Distribution of glutathione peroxidase-1 immunoreactive cells in pancreatic islets from type 1 diabetic donors and non-diabetic donors with and without islet cell autoantibodies is variable and independent of disease 

Distribution of glutathione peroxidase-1 immunoreactive cells in pancreatic islets from type 1 diabetic donors and non-diabetic donors with and without islet cell autoantibodies is variable and independent of disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Distribution of glutathione peroxidase-1 immunoreactive cells in pancreatic islets from type 1 diabetic donors and non-diabetic donors with and without islet cell autoantibodies is variable and independent of disease Kaaj Pala, Kevin Xueying Sun, Lars Krogvold, Knut Dahl-Jørgensen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5369709/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Mar, 2025 Read the published version in Cell and Tissue Research → Version 1 posted 10 You are reading this latest preprint version Abstract During type 1 diabetes (T1D), oxidative stress in beta cells may cause early dysfunction and initiate autoimmunity. Mouse islets express lower levels of reactive oxygen species (ROS) clearing enzymes, glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase in comparison with several other tissues. It remains unclear if human beta cells show a similar deficiency, particularly during T1D or show a higher degree oxidative resistance. We compared islet cell distributions and determined graded intensities of glutathione peroxidase1 (GPX1), a key enzyme which detoxifies hydrogen peroxide, by applying combined immunohistochemistry for GPX1, insulin and glucagon, in pancreatic sections from new-onset T1D (group 1), non-diabetic autoantibody-negative (group 2), non-diabetic autoantibody-positive (group 3) and long-term diabetic (group 4) donors. Islets from all study groups demonstrated either uniform but graded staining intensities for GPX1 in almost all islet cells or strong staining in selective islet cells with weaker intensities in the remaining cells. GPX1 was present in selective glucagon and in insulin cells also and in cells negative for both hormones, with stronger intensities in a higher percentage of glucagon than insulin cells. The enzyme was absent in a higher percentage of beta cells than glucagon cells independent of disease or autoantibody positivity. We conclude that a proportion of human beta cells and glucagon cells express GPX1 but show heterogeneity in its distribution and intensities, independent of disease or autoantibody status. Pharmacologic attempts to boost GPX1 level in human beta cells during early T1D or in isolated islets for transplantation may offer clinical benefits. Type 1 diabetes Glutathione peroxidase-1 Reactive oxygen species Beta cell damage Immunohistochemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Introduction During type 1 diabetes (T1D), beta cells in pancreatic islets are selectively and progressively destroyed by immune-mediated processes, culminating in an abrupt clinical onset (Atkinson et al, 2011 , 2014). Its diagnosis is preceded by an asymptomatic protracted phase lasting several months to years when subjects manifest variable degrees of beta cell dysfunction (Atkinson and Mirmira, 2023 , Evans-Molina, et al., 2018 ). Following onset, daily administration of insulin is mandatory to suppress hyperglycaemia. The immune destructive process is beta cell specific since the closely-located non-beta endocrine cells within the islet escape destruction, although glucagon cells display functional impairment (Atkinson and Mirmira, 2023 , Doliba, et al., 2022 , Yosten, 2018 ). The exquisite immune specificity for beta cells suggests that such cells may already harbour molecular signatures of vulnerability which may act not only as initiators, or conduits for the early immune response but also amplifiers of ongoing beta cell-directed immune destruction (Kracht, et al., 2017 , Mallone and Eizirik, 2020 , Mallone, et al., 2022 , Peters, et al., 2019 , Roep, et al., 2021 , Thompson, et al., 2023 ). However, a subset of beta cells in some subjects remain detectable many years after diagnosis (Keenan, et al., 2010 , Reddy, et al., 2021 ). Although low levels of reactive oxygen species (ROS) mediate critical physiological signalling pathways, accumulation of raised levels, if not cleared rapidly, can lead to major deleterious consequences affecting several organs, such as the heart, kidney, eye and brain, culminating in a variety of human disorders (Forman and Zhang, 2021 ). Elevated ROS injure cells by promoting degradation of cellular proteins, lipids and DNA. In addition they affect several adverse cell signalling pathways involving mammalian target of rapamycin complex 1 (mTORC1), c-jun N-terminal kinase (JNK) and also invoke inflammation by activating nuclear factor-kB (nF-κB) (Kulkarni, et al., 2022 ). Beta cells exhibit sustained high respiratory, metabolic, secretory and protein synthetic rates. High respiratory activity results in raised levels of ROS originating from the electron transport chain. However, little is known on how human beta cells eliminate excess levels of ROS efficiently. During T1D, early environmental agents, such as certain viruses and chemicals may also provoke deleterious events in beta cells leading to oxidative, nitrosative and endoplasmic reticulum (ER) stress (Amirruddin, et al., 2021 , Craig, et al., 2019 , Hasnain, et al., 2016 , Hober and Sauter, 2010 , Leenders, et al., 2021 , Mallone and Eizirik, 2020 , Roep, Thomaidou, van Tienhoven and Zaldumbide, 2021 ). Such stressors result in the formation of misfolded proteins, and neoantigens which trigger a clinically silent phase of beta cell dysfunction and self-directed immune reactivity prior to clinical diagnosis (Atkinson, et al., 2011 , Sims, et al., 2020 , Sims, et al., 2019 ). For example, recent studies have shown that ER stress within beta cells leads to translational errors in the reading frame of human insulin mRNA, resulting in the generation of defective polypeptides and formation of neo-epitopes, capable of recognition by circulating T cell populations which have escaped prior thymic deletion (Kracht, van Lummel, Nikolic, Joosten, Laban, van der Slik, van Veelen, Carlotti, de Koning, Hoeben, Zaldumbide and Roep, 2017, Roep, Thomaidou, van Tienhoven and Zaldumbide, 2021 ). In addition, neo-epitope formation from degraded citrullinated proteins such as glucose-regulated protein 78 following exposure of human islets to inflammatory molecules, can lead to immune responses against beta cells (Buitinga, et al., 2018 ). High levels of ROS, such as superoxide anions ( 1 O 2 ) and hydrogen peroxide (H 2 O 2 ), originate from the electron transport chain and to a lesser extent following catalysis by cytosolic and plasma membrane oxidoreductases (Nolfi-Donegan, et al., 2020 ). Excess levels of superoxide can dismutate either spontaneously to H 2 O 2 or following catalysis by superoxide dismutase (SOD). Hydrogen peroxide is converted to water and oxygen by glutathione peroxidases, catalase and peroxiredoxins (Bast, et al., 2002 , Handy and Loscalzo, 2022 , Stancill, et al., 2019 , Stancill and Corbett, 2021 , Wolf, et al., 2010 ). Mouse islets express lower levels of SOD, glutathione peroxidase (GPX) and catalase at the mRNA and protein level than other tissues such as liver and kidneys (Grankvist, et al., 1981 , Lenzen, 2008 , Tiedge, et al., 1997 , Tiedge, et al., 1999 ). During the prodromal phase of T1D and preceding early insulitis, islet cell autoantibodies against several antigens such as insulin, glutamic acid decarboxylase and zinc transporter-8 are detectable in sera of most subjects (Bogun, et al., 2020 , Campbell-Thompson, et al., 2016 , Krischer, et al., 2022 ). Whether excess uncleared ROS originating from beta cell mitochondrial respiration contributes to the ontogeny of islet cell autoantibodies and insulitis remains unclear. Glutathione peroxidase (GPX) is present in six isoforms. Isoforms 1 to 4 are homotetramers, containing selenium bound to cysteine in their active site and are referred as selenoproteins (Handy and Loscalzo, 2022 ). They play a key role in detoxifying H 2 O 2 and lipid peroxides in many tissues (Robertson and Harmon, 2007 , Tanaka, et al., 1999 ). Glutathione peroxidase catalysis occurs in the presence of reduced glutathione, a tripeptide, in a paired reaction involving glutathione reductase which regenerates reduced glutathione from oxidized glutathione (Handy and Loscalzo, 2022 ). However, whether the previously reported lower levels glutathione peroxidase-1 (GPX1) in mouse and human islets by Western blotting and enzyme