Biology subjects

Richardson, S. J.

Publications and source records attributed to Richardson, S. J..

12 recordsLinked to original sources

bioRxiv2026

Expression of non-neuronal Tau in humans and mice

BackgroundTau, encoded by the microtubule-associated protein tau gene (MAPT), is traditionally associated with neuronal function in maintaining cytoskeletal stability and regulating intracellular transport. Its dysregulation is implicated in a range of neurodegenerative disorders. Beyond its well-established high levels of expression in neurons of the central and peripheral nervous systems, increasing evidence indicates that Tau is also expressed in non-neuronal cells/tissues such as muscle, kidney, testis, and pancreas, albeit at much lower levels than in neurons. However, the assessment of low levels of non-neuronal Tau requires that the specificities of the anti-Tau antibodies used are fully validated. MethodsThree previously validated anti-Tau antibodies (Tau-12, Tau-1, RD3) were used to test Tau expression levels in 33 mouse and 66 human tissues using Food and Drug Administration-quality tissue microarrays in immunocytochemistry and western blot analyses. ResultsThe presence of Tau was confirmed in salivary gland, kidney, skeletal muscle, heart, pancreas and oesophagus in both human and mouse tissues. ConclusionsDysregulation of Tau protein expression and alterations in its post-translational modification is a causative factor in neurodegenerative disease. We have demonstrated a wider Tau expression in other tissues and cells, outside the brain, which may be dysregulated in other diseases.

cell biology↗

bioRxiv2025

Small things matter: Lack of extra-islet beta cells in Type 1 diabetes

Recent 3D analyses reported abundant, small beta-cell-rich endocrine objects (EOs) in the human pancreas. Here, we assessed morphological parameters of >262,000 EOs in pancreas sections from 220 donors with or without type 1 diabetes (T1D), ranging in age and disease duration. We observe many insulin (Ins)+/glucagon (Gluc)-EOs in donors without diabetes. Their relative contribution to the total endocrine area is greatest in early life (0-2y) but reduces thereafter. Strikingly, we show the virtual absence of Ins+Gluc- EOs in individuals with T1D, where only the medium and large EOs retain beta cells. We also report a lower EO density in T1D, especially in individuals diagnosed in early life. These findings suggest that extra-islet beta cells are impacted in the development of T1D, and their early loss is a characteristic feature. This new understanding has important implications for defining beta-cell mass, which may inform future screening and treatment strategies in T1D. HighlightsO_LIThe present extensive 2D studies confirm and extend recent 3D analyses of human pancreata, demonstrating that 50% of endocrine objects (EOs) are much smaller than classical islets of Langerhans and consist predominantly of beta cells (Ins+). C_LIO_LISmall insulin+ EOs comprise the largest proportion of the total endocrine area in early childhood and persist throughout the life course in donors without diabetes. C_LIO_LIThere is a shift towards larger EO size with increasing age, with the most pronounced changes in size occurring in the first few years of life. C_LIO_LISmall Ins+ EOs are virtually absent in individuals with type 1 diabetes, while the persisting EOs with beta cells are larger, suggesting a selective destruction of beta cells in small EOs. C_LIO_LIDevelopment of type 1 diabetes, particularly at an early age, is associated with fewer larger EOs in adulthood. This implies that the lack or early destruction of small Ins+ EOs may be detrimental to the generation of larger EOs. C_LI

pathology↗

bioRxiv2025

GGA2-depletion in beta cells by Enteroviruses causes Golgi acidification, premature activation of cathepsins and alters the MHC class I immunopeptidome

