Paddy Tighe

Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche

Background/aims: There is substantial evidence that mammalian epithelial stem cells are located within well defined niches. Although the corneoscleral limbus is acknowledged as the site of corneal epithelial stem cells no anatomical niche for such cells has yet been described. The authors undertook to re-evaluate the microanatomy of the limbus in order to identify possible sites that may represent a stem cell niche. Methods: Systematic serial 5–7 μm sections of human corneoscleral segments obtained from cadaver donors, were examined. The sections were stained with haematoxylin and eosin or toludine blue. Sections with specific areas of interest were further examined immunohistologically for the corneal epithelial marker cytokeratin 14 and the “stem cell” marker ABCG2 transporter protein. Results: Distinct anatomical extensions from the peripheral aspect of the limbal palisades were identified. These consist of a solid cord of cells extending peripherally or circumferentially. The cells stained positive for CK14 and ABCG2. Conclusions: A novel anatomical structure has been identified at the human limbus, which demonstrates characteristics of being a stem cell niche. The authors have termed this structure the limbal epithelial crypt.

Direct ex vivo comparison of the breadth and specificity of the T cells in the liver and peripheral blood of patients with chronic HCV infection

The role of intrahepatic lymphocytes in the control of hepatitis C virus (HCV) infection and the pathology associated with it is not understood; most studies of the immunology of this infection use peripheral blood lymphocyte populations. To address this further, we examined in detail the IHL from HCV‐infected patients and controls, focusing on the antigen‐specific CD8+ T lymphocyte component. Individual T cells from needle liver biopsies and peripheral blood were isolated from patients with chronic HCV infection and examined directly ex vivo. We used RT‐PCR spectratyping to compare the breadth of the T cell receptor usage in the liver in comparison with the peripheral blood, and applied MHC class I tetramer technology to investigate the numbers of HCV‐specific CD8+ cells in the two compartments. T cell receptor usage in the liver of HCV‐infected patients was broad, comparable with that in the peripheral blood of the same patients. A much higher proportion of liver CD8+ cells expressed receptors specific for HCV antigens compared with paired peripheral blood CD8+ cells. A greater proportion of the liver tetramer‐positive cells expressed the activation marker CD69, compared with those in the periphery or other CD8+ cells in the liver. In the course of chronic HCV infection, HCV‐specific CD8 cells, which have been recently activated, appear to accumulate specifically in the livers of infected patients but are present in much lower numbers in the peripheral circulation. Further studies are needed to determine the function of these cells and their role in protection and immunopathology.

The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergen-induced T cell polarization through modulation of IDO activity.

The mannose receptor (MR) is a C-type lectin expressed by dendritic cells (DCs). We have investigated the ability of MR to recognize glycosylated allergens. Using a gene silencing strategy, we have specifically inhibited the expression of MR on human monocyte-derived DCs. We show that MR mediates internalization of diverse allergens from mite (Der p 1 and Der p 2), dog (Can f 1), cockroach (Bla g 2), and peanut (Ara h 1) through their carbohydrate moieties. All of these allergens bind to the C-type lectin-like carbohydrate recognition domains 4–7 of MR. We have also assessed the contribution of MR to T cell polarization after allergen exposure. We show that silencing MR expression on monocyte-derived DCs reverses the Th2 cell polarization bias, driven by Der p 1 allergen exposure, through upregulation of IDO activity. In conclusion, our work demonstrates a major role for MR in glycoallergen recognition and in the development of Th2 responses.

Utility, reliability and reproducibility of immunoassay multiplex kits.

Multiplex technologies are becoming increasingly important in biomarker studies as they enable patterns of biomolecules to be examined, which provide a more comprehensive depiction of disease than individual biomarkers. They are crucial in deciphering these patterns, but it is essential that they are endorsed for reliability, reproducibility and precision. Here we outline the theoretical basis of a variety of multiplex technologies: Bead-based multiplex immunoassays (i.e. Cytometric Bead Arrays, Luminex™ and Bio-Plex Pro™), microtitre plate-based arrays (i.e. Mesoscale Discovery (MSD) and Quantsys BioSciences QPlex), Slide-based Arrays (i.e. FastQuant™) and reverse phase protein arrays. Their utility, reliability and reproducibility are discussed.

Clinicopathological significance of KU70/KU80, a key DNA damage repair protein in breast cancer.

Although the role of BRCA1 and the homologous recombination (HR) pathway in breast cancer (BC) has been extensively studied, the alternative repair pathway for DNA double-strand breaks (DSBs), non-homologous end-joining (NHEJ) remains to be defined. Ku proteins bind to DNA DSB ends and play a key role in NHEJ. In this study we aimed to assess the expression and biological significance of the KU70/KU80 heterodimer in the different molecular classes of BC. The expression of KU70/KU80 was assessed immunohistochemically in a well-characterised and annotated series of 1302 unselected invasive BC cases with a long-term follow-up together with 25 cases with known BRCA1 mutations. The results were correlated with clinicopathological parameters, other DNA repair proteins and patient outcome. The expression of KU70/KU80 protein was further evaluated in various BC cell lines using western blotting and reverse-phase protein microarray (RPPA). Nuclear KU70/KU80 expression was correlated with features of poor prognosis including higher histological grade, lymphovascular invasion, negative oestrogen receptor expression, basal-like phenotype, P53 and CHK1 positivity. KU70/KU80 was expressed in all BRCA1-associated tumours and showed an inverse correlation with nuclear BRCA1 protein and aberrant cytoplasmic RAD51 expression. RPPA confirmed these results and showed higher expression of KU70/KU80 in BRCA1-deficient cell line compared to BRCA1-proficient cell line. KU70/KU80 expression showed an association with disease-free interval; however, it was not an independent predictor of outcome. As a conclusion, KU70/KU80 may play a role in DNA DSBs repair in HR-deficient tumours. Further study of other NHEJ markers in sporadic BC is warranted.