assays are also an intrinsic feature of human beta cells in situ are unclear (Grankvist, Marklund and Täljedal, 1981 , Tonooka, et al., 2007 ). Little is known on the cellular expression of GPX1 in human pancreatic islets in donors with increasing duration of T1D and whether the expression patterns differ in non-diabetic cases with and without islet cell autoantibodies. In a brief study, immunohistochemical analysis of pancreatic sections from deceased non-diabetic human donors showed expression of GPX1 and catalase predominantly in selective glucagon cells and in some beta cells, with an absence in the remaining islet cell-types (Miki, et al., 2018 ). However, this study did not fully analyze the possible changing expression pattern of GPX1 in glucagon and beta cells from donors with variable duration of T1D, including new-onset cases. In addition, similar analysis was not undertaken by the same authors in non-diabetic donors with and without islet cell autoantibodies. Thus, there is an urgent need to investigate and compare the expression of GPX1 in persisting beta cells and other islet cell-types at and after diagnosis of T1D with non-diabetic cases with and without islet cell autoantibodies. The recent availability of rare pancreatic samples from cases with variable but precisely defined duration of T1D and non-diabetic donors with and without serum islet cell autoantibodies has provided us an impetus and rationale to undertake detailed studies on the expression of GPX1 in islets. In the present study, we have, therefore, utilized such unique samples and applied an optimized triple-label immunohistochemical protocol for comparing the localization of GPX1 in beta and alpha cells and estimate its level and relative expression in the two cell-types in high quality human pancreatic sections from diabetic and non-diabetic deceased donors, supplied by the Network for Pancreatic Organ Donors with Diabetes (nPOD) and the Exeter Archival Diabetes Biobank (EADB) (Morgan and Richardson, 2018 , Pugliese, et al., 2014 ). We have also analyzed sections of well-preserved surgically-retrieved rare pancreatic biopsies from four living volunteers with recent-onset T1D, supplied by the Diabetes Virus Detection (DiViD) Study (Krogvold, et al., 2014 ). Methods Subjects and pancreatic sections Paraffin-embedded pancreatic sections (5 µm) from formalin-fixed biopsy specimens from 4 living individuals with newly-diagnosed type 1 diabetes from DiViD, 22 formalin-fixed cadaveric pancreases supplied by nPOD, and 1 pancreas at post-mortem from EADB, United Kingdom were analysed (Krogvold, Edwin, Buanes, Ludvigsson, Korsgren, Hyöty, Frisk, Hanssen and Dahl-Jørgensen, 2014, Morgan and Richardson, 2018 , Pugliese, Yang, Kusmarteva, Heiple, Vendrame, Wasserfall, Rowe, Moraski, Ball, Jebson, Schatz, Gianani, Burke, Nierras, Staeva, Kaddis, Campbell-Thompson and Atkinson, 2014). In addition, sections prepared from formalin-fixed liver (University of Harbin, Harbin, China), breast (Auckland Tissue Biobank, University of Auckland) and lung (Department of Anatomy and Medical Imaging, University of Auckland) carcinoma were tested as positive controls for GPX1 immunostaining. Pancreatic sections from human cases were triple-immunostained for GPX1, insulin and glucagon and analysed. Please refer to “Acknowledgements” for ethical approval. The main study groups are summarised in Table 1 and donor demographics are detailed in the electronic supplementary material (ESM Table 1). Triple-label immunohistochemical protocol We employed a commercially available affinity-purified rabbit polyclonal antibody monospecific for human GPX1 (Abcam ab22604, supplied as 1 mg/ml IgG). This antibody has been validated by the suppliers as suitable for immunohistochemistry and Western blotting (refer below for further details on immunohistochemical protocols and to ESM Table 2 ). It has also been validated and employed by previous investigators for immunohistochemical localization of GPX1 in formalin-fixed human lung sections and Bouin’s fixed human pancreatic sections (Basnet, et al., 2019 , Miki, Ricordi, Sakuma, Yamamoto, Misawa, Mita, Molano, Vaziri, Pileggi and Ichii, 2018). Anti-GPX1 was titrated to a working dilution of 1:120 (final concentration: 8.33 µg/ml IgG), prior to its application to sections from formalin-fixed human pancreas. As negative controls, anti-GPX1 was replaced with diluent or normal rabbit IgG in the immunohistochemical procedure. In initial studies, immunohistochemical staining for GPX1 in pancreatic sections with species-specific secondary antibodies linked to anti-rabbit IgG-horseradish peroxidase polymer and subsequent reaction with diaminobenzidine hydrochloride (adopted in this study) was compared with donkey anti-rabbit IgG-Alexa 568 as secondary antibody. The two protocols yielded concordant cellular staining (see below under “Methods”). In addition, we stained pancreatic sections with a mixture rabbit anti-glucagon (same immunizing species as GPX1 antibody), guinea pig anti-insulin and mouse anti-CD45. Immunohistochemical specificity of anti-GPX1 was also evaluated in formalin-fixed paraffin sections from liver, breast and lung cancer, known to express GPX1 (Basnet, Tian, Ganesh, Huang, Macalinao, Brogi, Finley and Massagué, 2019, Woolston, et al., 2011 ). Please see ESM Fig. 1 and ESM Fig. 2 under “Results”. The immunohistochemical protocol for GPX1 applied in this study was slightly modified from our previous procedures for interleukin-1β and inducible nitric oxide synthase (Reddy, et al., 2018 , Reddy, Krogvold, Martin, Sun, Martin, Al-Ani and Dahl-Jørgensen, 2021). Relevant immunohistochemical steps, reagents and incubation times are summarised in ESM Table 2 . Thus, sections were deparaffinized, rehydrated and subjected to antigen retrieval with citrate buffer, pH 6, containing 0.05% (vol/vol) Tween-20 (Sigma-Aldrich, Darmstadt, Germany), cooled in distilled water and permeabilized in 0.4% (vol/vol) Triton-X-100 in phosphate-buffered saline (PBS), pH 7.4, on ice for 30 min. After washing sections in PBS for 10 min, they were exposed to 3% (vol/vol) H 2 O 2 in PBS for 15 min. Following two consecutive 5- minute washing steps in PBS, sections were blocked with 10% (vol./vol.) normal goat serum constituted in low protein blocking solution (EBioSciences, San Diego, CA, USA) for 1 h at 37 ᵒ C, followed by incubation with rabbit anti-GPX1 (1:120, diluted in antibody diluent from Cell Signaling Technology, Danvers, MA, USA) for 16–18 h at 4 ᵒ C. After a wash step, sections were incubated with anti-rabbit IgG horseradish peroxidase polymer (Cell Signaling Technology), diluted 1:1 with 0.1% vol/vol Tween-20 in PBS (wash buffer). After further washing, sections were reacted with 3,3'-diaminobenzidine (DAB) substrate (freshly-prepared in accordance with the manufacturer’s instructions; catalogue number: 8059; Cell Signaling Technology) for 5–6 minutes. Following monitoring of peroxidase-catalysed DAB-product, by bright field microscopy, reaction was terminated in excess distilled water. Sections were then briefly immersed in wash buffer and incubated with a mixture of guinea pig anti-insulin and mouse anti-glucagon (both at 1:600 dilution in antibody diluent from Cell Signaling) and incubated for 90 minutes at 37 ᵒ C. Sections were washed in wash buffer and reacted with a mixture of highly cross-absorbed species-specific donkey anti-guinea pig IgG-Alexa 488 (1:300) and donkey anti-mouse IgG-Alexa 568 (1:600), diluted in 0.2% vol/vol Tween-20 in PBS for 1 h at 37 ᵒ C. Following washing, sections were mounted with CitiFluor mountant for microscopic analysis. Image acquisition Sections were examined with a Nikon Eclipse-epifluorescence-bright field microscope and digital images were recorded and saved as Tiffs. Groups 2 and 3 were blinded to the investigator, but not Groups 1 and 4, where many islets from all cases were either insulin-negative or showed reduced number of residual beta cells. Such distinct pathologies displayed by all cases from the two diabetic groups did not permit sample blinding. However, case characteristics such as duration of T1D and clinical information was blinded to the investigator. From each section, all islets with approximately ≥ 20 endocrine cells were imaged for the presence of GPX1 by bright field microscopy. Uniform gain and exposure settings in the Nikon NIS Imaging Software with identical objective lens magnification of 40x with a numerical aperture of 0.75 were applied to all sections for ensuring consistency in image intensities and magnification. Following image acquisition by bright field, the same islets were co-imaged for insulin and glucagon staining by fluorescence microscopy