Enteroviruses (EVs), such as Coxsackievirus B5 (CVB5), are linked to pancreatic beta cell autoimmunity in type 1 diabetes (T1D). CVB5 infection of beta cells blocks their cap-dependent translation but spares cap-independent translation of insulin secretory granule (SG) cargoes, including major T1D autoantigens. Despite this, insulin SG stores are depleted. We show that CVB5 protease 2A rapidly depletes Golgi-associated sorting factor GGA2 in beta cells due to its short half-life. Immunostaining of pancreas sections from recent-onset T1D donors confirmed reduced GGA2 in beta cells expressing EV capsid protein VP1. Both GGA2 depletion and CVB5 2A expression impair SG biogenesis at the TGN without affecting MHC class I trafficking. They also disrupt the sorting of vacuolar ATPase and cathepsins, leading to TGN acidification and premature lysosomal hydrolase activation, which typically generate peptides presented in HLA-II alleles. Notably, the unique immunopeptidome of EV-infected, GGA2-depleted ECN90 beta-like cells is mainly presented via HLA-B alleles with an average isoelectric point (pI) of [~]5, compared to a pI of [~]7 of the prevailing HLA-I A allele immunopeptidome of non-infected cells. We propose that delayed sorting at the acidified TGN promotes cathepsin-mediated processing of in-transit secretory proteins, including SG cargoes, thereby generating novel peptides that may replace HLA class I antigens due to the acidic environment. This novel cross-presentation mechanism could have implications for T1D pathogenesis and antigen presentation in general.

cell biology↗

bioRxiv2025

Characterisation of native human pancreatic mesenchymal stromal cells in type 1 diabetes

Aims/hypothesisCulture-expanded mesenchymal stromal cells (MSCs) reduce immune cell activation and improve islet functional survival. However, little is known about native human pancreatic MSCs (npMSCs) in health or how they are altered in type 1 diabetes. Here, we determined the number, density and islet-protective phenotype of npMSCs in situ in control individuals and those with type 1 diabetes. MethodsMultiplex immunohistochemistry was used to identify npMSCs (CD90+/CD105+/CD73+/CD31-/CD45-/CD34-) in human pancreas sections from 38 donors (Network for Pancreatic Organ Donors with Diabetes and Exeter Archival Diabetes Biobank). Donors were categorised as either < 13 years at type 1 diabetes diagnosis (n = 8) or [≥] 13 years at type 1 diabetes diagnosis (n = 11) or were age- and sex-matched individuals without diabetes. Consecutive sections were immunostained with antisera against insulin, glucagon, and the established islet-protective and immunomodulatory factors Annexin A1 (ANXA1) and indoleamine 2,3-Dioxygenase-1. Whole-slide scans were acquired and npMSCs either inside or at the periphery (within 10 {micro}m) of islets were quantified on an individual-islet basis. We identified 53,375 npMSCs and performed an analysis of 26,376 individual islets. Culture-expanded MSCs were exposed to cytokines and viability and proliferation was assessed by flow cytometry. ResultsnpMSC were identified in situ in the human pancreas where they wrap around the islet periphery in an expected spindle-like morphology. ANXA1 was expressed by 33.2% of npMSCs and was expressed constitutively among individuals with or without diabetes. The density of both intraislet npMSCs and npMSCs within 10 {micro}m of the islet periphery was increased for insulin-containing islets in individuals with type 1 diabetes compared to individuals without diabetes (p < 0.001). npMSC density within 10 {micro}m of the islet periphery was preferentially increased in individuals [≥] 13 years at type 1 diabetes diagnosis compared to individuals < 13 years at type 1 diabetes diagnosis (p < 0.001). npMSC density was reduced around insulin-deficient islets compared to insulin-containing islets in individuals with diabetes (p < 0.001), consistent with an islet-protective role for npMSCs. Exposure of culture-expanded MSCs to an aggressive cytokine combination led to increased cell death and reduced proliferation. Conclusion/interpretationnpMSCs express ANXA1 constitutively suggesting an islet-protective role in health. The density of npMSCs was increased around insulin-containing islets and lost around insulin-deficient islets in individuals with type 1 diabetes which aligns with this hypothesis. npMSC density at the periphery of insulin-containing islets was preferentially higher in individuals with later onset type 1 diabetes, correlating with a less intense immune cell infiltration. The reduced ability of npMSCs to survive in the more intense pro-inflammatory environment around islets in younger onset type 1 diabetes may contribute to the rapid rate of beta cell loss in these individuals. Research in context What is already known about the subject?* Culture-expanded mesenchymal stromal cells (MSCs) reduce immune cell activation and improve islet functional survival. * Therapeutically administered MSCs migrate to sites of injury and preserve endogenous C-peptide in individuals with newly diagnosed type 1 diabetes. What is the key question?* Is the number, distribution and/or cytoprotective and immunomodulatory phenotype of native pancreatic MSCs (npMSCs) altered in type 1 diabetes? What are the new findings?* npMSCs express typical markers of isolated, culture-expanded MSCs and their number and density is increased within and at the periphery of insulin-containing islets in individuals with type 1 diabetes. * npMSCs are more abundant at the periphery of islets in individuals with later onset type 1 diabetes, correlating with a less intense immune cell infiltration. * MSCs are less able to survive an aggressive inflammatory environment such as that arising from the enhanced islet immune cell infiltration in individuals with younger onset type 1 diabetes How might this impact on clinical practice in the foreseeable future?* Therapeutically administered MSCs are likely to be more effective at preserving endogenous beta cell mass in individuals with later onset type 1 diabetes where MSCs have a greater ability to survive and exert their therapeutic functions.

