assay performance for these HTP protein batches was an accurate reflection of the performance of R&D or commercial batches. A HTP discovery platform for the identification and optimal production of tumor- associated antigens which detects autoantibodies has been developed and validated. The most favorable conditions for the exposure of immunogenic epitopes were assessed to produce discriminatory proteins for use in a commercial ELISA. This process is rapid and cost-effective compared to standard cloning and screening technologies and enables rapid advancement in the field of autoantibody assay discovery. This approach will significantly reduce timescale and costs for developing similar panels of autoantibody assays for the detection of other cancer types with the ultimate aim of improved overall survival due to early diagnosis and treatment.

Migration of precursors of intraepithelial lymphocytes (IELs) from human colonic lamina propria

Migration of Precursors of Intraepithelial Lymphocytes (IELs) from Human Colonic Lamina Propria. Wael Ellabban, Div of Gastroenterology, Univ of Nottingham, Nottingham United Kingdom; Patrick Tighe, Div of Immunology, Univ of Nottingham, Nottingham United Kingdom; Brian McKaig, Div of Gastroenterology, Univ of Nottingham, Nottingham United Kingdom; Adrian Robins, Alison Gafvin, Herbert F. Sewell, Div of Immunology, Univ of Nottingham, Nottingham United Kingdom; Yashwant R. Mahida, Div of Gastroenterology, Univ of Nottingham, Nottingham United Kingdom