equipped with appropriate narrow band filters. Each of the specific image sets from multiple acquisitions (GPX1, ±insulin and glucagon) were merged using Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA). For image display in combination with insulin and glucagon staining by fluorescence, GPX-1 immunostained cells (initially brown) were converted to a blue fluorescent pseudocolour for photographic display. Image analysis Insulin-positive and -negative islets in all donors were enumerated. Following triple staining (GPX1 ± insulin + glucagon), the overall cellular intensity for GPX1 (brown) and its distribution pattern in all islets with more than 20 estimated cells were graded visually following display of acquired images by Photoshop and magnification for determining distribution of cells as follows. Islets with very weak staining in almost all islet cells were assigned grade 1, those with overall weak staining as grade 2 and those with moderate to strong staining in most islet cells were assigned grade 3 (Pattern 1). In addition, islets with strong GPX1 staining in only a few selective islet cells with negative staining in a majority of islet cells were assigned grade 4, while those with strong staining in a minority of islet cells accompanied by weak staining in a majority of islet cells were assigned grade 5. Islets showing strong staining in a minority of cells coupled with moderate staining in a majority of islet cells were assigned “grade 6”. Grades 4–6 were defined as “Pattern 2”. The grading system for the two patterns was based on a visual guide shown in Fig. 1 . We also graded the intensities of GPX1 staining in each insulin and glucagon cell located in 10 randomly selected islets per case by viewing, magnifying and merging image sets in Photoshop, to ascertain the precise identity of the two cell-types, GPX-1 positivity, grading and enumeration. Staining for GPX1 in each insulin and glucagon cell from 10 islets per case was scored as either negative, weak, moderate or strong in accordance with a visual template guide depicted in Fig. 2 . In insulin-negative islets from diabetic cases the various intensity grades for GPX1 in glucagon cells. Data Analysis Results were tabulated and represented as bar graphs for each donor and analysed for: Number of islets imaged per section Number of islets per case positive and negative for insulin Percentage of all islets including insulin-positive and negative islets from each donor with GPX-1 positive cells and their intensities, graded on a scale from 1–3 (Pattern 1; Fig. 1 ) 4–6 (Pattern 2; Fig. 1 ) Percentage of insulin and glucagon cells in 10 randomly selected islets from each case with various intensities of GPX-1 staining, graded on a scale of negative, weak, moderate and strong, indicated in Fig. 2 Total percentage of insulin and glucagon cells per group with various grades of GPX1 staining as specified under (d). For each group, the total percentages were calculated from the number of insulin- and glucagon-positive cells in 10 islets per case Following image acquisition and analysis, sections were counterstained with haematoxylin to reveal islet histology and its surrounding area. Statistical analysis A student t-test in the excel of Microsoft Office 2016 was used for statistical analysis. P < 0.05 was considered statistically significant. Results Immunohistochemical specificity of rabbit anti-GPX-1 In the immunohistochemical procedure for GPX-1, substitution of anti-rabbit GPX-1 with antibody diluent or normal rabbit IgG did not show staining of islets cells and the surrounding region (ESM Fig. 1 a-f). Incubation of a pancreatic section with rabbit anti-glucagon (also a rabbit antibody as for GPX1) in combination with guinea pig anti-insulin and mouse anti-CD45 (antibody of mouse origin as for glucagon) showed the expected staining of alpha cells, beta cells and CD45-positive cells, respectively (ESM Fig. 1 g-i). Addition of anti-GPX1 to separate pancreatic sections showed staining of several islet cells following incubation with anti-rabbit IgG horseradish peroxidase polymer and reaction with 3,3’diaminobenzidine (DAB) substrate (ESM Fig. 1 j-l) or donkey anti-rabbit IgG-Alexa 568 (ESM Fig. 1 m-o). Previous reports demonstrated that anti-GPX-1 (Abcam, ab22604; employed in this study) stained distinct cells in formalin-fixed tissue sections from lung carcinoma (Basnet, Tian, Ganesh, Huang, Macalinao, Brogi, Finley and Massagué, 2019). The corresponding transcript was also demonstrated in multiple tissues including breast cancer (Chu, et al., 1992 , Woolston, Al-Attar, Storr, Ellis, Morgan and Martin, 2011 ). Here we confirm that anti-GPX1 stained cells in sections of formalin-fixed human breast, liver, lung and pancreas (ESM Fig. 2 a-c; Fig. 4 ). Percentage of insulin-positive islets Please refer to the demographic data for characteristics of each donor in ESM Table 1. The percentage of insulin-positive islets per donor from the 4 study groups is shown in Fig. 3 a-d (values per case are itemized in the second column of ESM Tables 3–6). The number of islets analysed per case is indicated above each bar. In the non-diabetic autoantibody-negative and positive groups, all islets were insulin-positive (Fig. 3 b, c), but were highly variable in diabetic donors (Fig. 3 a,d). Islets from DiViD donor 1 and nPOD 6088, 6045 and 6220 (long-standing diabetes), were insulin-negative. In DiViD donors 2, 3 and 4, the percentages were 34.15%, 58.44% and 21.43%, respectively. Among the long-term diabetic donors (Group 4), the highest percentages were in case E560 (55.7%), nPOD 6211 (21.25%) and nPOD 6070 (29.76%). There was a marked reduction in the number of insulin-positive islets in nPOD 6245 and nPOD 6262, being absent in cases 6088 and 6220 (Fig. 3 d). Distribution and localization of GPX1 in islet cells We observed inter-islet variability in the distribution and staining intensities of GPX1-positive islet cells in each donor and among different donors. Additional immunoreactivity was observed in selective pancreatic ductal cells, some of which were glucagon-positive (ESM Fig. 3 ). Some cells negative for insulin and glucagon within the intra-islet vascular space and external to the islet showed GPX1 positivity (Fig. 8 d-f). It was only weakly expressed in pancreatic acinar cells. In pancreatic islets, two major patterns of GPX1 expression were observed and defined as pattern 1 or 2 (Fig. 1 a-f). Examples of the overall distribution of GPX1-positive cells in selected islets from various donors are depicted as assembled micrographs, where the first column shows the presence of GPX1 cells alone within islets and the surrounding regions, the second column displays merged images of cells positive for GPX1 (brown GPX1 cells converted to a blue pseudocolour), insulin (green) and glucagon (red), while the third column shows corresponding fields after counterstaining with haematoxylin. Group 1 (newly-diagnosed cases from DiViD): In DiViD case 1, all islets were devoid of beta cells, with strong expression of GPX1 in a majority of glucagon cells which occupied almost the entire islet area (Fig. 4 a-f). In DiViD case 2, GPX1 was weakly expressed in glucagon cells and in the remaining beta cells; islets devoid of beta cells showed GPX1 immunoreactivity in selective glucagon cells and in some cells negative for insulin and glucagon (Fig. 4 g-l). In DiViD cases 3 and 4, GPX1 was strongly as well as weakly expressed in glucagon cells, in association with positivity in selective insulin cells (Fig. 5 a-l). In insulin-negative islets, it was also observed in almost all islet cells, with many corresponding to glucagon cells (Fig. 5 d-f, j-l). It was also seen in some islet cells negative for insulin and glucagon (Fig. 5 j-l). Group 2 (Non-diabetic autoantibody-negative cases): In nPOD 6289, 6234 and 6160, weak to moderate GPX1 intensities were present in almost all islet cells, while only some islets showed slightly stronger expression in selective insulin and glucagon cells (Fig. 6 a-l; Fig. 7 a-f). However, in nPOD 6178, a majority of islets showed strong expression of GPX1 in only a minority of glucagon cells, with either an absence or weak to moderate staining in the remaining glucagon cells; only weak or negative expression was present in selective insulin cells (Fig. 7 g-l). A similar pattern was observed in nPOD 6229 (ESM Fig. 4 g-l). In nPOD 6401, 6055 and 6048, moderate to strong staining for GPX1 was present in entire islet cells and was colocalized in glucagon and insulin cells (ESM Fig. 4 a-f; Fig. 8 a-l) while in nPOD 6369 strong expression was observed in selective glucagon cells with moderate expression in most glucagon and insulin