physiology↗

bioRxiv2025

Morpho-functional timeline of progressive cystic fibrosis pancreatic exocrine and endocrine pathology derived from semi-quantitative scoring and AI-driven quantitativeimage analysis

Cystic fibrosis (CF) is associated with pancreatic exocrine insufficiency (PEI) early in life and diabetes in up to 50% of adults. The underlying CF-related sequential changes within the pancreas associated with exocrine and endocrine insufficiency remain incompletely understood due to scarcity of available human tissue, protracted disease course and absence of established robust and reproducible analytical approaches. This study aimed to develop and apply a systematic analysis cross-sectionally to CF pancreatic tissue samples from donors over a wide age range to construct a timeline related to the main exocrine and endocrine changes underlying progressive disease. Based on a histopathological semi-quantitative scoring system and AI-driven quantitative image analysis pancreatic changes were individually evaluated and classified according to three patterns: fibrotic; fibrotic and lipotic; and lipoatrophic. This systematic evaluation was applied to 29 CF and 58 control donors without pancreatic disease. Rapid loss of acinar tissue with virtually complete absence by the age of 7 years was confirmed, mirrored by fatty tissue replacement - changes underlying PEI and likely preceding progression towards diabetes. Ductal blockage by thickened secretions was associated with increasing ductal dilatation accompanied by peri-ductal fibrosis, followed by ductal loss with involution of associated fibrosis in parallel with increasing adipocyte proportional area (PA). Remaining ducts were relatively small surrounded by residual fibrosis. Islets became increasingly clustered initially surrounded by activated pancreatic stellate cells (PSCs) and fibrosis and then disorganised by interposing fibrotic tissue between endocrine cell regions and surrounded by residual collagen stranding in a lipoatrophic pancreas. Overall islet mass was not significantly reduced but {beta}-cell PA was significantly reduced from birth without further loss over time. We concluded that the natural history of pancreatic CF progresses inexorably from peri-ductal fibrosis to global fat replacement with relatively well-maintained islet mass but PSC-associated fibrotic islet remodelling circumstantially implicated in {beta}-cell failure.

pathology↗

bioRxiv2023

Developmentally dynamic changes in DNA methylation in the human pancreas

Development of the human pancreas requires the precise temporal control of gene expression via epigenetic mechanisms and the binding of key transcription factors. We quantified genome-wide patterns of DNA methylation in human fetal pancreatic samples from donors aged 6 to 21 post-conception weeks. We found dramatic changes in DNA methylation across pancreas development, with >21% of sites characterized as developmental differentially methylated positions (dDMPs) including many annotated to genes associated with monogenic diabetes. An analysis of DNA methylation in postnatal pancreas tissue showed that the dramatic temporal changes in DNA methylation occurring in the developing pancreas are largely limited to the prenatal period. Significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small proportion of sites showing sex-specific DNA methylation trajectories across pancreas development. Pancreas dDMPs were not distributed equally across the genome, and were depleted in regulatory domains characterized by open chromatin and the binding of known pancreatic development transcription factors. Finally, we compared our pancreas dDMPs to previous findings from the human brain, identifying evidence for tissue-specific developmental changes in DNA methylation. To our knowledge, this represents the most extensive exploration of DNA methylation patterns during human fetal pancreas development, confirming the prenatal period as a time of major epigenomic plasticity.