SUMOylation proteins in breast cancer

Small Ubiquitin-like Modifier proteins (or SUMO) modify the function of protein substrates involved in various cellular processes including DNA damage response (DDR). It is becoming apparent that dysregulated SUMO contribute to carcinogenesis by affecting post-transcriptional modification of key proteins. It is hypothesised that SUMO contributes to the aggressive nature of breast cancer particularly those associated with features similar to breast carcinoma arising in patients with BRCA1 germline mutations. This study aims to assess the clinical and biological significance of three members of SUMO in a well-characterised annotated series of BC with emphasis on DDR. The study cohort comprised primary operable invasive BC including tumours from patients with known BRCA1 germline mutations. SUMO proteins PIAS1, PIAS4 and UBC9 were assessed using immunohistochemistry utilising tissue microarray technology. Additionally, their expression was assessed using reverse phase protein microarray utilising different cell lines. PIAS1 and UBC9 showed cytoplasmic and/or nuclear expression while PIAS4 was detected only in the nuclei. There was a correlation between subcellular localisation and expression of the nuclear transport protein KPNA2. Tumours showing positive nuclear/negative cytoplasmic expression of SUMO featured good prognostic characteristics including lower histologic grade and had a good outcome. Strong correlation with DDR-related proteins including BRCA1, Rad51, ATM, CHK1, DNA-PK and KU70/KU80 was observed. Correlation with ER and BRCA1 was confirmed using RPPA on cell lines. SUMO proteins seem to play important role in BC. Not only expression but also subcellular location is associated with BC phenotype.

Immunocytochemical study of Peyer's patches follicular-associated epithelium in the marsupial, Didelphis albiventris

The lack of probes defining leukocyte subpopulations has restricted ontogenetic studies of the opossum gut. We report for the first time the organization of the gut cellular immune components using species cross-reactive antibodies. Mouse monoclonal antibodies against human HLA-DR were used together with immunocytochemistry to demonstrate MHC class II-like antigens in the opossum Peyers patches (PP). Positive staining was obtained in the M cell and enterocytes comprising the follicular-associated epithelium (FAE). Rabbit polyclonal antibody against human CD3 stained opossum thymocytes and T-cell dependent areas of spleen, lymph node, and PP interfollicular zones, but failed to stain intraepithelial lymphocytes in the FAE. In contrast rabbit polyclonal antibody against human IgA stained B-cell immunocytes and plasma cells present in the M-cell lateral invaginations. It is surmised that B-cell activation could occur in the opossum M-cell niches by thymus independent antigens, bypassing T-helper-cell function.

SMAD4 loss enables EGF, TGFβ1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells

Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor β1 (TGFβ1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFβ1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFβ1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/β-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFβ1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFβ1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/β-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFβ1 and S100A8/A9 mainly inhibit NF-κB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner.

The intracellular signalling pathway signature (the signalome) in PBMCs in the presence of a common TRAPS-associated genetic variant, TNFRSF1A p.(Arg121Gln) (legacy p.R92Q) is distinct from normal PBMCs and from other pathogenic variants

Question Like other disorders with a strong genetic association, a growing sequence dataset from various autoinflammatory syndrome patients continues to identify many variants of uncertain significance (VUS), i.e. missense and intronic variants or small insertions/deletions, for which the impact on protein function and pathways, and therefore the clinical significance, is unknown. Since it is unclear how these variants are associated with an increased risk of autoinflammatory disease, the clinical management of carriers of VUS is complicated. Therefore, there is a strong demand for reliable tests to rapidly assess the clinical significance of VUS, providing carriers of these variants with the necessary information to make an informed clinical decision and refining treatment by stratified therapy strategies. In many cases, a novel VUS is not common enough to evaluate its significance. Common variants with apparently variable penetrance can be more accessible as a model to test functional aspects. A missense variant in TNFRSF1A rs41495584 (“p.R92Q”) is the most commonly identified variant associated with TRAPS within our mainly Caucasian UK patient population. The aim of this study was to comprehensively examine the intracellular signalling pathways that are affected by the presence of p.R92Q, in comparison to normal cells and to wellknown symptomatic variants such as p.C33Y. Methods PBMCs were collected from patients and healthy volunteers with informed consent. Reverse-phase protein microarray was applied to examine a large number of signalling molecules and inflammatory cytokines, using a feature subset selection process to identify distinctive subsets.

Altered cytokine pattern and inflammatory pathways in monogenic and complex autoinflammatory diseases

Autoinflammatory disorders (AIDs) are a group of innate immunity-related diseases, characterized by seemingly unprovoked inflammatory episodes mainly involving skin, eyes and joints. They can be categorized into hereditary monogenic and multifactorial complex disorders. The inflammatory mechanisms underlying both monogenic and complex AIDs are not completely understood.