cells (ESM Fig. 5 a-f). Group 3 (Non-diabetic autoantibody-positive cases): In nPOD 6424 and 6267 (with 2 autoantibodies), an overall weak GPX staining was present in most islet cells (Fig. 9 a-l). Occasional strong GPX1-expressing cells within the intra-islet space were also present (Fig. 9 g-l). In nPOD 6301 and 6310 (single autoantibody-positive), selective insulin and glucagon cells showed moderate to strong GPX1 staining, while a majority showed weaker expression (Fig. 10 a-l). An overall weak to moderate expression was present in most islet cells in nPOD 6167 and 6158 (with two autoantibodies; ESM Fig. 6 a-l). Group 4 (Long-term diabetic cases): In E560 (1.5 years of T1D), moderate to strong expression of GPX1 was present in selective glucagon and some remaining insulin cells and in cells negative for both cell-types (Fig. 11 a-f). In nPOD 6211 (4 years of T1D), islet cells showed either weak or stronger expression in selective glucagon and remaining insulin cells (Fig. 11 g-l). A similar pattern was also present in nPOD 6088 (4 years of T1D; Fig. 12 a-f) and nPOD 6070 (7 years of T1D; Fig. 12 g-l). In nPOD 6245 (7 years of T1D), most islet cells showed an overall weak expression (Fig. 13 a-f). A similar cellular pattern was observed in some islets from nPOD 6045 (8 years of T1D). However, in this donor, several cells in specific islets negative for insulin and glucagon, showed unusually strong staining intensities for GPX1 (Fig. 13 j-l). In nPOD 6262 (8 years of T1D) and nPOD 6220 (11 years of T1D), islets were negative for insulin, but showed strong expression of GPX1 in selective glucagon cells, with moderate expression in the remaining islet cells (ESM Fig. 7 a-l). Analysis of overall GPX1 staining patterns in islets in the 4 study groups The overall cellular pattern of GPX1 expression in islets and its staining intensities in islet cells and in insulin-negative and positive islets, were quantified based on a visual grading system depicted in Fig. 1 (Pattern 1 (grades 1–3) and Pattern 2 (grades 4–6)). The various grades are represented graphically in Fig. 14 a, b and values listed in ESM Tables 3–6. The percentage of islets with combined grades of 1, 2 and 3 (pattern 1) versus 4, 5 and 6 (pattern 2) are summarised in Table 2 . In groups 1, 2 and 3, differences between the two patterns reached statistical significance (p = 0.035, 0.006 and 0.008, respectively but not in group 4 (p = 0.071) Gradings for GPX1 in insulin-positive and negative islets (Groups 1 and 4) are represented as segmented bar graphs in Fig. 14 b and tabulated in ESM Tables 7–10. The variable percentages of islets with the two patterns probably reflect re-distribution of islet cells during beta cell loss. In group 1 (newly-diagnosed diabetic group), there was no significant difference between the percentage of insulin-positive islets with pattern 1 and pattern 2 (p = 0.09) and in insulin-negative islets (p = 0.80). However, in Group 4 (long-term diabetic group) there was a significant difference between the two patterns in insulin-positive islets (p = 0.04) but not in insulin-negative islets (p = 0.06). Analysis of GPX1 staining intensities in individual insulin and glucagon cells in 10 randomly selected islets per case from all study groups (refer to Fig. 15 and ESM Tables 11–15) Group 1 (newly-diagnosed cases): Islets from case 1 were insulin-negative, with strong GPX1 staining in approximately 90% of glucagon cells, while in case 2, 80% of beta cells had weak staining and almost 55% of glucagon cells had moderate to strong staining. In case 3, 77% of insulin cells had negative GPX1 staining, while almost 90% glucagon cells had moderate to strong staining for GPX1. In case 4, almost all insulin cells and glucagon cells showed moderate GPX1 staining intensities. Group 2 (non-diabetic autoantibody-negative cases): In cases 6289, 6160, 6055 and 6229, many insulin cells showed weak GPX1 staining while in case 6178, 97% of such cells were negative with only 24% of glucagon cells with moderate to strong staining. In case 6401, > 78% of insulin cells had moderate GPX1 intensities. More than 80% of glucagon cells in cases 6401 and 6369 had moderate GPX1 intensity while in the remaining cases the intensities in the same cell-types were more variable. Group 3 (non-diabetic autoantibody-positive cases): In cases 6424, 6301 and 6158, more than 65% of insulin cells showed moderate GPX1 staining intensities while in cases 6267, 6310 and 6167, 99%, 62% and 56%, respectively showed weak intensities. In cases 6424, 6301, 6310 and 6158, moderate intensities were present in 68%, 88%, 64% and 94%, of glucagon cells, respectively. In cases 6267 and 6167, weak intensities were present in 73% and 56% of glucagon cells, respectively. In all 6 cases, strong intensities were observed in a minority of glucagon cells (< 15% of glucagon cells), with staining intensities being independent of the presence of single or double autoantibodies. In groups 1, 2 and 3, differences between the percentages of insulin cells versus glucagon cells with negative, weak and moderate GPX1 staining intensities did not reach statistical difference. However, there was a significant difference in Group 2 between insulin and glucagon cells with strong GPX1 staining (p = 0.03). Group 4 (long-term diabetic cases): Four of 8 cases from this group, showed an absence of insulin cells in 10 randomly selected islets per case. In cases 6211, 6070, and 6245 more than 50% of insulin cells showed weak staining intensities for GPX1 while in case E560, approximately 75% of insulin cells displayed moderate staining intensities. Weak intensities were displayed in a majority of the remaining insulin cells (6211: 50.55%; 6070: 76.6% and 6245: 83.5%). In 7/8 cases from the same group, more than 50% of glucagon cells per case showed moderate staining intensities while in the remaining case (E560: 1.5 years of T1D), 75% glucagon cells showed moderate-strong intensities. In this group, there was a significant difference in the percentages of insulin cells versus glucagon cells with moderate to strong GPX1 staining intensities (p = 0.003). Cumulative GPX1 staining intensities per study group (refer to ESM Table 15 and Fig. 16 ): GPX1 staining in Group 1 was absent in a higher percentage of insulin cells (30.34%) than glucagon cells (0.2%). A similar difference was seen in groups 2 and 3 while in group 4 (long-term diabetic group), almost all insulin and glucagon cells showed weak to strong GPX1 staining intensities. In all 4 groups moderate to strong intensities were observed in higher percentage of glucagon cells than beta cells (refer to ESM Table 15). In Group 4, there was a significant difference between the percentage of beta cells and glucagon cells with moderate GPX1 staining intensity (p = 0.005). Discussion Earlier results from experimental mice show an intrinsic absence or lower expression levels of the three important ROS clearing enzymes, namely GPX1, catalase and SOD in islets (Grankvist, Marklund and Täljedal, 1981 , Harmon, et al., 2009 , Lenzen, 2008 , Tiedge, Lortz, Drinkgern and Lenzen, 1997 , Tiedge, Lortz, Munday and Lenzen, 1999 ). If such findings hold true for human islets, one may conclude that human beta cells may also fail to clear excess ROS. However, low levels of expression of the three enzymes selectively in beta cells of mice have not been studied in detail. If such deficits are present in human beta cells, the resulting redox imbalance may activate critical immune signals in these cells prior to and during human T1D (Gerber and Rutter, 2017 , Leenders, Groen, de Graaf, Engelse, Rabelink, de Koning and Carlotti, 2021, Mallone, Halliez, Rui and Herold, 2022 , Newsholme, et al., 2019 , Robertson and Harmon, 2006 , Robertson and Harmon, 2007 ). Despite such void, animal findings have led to the evaluation of several therapeutic approaches targeted at boosting levels of SOD and/or GPX1 or the use of low molecular weight mimetics to suppress excess ROS in beta cell lines and in experimental animals afflicted with various pathologies (Bertera, et al., 2003 , Forman and Zhang, 2021 , Harmon, Bogdani, Parazzoli, Mak, Oseid, Berghmans, Leboeuf and Robertson, 2009, Mallone, Halliez, Rui and Herold, 2022 , Tiedge, Lortz, Munday and Lenzen, 1999 ). Although such efforts have yielded little success, more recent ploys incorporating specific and targeted payload delivery systems and techniques for up-regulating transcription factors that activate specific genes encoding anti-oxidant enzymes are in progress (Poprac, et al., 2017 ). The availability of rare pancreatic sections from clinically well-defined diabetic and non-diabetic donors has permitted us to develop