genomics↗

bioRxiv2023

Interferon-α promotes neo-antigen formation andpreferential HLA-B-restricted antigen presentation in pancreatic β-cells

Interferon (IFN)- is the earliest cytokine signature observed in individuals at risk for type 1 diabetes (T1D), but its effect on the repertoire of HLA Class I (HLA-I)-bound peptides presented by pancreatic {beta}-cells is unknown. Using immunopeptidomics, we characterized the peptide/HLA-I presentation in in-vitro resting and IFN--exposed {beta}-cells. IFN- increased HLA-I expression and peptide presentation, including neo-sequences derived from alternative mRNA splicing, post-translational modifications - notably glutathionylation - and protein cis-splicing. This antigenic landscape relied on processing by both the constitutive and immune proteasome. The resting {beta}-cell immunopeptidome was dominated by HLA-A-restricted ligands. However, IFN- only marginally upregulated HLA-A and largely favored HLA-B, translating into a major increase in HLA-B-restricted peptides and into an increased activation of HLA-B-restricted vs. HLA-A-restricted CD8+ T-cells. A preferential HLA-B hyper-expression was also observed in the islets of T1D vs. non-diabetic donors, and we identified islet-infiltrating CD8+ T-cells from T1D donors reactive to HLA-B-restricted granule peptides. Thus, the inflammatory milieu of insulitis may skew the autoimmune response toward epitopes presented by HLA-B, hence recruiting a distinct T-cell repertoire that may be relevant to T1D pathogenesis.

immunology↗

bioRxiv2023

Coxsackievirus infection induces direct pancreatic β-cell killing but poor anti-viral CD8+ T-cell responses

Coxsackievirus B (CVB) infection of pancreatic {beta} cells is associated with {beta}-cell autoimmunity. We investigated how CVB impacts human {beta} cells and anti-CVB T-cell responses. {beta} cells were efficiently infected by CVB in vitro, downregulated HLA Class I and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized only a fraction of these peptides, and only another sub-fraction was targeted by effector/memory T cells that expressed the exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with the {beta}-cell antigen GAD. Infected {beta} cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Thus, our in-vitro and ex-vivo data highlight limited T-cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and non-structural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.

immunology↗

bioRxiv2023

Interferons are the key cytokines acting on pancreatic islets in type 1 diabetes

The pro-inflammatory cytokines IFN, IFN{gamma}, IL-1{beta} and TNF may contribute to innate and adaptive immune responses during islet inflammation (insulitis) in type 1 diabetes (T1D). We used deep RNA-sequencing analysis to characterize the response of human pancreatic beta cells to each cytokine individually and compared the signatures obtained with those present in islets of individuals affected by T1D. IFN and IFN{gamma} had a much greater impact on the beta cell transcriptome when compared to IL-1{beta} and TNF. The IFN-induced gene signatures have a strong correlation with those observed in beta cells from T1D patients, and the level of expression of specific IFN-stimulated genes is positively correlated with proteins present in islets of these individuals, regulating beta cell responses to "danger signals" such as viral infections. These data suggest that IFN and IFN{gamma} are the central cytokines at the islet level in T1D, contributing to the triggering and amplification of autoimmunity.

immunology↗

bioRxiv2023

Validation of Tau Antibodies for Use in Western Blotting and Immunohistochemistry