and apply a validated immunohistochemical protocol for the analysis of GPX1 expression in combination with insulin and glucagon and compare its distribution and staining intensities in donors with T1D and in autoantibody-positive and -negative non-diabetic donors. The specificity of anti-GPX1 was confirmed in this study by demonstrating positive staining in human liver, lung and breast carcinoma, omission of anti-GPX1 in the immunohistochemical procedure and the use of GPX1 antibody from the same supplier by others (Basnet, Tian, Ganesh, Huang, Macalinao, Brogi, Finley and Massagué, 2019, Chu, Esworthy, Doroshow, Doan and Liu, 1992 , Miki, Ricordi, Sakuma, Yamamoto, Misawa, Mita, Molano, Vaziri, Pileggi and Ichii, 2018). We also validated the expression of GPX1 in human pancreatic sections by employing an immunofluorescence technique. Staining of the enzyme in some ductal cells, a proportion of which co-localized with glucagon is novel. Glutathione peroxidase-1 immunoreactivity in insulin- and glucagon-negative islet cells, shown here, likely represents somatostatin and/or pancreatic polypeptide hormone and ghrelin cells or non-endocrine cells within the intra-islet vascular region, such as endothelial cells or immune cells. Thus, we show that the distribution of GPX1 in the human pancreatic islet cells is more widespread than previously reported by others (Miki, Ricordi, Sakuma, Yamamoto, Misawa, Mita, Molano, Vaziri, Pileggi and Ichii, 2018). Our observation of two major cellular patterns of GPX1 immunoreactivity in islet cells (Pattern 1 and Pattern 2) has not been observed previously. Different percentages of islets with the two staining patterns, with varying intensities were observed in all donors, irrespective of diabetes status or the presence or absence of beta cells. In the 2 non-diabetic autoantibody-positive and negative groups (Groups 2 and 3), Pattern 1 was present in a majority of cases. Differences in the frequency of the 2 distribution patterns in newly-diagnosed and long-term diabetic groups (Groups 1 and 4) may have been due to the presence or absence of residual beta cells in the islets, variability in the expression of the GPX1 in non-beta cells, including glucagon cells or spatial re-distribution of endocrine cells in beta cell-negative pseudoatrophic islets. We highlight that various percentages of beta cells do express GPX1 in all study groups, with moderate to strong staining intensities in a higher percentage of glucagon cells than beta cells, except in nPOD case 6178, where a majority of insulin and glucagon cells were negative for GPX1. These observations suggest that in most cases many beta cells express weaker or negative staining intensities for GPX1 than glucagon cells and may, therefore, be ill-equipped to clear excess H 2 O 2 , conferring selective vulnerability. Heterogeneity of intra-islet staining intensities in insulin and glucagon cells with islet-to-islet variability within the same donor may suggest differing rates of intracellular storage of GPX1 and its turnover within the same donor pancreas. Previous findings which show intra-islet functional heterogeneity, such as asynchronous secretion of insulin in response to glucose and senescence lend support to our current findings (Farack, et al., 2019 , Thompson, Pipella, Rutter, Gaisano and Santamaria, 2023 , Van Schravendijk, et al., 1992 ). More recent studies in mice suggest that beta cells are equipped with other anti-oxidant systems to scavenge excess ROS, and in particular H 2 O 2 . These include various isoforms of peroxiredoxin, thioredoxin and thioredoxin reductase (Stancill, Broniowska, Oleson, Naatz and Corbett, 2019 , Stancill and Corbett, 2021 ). However, whether such findings hold true for human islets remains to be proven. In support, earlier studies have shown that human islets are more resistant to oxidant-generating beta cell toxins such as alloxan, streptozotocin, nitroprusside or cytokine-induced injury than mouse islets (Welsh, et al., 1995 ). In the same studies, enzyme activities of catalase and SOD were significantly lower in islets from mice than humans. More recent work suggests that high levels of thioredoxin-interacting protein (TXNIP) in beta cells may inhibit the ability of thioredoxin in the peroxiredoxin-thioredoxin system to detoxify H 2 O 2 (Ovalle, et al., 2018 ). The latter findings have led to an ongoing clinical trial in adults with recent-onset T1D, involving the use of verapamil, a known anti-hypertensive calcium channel blocker, in suppressing TXNIP levels and improving beta cell function (Ovalle, Grimes, Xu, Patel, Grayson, Thielen, Li and Shalev, 2018) (clinical trial reg. no. NCT0455151, ClinicalTrials.gov). We are cognisant of some of the limitations of the present study. For example, the use of immunohistochemistry as the sole experimental approach may have some caveats. However, the availability of pancreatic sections only precluded us from undertaking additional confirmatory studies such as Western blotting or proteomic analysis of isolated human islets for assessing GPX1 levels. However, Western blotting has inherent limitations since it involves disruption of islet architecture and subsequent loss of spatial and cell-type distribution and identification of proteins under study, and we propose that immunohistochemical approach remains compatible with the aims of the present study. We recognize that this study involved analysis of a smaller sample size due to the limited availability of rare human samples with inter-individual heterogeneity and variations in sample acquisition. Therefore, generalizations of our findings to all individuals with new-onset and long-term T1D require some caution. We recognize that while DiViD donors were in better diabetic control during and prior to biopsy, deceased donors from cadaveric and autopsy pancreas may have experienced variable degrees of unavoidable metabolic fluctuations, hyperglycaemia-driven oxidative and nitrosative stress and cold ischaemia before pancreas sampling. Prior medications during hospitalisation, patient stress and variable but unavoidable delays between death and tissue procurement may have also influenced the dynamics, expression pattern and staining intensities of GPX1. Since our immunohistochemical approach does not discriminate between enzymatically active and inactive forms of GPX1, monomeric and tetrameric forms and gene variants, caution also needs to be exercised in ascribing a physiological role of GPX1 solely based on immunohistochemical approaches (Mohammedi, et al., 2016 ). We also recognize that although our studies are cross-sectional and reflect a static snapshot of GPX1 expressing cells in human pancreatic sections, they are nevertheless informative and provide clues on beta cell stress. Other limitations include the possibility of regional differences in the frequency, distribution and intensity of GPX1-positive cells within various islets from the same donor. However, in this study, we report our results following careful analysis of a large number of separate islets with various cross-sectional areas per donor, providing scientific rigour. Future non-invasive deep tissue multiplex imaging of a panel of anti-oxidant enzymes in islets of diabetic and non-diabetic cases may provide more precise and mechanistic information on the distribution and vulnerability of human beta cells to excess levels of ROS, how they impact on the early origins of beta cell destruction and whether they show adaptive protective mechanisms when exposed to raised levels of ROS. The present study conducted in rare human pancreatic tissues, unveils scientific perspectives and offers new and previously unreported findings on the cellular patterns of GPX1 expression. Contrary to recent findings, we report that the expression of the enzyme is seen with varying intensities in not only in selective glucagon cells but also in a lower percentage of beta cells in both non-diabetic and diabetic human donors. Differences in intensities of GPX1 expression in intra- and inter-islet beta cells among the study groups, implies different rates GPX1 biosynthesis and turnover in individual beta cells and their selective vulnerability to H 2 O 2− mediated oxidative injury. Contrary to previous reports in experimental mice, our current findings demonstrating GPX1 positivity in beta cells, albeit in lower numbers, are novel and suggest that a proportion of human beta cells have the capacity to clear GPX1-mediated excess H 2 O 2 . Of note, the distribution of GPX1 immunoreactivity in islet cells showed two distinct patterns of staining, namely whole islet cell staining and selective islet cell staining in a minority of islet cells. The heterogeneity of GPX1 staining in beta cells is consistent with recent observations that demonstrate their asynchronous destruction during T1D (Thompson, Pipella, Rutter, Gaisano and Santamaria, 2023 ). Targeted delivery of GPX1 or its mimetics to beta cells at the early stages of T1D or pharmacological attempts to boost GPX-1 via the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), and other beta cell protective candidates may mitigate further destruction of beta cells (Forman and Zhang, 2021 ). Declarations Supplementary information The online version contains supplementary material. Author contributions SR conceived and designed the experimental studies, carried out a considerable portion of them, acquired and analysed the data, wrote, and revised the manuscript critically for publication. He also led and directed the study. KP also carried out considerable portion of the experimental studies and compiled numerous microscopic images into plates and prepared all tables and edited the manuscript. KS, KD-J and LK made a significant contribution towards editing and revising the manuscript for intellectual content. KS also carried out statistical analysis and prepared all bar graphs and edited the manuscript. All authors have given their final approval of the version to be published. SR is the guarantor of the work. Data availability Electronic supplementary material is available online. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Informed consent Informed consent for donation and analysis of pancreatic biopsies from DiViD donors were obtained by the DiViD Study Group from the Norwegian Government’s Regional Ethics Committee. Ethical approval Ethical approval for conducting this study was granted by the New Zealand Ministry of Health and Disability Ethics Committee (Ethics reference: 2023 PR 2121). Harbin Medical University (China), Auckland Tissue Bank and the Department of Anatomy of this faculty provided control tissue sections from the breast, liver and lung and were also covered by the above New Zealand ethics committee approval. Approval for biopsy procurement from new-onset type 1 diabetes donors was granted by the Norwegian Government’s Regional Ethics Committee following informed consent. Ethical approval for the analysis of pancreatic sections from deceased nPOD donors was granted to nPOD. Ethical approval for the analysis of sections from post-mortem pancreas for EADB (http://foulis.vub.ac.be) was granted by the West of Scotland Research Committee 4 ((WoSREC4; 15//WS/0258) to Professors S Richardson and N Morgan, University of Exeter. Therefore, all procedures performed involving human tissue donors were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Disclaimer The content and views expressed in this publication are the responsibility of the authors and do not reflect an official view of nPOD, DiViD and EADB. This research is dedicated to the Late Sir Robert B Elliott, Former Professor of Paediatrics and Child Health Research, University of Auckland, Auckland, New Zealand References Amirruddin NS, Tan WX, Tan YS, Gardner DS, Bee YM, Verma CS, Hoon S, Lee KO, Teo AKK (2021) Progressive endoplasmic reticulum stress over time due to human insulin gene mutation contributes to pancreatic beta cell dysfunction. 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Comparison of mean percentages of islets with GPX1 staining per study group Study groups No. of cases Pattern 1 Pattern 2 P values Group 1 4 24.77 75.24 0.035* Group 2 9 65.93 34.07 0.006* Group 3 6 68.01 31.99 0.008* Group 4 8 64.04 35.97 0.1071 Note: The percentages of islets with GPX1 staining were combined grades of 1, 2 and 3 (pattern 1) versus 4, 5 and 6 (pattern 2). The mean values were compared by using a student t-test per study group. “*” indicates a significant difference. Additional Declarations No competing interests reported. Supplementary Files SECONDSUBSumpplementarymaterial1.pdf Cite Share Download PDF Status: Published Journal Publication published 10 Mar, 2025 Read the published version in Cell and Tissue Research → Version 1 posted Editorial decision: Revision requested 18 Dec, 2024 Reviews received at journal 17 Dec, 2024 Reviewers agreed at journal 13 Dec, 2024 Reviews received at journal 13 Nov, 2024 Reviewers agreed at journal 05 Nov, 2024 Reviewers agreed at journal 05 Nov, 2024 Reviewers invited by journal 05 Nov, 2024 Editor assigned by journal 04 Nov, 2024 Submission checks completed at journal 04 Nov, 2024 First submitted to journal 31 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5369709","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":377875992,"identity":"97ab7d54-8878-4106-b94c-554a297f1978","order_by":0,"name":"Kaaj Pala","email":"","orcid":"","institution":"University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Kaaj","middleName":"","lastName":"Pala","suffix":""},{"id":377875993,"identity":"75764d3d-97d0-4c5e-b25e-f53c67d0b1f1","order_by":1,"name":"Kevin Xueying Sun","email":"","orcid":"","institution":"University of Auckland","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"Xueying","lastName":"Sun","suffix":""},{"id":377875994,"identity":"4ba4bc67-b049-4143-9976-8b4c1742e670","order_by":2,"name":"Lars Krogvold","email":"","orcid":"","institution":"Oslo University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lars","middleName":"","lastName":"Krogvold","suffix":""},{"id":377875995,"identity":"0d234d92-ad2b-49fd-8796-90e18e26f9a3","order_by":3,"name":"Knut Dahl-Jørgensen","email":"","orcid":"","institution":"University of Oslo","correspondingAuthor":false,"prefix":"","firstName":"Knut","middleName":"","lastName":"Dahl-Jørgensen","suffix":""},{"id":377875996,"identity":"54a5dd57-ce51-4803-9666-34279ff8630d","order_by":4,"name":"Shiva Reddy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYLACHjYGOSDFzPCAgYGxgVgtxmAtCaRoSWwgWot8A+/DB2/KDqf3z0h+bJBQc0e2gf3wA+aCP7i1GBxgNzacc+5w7owbacYJCceeGTfwpBkwz+DBo4WBjU2at+1wbsONHOYDCWyHgS7MYWDmkcDnMDb230At6fJgLf+AWvjfALUY4PHMATY2ZqCWBAOgloTENqAWCZAtCXgcdpiNWXLOuXTDjWeeGRsk9h02bpN4ZnCY5wAeh7W3MX54U2YtL3c8+bHEh2+HZfv5kx8+5sETYsDIAIFmhAAbyLV4NMBAHRFqRsEoGAWjYMQCAIF4TltlD+McAAAAAElFTkSuQmCC","orcid":"","institution":"University of Auckland","correspondingAuthor":true,"prefix":"","firstName":"Shiva","middleName":"","lastName":"Reddy","suffix":""}],"badges":[],"createdAt":"2024-11-01 01:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5369709/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5369709/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00441-025-03955-5","type":"published","date":"2025-03-10T15:57:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69093688,"identity":"8d95167d-f4dc-46a7-8eb8-991a609f6ca3","added_by":"auto","created_at":"2024-11-15 14:11:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32428132,"visible":true,"origin":"","legend":"\u003cp\u003eGrading system employed for estimating overall GPX1staining intensities in various islets. The overall staining intensities in islet cells were graded on a score of 1-6, following a display of microscopic digital images of islets in Photoshop. Intensity scores in islets and the pattern of GPX1 staining were assigned as follows: (a) 1 = very weak staining of most islet cells; (b) 2 = moderate staining of most islet cells; (c) 3 = strong staining of most islet cells; (d) 4 = strong staining of a minority of islet cells (black arrows) with the majority showing negative staining in most islet cells (green arrows); (e) 5 = strong staining of a minority of islet cells (black arrows), with a majority showing weak staining in most islet cells (yellow arrows); (f) 6 = strong staining of a minority of islet cells (black arrows) with the majority of cells showing moderate staining in most islet cells (red arrows). Islet boundaries are indicated by black dashes. Scale bar in (f), 50 µm, applies to all micrographs. Grades 1-3 and 4-6 were assigned Pattern 1 and Pattern 2, respectively.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/948e721f64c2f9b9deb25569.png"},{"id":69093687,"identity":"254a9d72-a0b7-43d1-b618-7ea35370ea08","added_by":"auto","created_at":"2024-11-15 14:11:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12747095,"visible":true,"origin":"","legend":"\u003cp\u003eImages of 2 islet sections employed as a visual guide for grading differing cellular intensities of GPX1 staining in insulin and glucagon cells. Images of 10 randomly selected islets from each case triple-stained for GPX1, insulin and glucagon were merged in Photoshop and graded for GPX1 intensities in insulin and glucagon cells separately on a scale of negative (green arrows), weak (yellow arrows), moderate (red arrows) and strong (black arrows). Islet boundaries are indicated by black dashes. Scale bar in (b), 50 µm, applies to all micrographs.