BackgroundThe microtubule-associated protein Tau has attracted diverse and increasing research interest, with Tau being mentioned in the title/abstract of nearly 34,000 PubMed-indexed publications to date. To accelerate studies into Tau biology, the characterisation of its multiple proteoforms, including disease-relevant post-translational modifications (PTMs), and its role in neurodegeneration, a multitude of Tau-targeting antibodies have been developed, with hundreds of distinct antibody clones currently available for purchase. Nonetheless, concerns over antibody specificity and limited understanding of the performance of many of these reagents has hindered research. MethodsWe have employed a range of techniques in combination with samples of murine and human origin to characterise the performance and specificity of 53 commercially-available Tau antibodies by Western blot, and a subset of these, 35 antibodies, in immunohistochemistry. ResultsContinued expression of residual protein was found in presumptive Tau "knockout" human cells and further confirmed through mass-spectrometry proteomics, providing evidence of Tau isoforms generated by exon skipping. Importantly, many total and isoform-specific antibodies failed to detect this residual Tau, as well as Tau expressed at low, endogenous levels, thus highlighting the importance of antibody choice. Our data further reveal that the binding of several "total" Tau antibodies, which are assumed to detect Tau independently of post-translational modifications, was partially inhibited by phosphorylation. Many antibodies also displayed non-specific cross-reactivity, with some total and phospho-Tau antibodies cross-reacting with MAP2 isoforms, while the "oligomer-specific" T22 antibody detected monomeric Tau on Western blot. Regardless of their specificity, with one exception, the phospho-Tau antibodies tested were found to not detect the unphosphorylated protein. ConclusionsWe identify Tau antibodies across all categories (total, PTM-dependent and isoform-specific) that can be employed in Western blot and/or immunohistochemistry applications to reliably detect even low levels of Tau expression with high specificity. This is of particular importance for studying Tau in non-neuronal cells and peripheral tissues, as well as for the confident validation of knockout cells and/or animal models. This work represents an extensive resource that serves as a point of reference for future studies. Our findings may also aid in the re-interpretation of existing data and improve reproducibility of Tau research.

neuroscience↗

bioRxiv2019

Vitamin D-binding protein is required for the maintenance of α-cell function and glucagon secretion

Vitamin D-binding protein (DBP) or GC-globulin carries vitamin D metabolites from the circulation to target tissues. DBP expression is highly-localized to the liver and pancreatic -cells. While DBP serum levels, gene polymorphisms and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates -cell morphology, -cell function and glucagon secretion. Deletion of DBP led to smaller and hyperplastic -cells, altered Na+ channel conductance, impaired -cell activation by low glucose, and reduced rates of glucagon secretion. Mechanistically, this involved reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects were also seen in {beta}-cell and {delta}-cell function. Immunostaining of human pancreata revealed generalized loss of DBP expression as a feature of late-onset and longstanding, but not early-onset type 1 diabetes. Thus, DBP is a critical regulator of -cell phenotype, with implications for diabetes pathogenesis. HIGHLIGHTSO_LIDBP expression is highly-localized to mouse and human -cells C_LIO_LILoss of DBP increases -cell number, but decreases -cell size C_LIO_LI-cells in DBP knockout islets are dysfunctional and secrete less glucagon C_LIO_LIDBP expression is decreased in -cells of donors with late-onset or longstanding type 1 diabetes C_LI

physiology↗

bioRxiv2018

Detection of enterovirus protein and RNA in multiple tissues from nPOD organ donors with type 1 diabetes

Epidemiological studies have shown an association between enterovirus (EV) infections and type 1 diabetes (T1D), and EV protein has been detected in the pancreatic islets of T1D patients. Here we correlated the detection of EVs in lymphoid tissues (spleen and pancreatic lymph nodes) and small intestinal mucosa to the virus detection in the pancreas of T1D, autoantibody-positive (aab+) and non-diabetic control organ donors of the Network for Pancreatic Organ Donors with Diabetes (nPOD) study. Formalin-fixed paraffin-embedded tissue samples were screened for insulin and EV protein using immunohistochemistry, and frozen tissue for EV genome using RT-PCR. The presence of EV protein in the pancreatic islets correlated with the presence of insulin-positive cells. Altogether 62 % of T1D and aab+ donors were positive for EV protein in pancreatic islets (only insulin-positive donors included), 40 % in duodenum and 32 % in spleen, compared to 33 %, 14 %, and 27 % of non-diabetic controls. Pancreatic lymph nodes were positive for EV protein in 60 % of T1D and aab+ cases. T1D and aab+ donors were more frequently VP1-positive in multiple organs than control donors (39 % vs. 11 %; including only insulin-positive donors). EV RNA was found in selected donors and from multiple tissue types except for duodenum, and individual T1D and aab+ donors were EV RNA-positive in multiple organs. The role of extra-pancreatic organs and their interplay with EV in T1D pathogenesis remains to be solved, but we hypothesize that these organs may serve as a reservoir for the virus which may reside in these tissues in a slow-replicating persistent form.

epidemiology↗