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/e964c07e129a694dae0ef86c.png"},{"id":69093686,"identity":"34f11008-3963-4c22-9b06-fc187548de93","added_by":"auto","created_at":"2024-11-15 14:11:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":14415201,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of insulin-positive islets (green bars) in pancreatic sections from donors belonging to groups 1 (\u003cstrong\u003ea\u003c/strong\u003e), 2, (\u003cstrong\u003eb\u003c/strong\u003e), 3 (\u003cstrong\u003ec\u003c/strong\u003e) and 4 (\u003cstrong\u003ed\u003c/strong\u003e).\u003cstrong\u003e \u003c/strong\u003eThe total number of islets examined in each donor from groups 1-4 is indicated above each bar and in the second column of ESM Tables 3 (Group 1), 4 (Group 2), 5 (Group 3) and 6 (Group 4). In Group 1 and 4, the number within brackets adjacent to cases denotes duration of diabetes in weeks for Group 1 and in years for Group 4. GADA, anti-glutamic acid decarboxylase; IA-2A, anti-insulinoma associated antigen; IAA, insulin autoantibodies; ZnT8A, anti-zinc transporter 8.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/1dc1259e905ffdbf5bc98e93.png"},{"id":69093691,"identity":"5460680f-0163-4a8f-8f07-8c8742882920","added_by":"auto","created_at":"2024-11-15 14:11:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":86865301,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of pancreas sections from DiViD donors 1 (a-f, diabetes duration 4 weeks) and 2 (g-l, diabetes duration 3 weeks). Left panel shows GPX1-positive cells (brown) in islets or occasionally outside the islet. The middle panel shows corresponding GPX1-positive cells in blue, merged with beta (green) and alpha (red) cells, following co-staining. The third panel shows corresponding fields after counterstaining with haematoxylin, where islet boundaries are indicated by black dashes. Magenta arrows indicate GPX1 in alpha cells and yellow arrows in beta cells while white arrows in cells negative for insulin and glucagon; red arrowheads indicate GPX1 staining in some insulin- and glucagon-negative cells in the exocrine region. Scale bar in (j), 50 µm, applies to all micrographs. DiViD, Diabetes Virus Detection Study; GLU, glucagon; GPX1, glutathione peroxidase1; INS, insulin\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/57fe8e9b48768d9d5b428254.png"},{"id":69093997,"identity":"15c2a827-474c-4625-83fe-7514ceb1ee29","added_by":"auto","created_at":"2024-11-15 14:19:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":96020870,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of pancreas sections from DiViD donors 3 (a-f, diabetes duration 4 weeks) and 4 (g-l, diabetes duration 3 weeks). Left panel shows GPX1-positive cells (brown) in islets or occasionally outside the islet. The middle panel shows corresponding GPX1-positive cells in blue, merged with beta (green) and alpha (red) cells, following co-staining. The third panel shows corresponding fields after counterstaining with haematoxylin, where islet boundaries are indicated by black dashes. Magenta arrows indicate GPX1 in alpha cells and yellow arrows in beta cells while white arrows in cells negative for insulin and glucagon; red arrowheads indicate GPX1 staining in some insulin- and glucagon-negative cells in the exocrine region. Scale bar in (j), 50 µm, applies to all micrographs. DiViD, Diabetes Virus Detection Study; GLU, glucagon; GPX1, glutathione peroxidase1; INS, insulin\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/4a1a4b999393ef7c88fe5385.png"},{"id":69093996,"identity":"2cbfbf06-3379-4440-b2ab-9a8f26f9a245","added_by":"auto","created_at":"2024-11-15 14:19:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":77476835,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of pancreas sections from non-diabetic autoantibody-negative nPOD donors 6289 (a-f) and 6234 (g-l). Left panel shows GPX1-positive cells (brown) in islets or occasionally outside the islet. The middle panel shows corresponding GPX1-positive cells in blue, merged with beta (green) and alpha (red) cells, following co-staining. The third panel shows corresponding fields after counterstaining with haematoxylin, where islet boundaries are indicated by black dashes. Magenta arrows indicate GPX1 in alpha cells and yellow arrows in beta cells while white arrows in cells negative for insulin and glucagon; red arrowheads indicate GPX1 staining in some insulin- and glucagon-negative cells in the exocrine region. Scale bar in (j), 50 µm, applies to all micrographs. GLU, glucagon; GPX1, glutathione peroxidase1; INS, insulin; nPOD, Network for Pancreatic Organ Donors with Diabetes\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/818ad7afcb672ed8fa0e101d.png"},{"id":69093693,"identity":"1432915d-43ad-4ba3-b178-9fc94c1b3f57","added_by":"auto","created_at":"2024-11-15 14:11:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":73025533,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of pancreas sections from non-diabetic autoantibody-negative nPOD donors 6160 (a-f) and 6178 (g-l). Left panel shows GPX1-positive cells (brown) in islets or occasionally outside the islet. The middle panel shows corresponding GPX1-positive cells in blue, merged with beta (green) and alpha (red) cells, following co-staining. The third panel shows corresponding fields after counterstaining with haematoxylin, where islet boundaries are indicated by black dashes. Magenta arrows indicate GPX1 in alpha cells and yellow arrows in beta cells while white arrows in cells negative for insulin and glucagon; red arrowheads indicate GPX1 staining in some insulin- and glucagon-negative cells in the exocrine region. Scale bar in (j), 50 µm, applies to all micrographs. GLU, glucagon; GPX1, glutathione peroxidase1; INS, insulin; nPOD, Network for Pancreatic Organ Donors with Diabetes\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/6b8dc4636c98bab22919e9fc.png"},{"id":69093696,"identity":"f77a7459-68e7-4e07-a8ee-4f6ff7b77740","added_by":"auto","created_at":"2024-11-15 14:11:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":83971758,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of pancreas sections from non-diabetic autoantibody-negative nPOD donors 6401 (a-f) and 6055 (g-l). Left panel shows GPX1-positive cells (brown) in islets and occasionally outside the islet. The middle panel shows corresponding GPX1-positive cells in blue, merged with beta (green) and alpha (red) cells, following co-staining. The third panel shows corresponding fields after counterstaining with haematoxylin, where islet boundaries are indicated by black dashes. Magenta arrows indicate GPX1 in alpha cells and yellow areas in beta cells while white arrows in cells negative for insulin and glucagon. Red arrowheads indicate GPX1 staining in some cells in exocrine regions while in (a-f) white arrowheads indicate localization of GPX1 in ductal cells. Scale bar in (j), 50 µm, applies to all micrographs. GLU, glucagon; GPX1, glutathione peroxidase1; INS, insulin; nPOD, Network for Pancreatic Organ Donors with Diabetes\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/bf4bfbf34229e1f6d7ea24e8.png"},{"id":69093689,"identity":"92b1cde1-098f-4d78-a15e-7ef3f550345b","added_by":"auto","created_at":"2024-11-15 14:11:52","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":22644892,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of pancreas sections from non-diabetic autoantibody-positive nPOD donors 6424 (a-f) and 6267 (g-l). Left panel shows GPX1-positive cells (brown) in islets and occasionally outside the islet. The middle panel shows corresponding GPX1-positive cells in blue, merged with beta (green) and alpha (red) cells, following co-staining. The third panel shows corresponding fields after counterstaining with haematoxylin, where islet boundaries are indicated by black dashes. Magenta arrows indicate GPX1 in alpha cells and yellow arrows in beta cells while white arrows in cells negative for insulin and glucagon; red arrowheads indicate GPX1 staining in some cells in exocrine regions negative for insulin and glucagon. Scale bar in (j), 50 µm, applies to all micrographs. AAb, autoantibodies; GLU, glucagon; GPX1, glutathione peroxidase1; INS, insulin; nPOD, Network for Pancreatic Organ Donors with Diabetes\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/30347d7ceef8f155497b8469.png"},{"id":69093699,"identity":"f13aee7d-d630-4909-8766-76e8d131143e","added_by":"auto","created_at":"2024-11-15 14:11:54","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":131094389,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of pancreas sections from non-diabetic autoantibody-positive nPOD donors 6301 (a-f) and 6310 (g-l). Left panel shows GPX1-positive cells (brown) in islets or occasionally outside the islet. The middle panel shows corresponding GPX1-positive cells in blue, merged with beta (green) and alpha (red) cells, following co-staining. The third panel shows corresponding fields after counterstaining with haematoxylin, where islet boundaries are indicated by black dashes. Magenta arrows indicate GPX1 in alpha cells and yellow arrows in beta cells while white arrows in cells negative for insulin and glucagon; red arrowheads indicate GPX1 staining in some insulin- and glucagon-negative cells in the exocrine region. Scale bar in (j), 50 µm, applies to all micrographs. AAb, autoantibodies; GLU, glucagon; GPX1, glutathione peroxidase1; INS, insulin; nPOD, Network for Pancreatic Organ Donors with Diabetes\u003c/p\u003e","description":"","filename":"Fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/568d605d7f4de489ab5f05ac.png"},{"id":69093702,"identity":"2b343321-beeb-4428-a438-71d7ae1b1a6f","added_by":"auto","created_at":"2024-11-15 14:11:55","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":113226196,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of pancreas sections from long-term diabetic donors E560 (a-f; diabetes duration: 1.5 years) and nPOD 6211 (g-l; diabetes duration: 4 years). Left panel shows GPX1-positive cells (brown) in islets or occasionally outside the islet. The middle panel shows corresponding GPX1-positive cells in blue, merged with beta (green) and alpha (red) cells, following co-staining. The third panel shows corresponding fields after counterstaining with haematoxylin, where islet boundaries are indicated by black dashes. Duration of diabetes in years is specified in (a, g). Magenta arrows indicate GPX1 in alpha cells and yellow arrows in beta cells while white arrows in cells negative for insulin and glucagon; red arrowheads indicate GPX1 staining in some insulin- and glucagon-negative cells in the exocrine region. Duration of diabetes in years is indicated in (a) and (g). Scale bar in (j), 50 µm, applies to all micrographs. GLU, glucagon; GPX1, glutathione peroxidase1; INS, insulin; nPOD, Network for Pancreatic Organ Donors with Diabetes; T1D, type 1 diabetes\u003c/p\u003e","description":"","filename":"Fig11.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/31648b2f320a11687d518edf.png"},{"id":69093701,"identity":"b4a52f90-9588-4dc6-af6c-2d67821c874b","added_by":"auto","created_at":"2024-11-15 14:11:54","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":122220177,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of pancreas sections from long-term diabetic donors nPOD 6088 (a-f; diabetes duration: 5 years) and nPOD 6070 (g-l; diabetes duration:7 years). Left panel shows GPX1-positive cells (brown) in islets or occasionally outside the islet. The middle panel shows corresponding GPX1-positive cells in blue, merged with beta (green) and alpha (red) cells, following co-staining. The third panel shows corresponding fields after counterstaining with haematoxylin, where islet boundaries are indicated by black dashes. Duration of diabetes in years is specified in (a, g). Magenta arrows indicate GPX1 in alpha cells and yellow arrows in beta cells while white arrows in cells negative for insulin and glucagon; red arrowheads indicate GPX1 staining in some insulin- and glucagon-negative cells in the exocrine region. Duration of diabetes in years is indicated in (a) and (g). Scale bar in (j), 50 µm, applies to all micrographs. GLU, glucagon; GPX1, glutathione peroxidase1; INS, insulin; nPOD, Network for Pancreatic Organ Donors with Diabetes, T1D, type 1 diabetes\u003c/p\u003e","description":"","filename":"Fig12.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/f7f32f528d67908d4525e7a6.png"},{"id":69093700,"identity":"31263a4a-fa81-48dd-8db8-55dcfa5ebeaa","added_by":"auto","created_at":"2024-11-15 14:11:54","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":121533219,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of pancreas sections from long-term diabetic donors nPOD 6245 (a-f; duration of diabetes: 7 years) and nPOD 6045 (g-l, duration of diabetes: 8 years). Left panel shows GPX1-positive cells (brown) in islets or occasionally outside the islet. The middle panel shows corresponding GPX1-positive cells in blue, merged with beta (green) and alpha (red) cells, following co-staining. The third panel shows corresponding fields after counterstaining with haematoxylin, where islet boundaries are indicated by black dashes. Duration of diabetes in years is indicated in (a, g). Magenta arrows indicate GPX1 in alpha cells and yellow arrows in beta cells while white arrows in cells negative for insulin and glucagon; red arrowheads indicate GPX1 staining in some insulin- and glucagon-negative cells in the exocrine region. Duration of diabetes in years is indicated in (a) and (g). Scale bar in (j), 50 µm, applies to all micrographs. GLU, glucagon; GPX1, glutathione peroxidase1; INS, insulin; nPOD, Network for Pancreatic Organ Donors with Diabetes, T1D, type 1 diabetes\u003c/p\u003e","description":"","filename":"Fig13.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/da3aa26ced2a8c2531bad0c5.png"},{"id":69093694,"identity":"500dabbb-6d04-4ef1-b624-ca1369c3fd1f","added_by":"auto","created_at":"2024-11-15 14:11:52","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":4366947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003ePercentages of all islets per donor in the 4 study groups with graded staining intensities of GPX1 ranging from 1-3 (pattern 1) and 4-6 (pattern2) shown as adjacent bars for each case (refer to Tables 3-6 for individual values). \u003cstrong\u003e(b)\u003c/strong\u003ePercentages of insulin-positive and -negative islets with graded GPX1 staining intensities of 1-3 and 4-6 are shown as adjacent bars in diabetic cases (Groups 1 and 4; individual values are shown in Tables 7-10). In Group 1 and 4, the number within brackets adjacent to cases denotes duration of diabetes in weeks for Group 1 and in years for Group 4. GADA, anti-glutamic acid decarboxylase; IA-2A, anti-insulinoma associated antigen; IAA, insulin autoantibodies; ZnT8A, anti-zinc transporter 8\u003c/p\u003e","description":"","filename":"Fig14.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/46da966c2099695708cb82bb.png"},{"id":69093698,"identity":"dff6cb3e-298c-423a-8842-19ffcf63f2e4","added_by":"auto","created_at":"2024-11-15 14:11:53","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":17915951,"visible":true,"origin":"","legend":"\u003cp\u003ePercentages of beta cells and glucagon cells in 10 randomly selected islets per case showing GPX1 staining intensities graded on a scale of negative, weak, moderate and strong. Percentages are shown as segments within each bar (refer to Tables 10-14 for individual values). Total number of insulin and glucagon cells analysed for each case are shown above each bar. In Group 1, the number within brackets adjacent to cases denotes duration of diabetes in weeks and in years for Group 4. GADA, anti-glutamic acid decarboxylase; IA-2A, anti-insulinoma associated antigen; IAA, insulin autoantibodies; ZnT8A, anti-zinc transporter 8\u003c/p\u003e","description":"","filename":"Fig15.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/fc192bbc68f75c356b3082cd.png"},{"id":69093697,"identity":"2fbadc01-fe27-42c9-b56c-7903465dd86b","added_by":"auto","created_at":"2024-11-15 14:11:53","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":9317048,"visible":true,"origin":"","legend":"\u003cp\u003eCumulative percentages of beta cells (left bar graphs) and glucagon cells (right bar graphs) in the 4 study groups showing GPX1 staining intensities graded on a scale of negative, weak, moderate and strong. Percentages are shown as segments within each bar. Note: Cumulative percentages were calculated as a sum of all beta or glucagon cells in each study group with graded intensities. 10 randomly selected islets per case were analysed. Refer to Table 15 for cumulative percentages for each group.\u003c/p\u003e","description":"","filename":"Fig16.png","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/61a0035ed6098c27f5501e59.png"},{"id":69093690,"identity":"4d70fa4c-6ac8-4fd6-be6d-58a1eea40671","added_by":"auto","created_at":"2024-11-15 14:11:52","extension":"pdf","order_by":20,"title":"","display":"","copyAsset":false,"role":"supplement","size":5030931,"visible":true,"origin":"","legend":"","description":"","filename":"SECONDSUBSumpplementarymaterial1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5369709/v1/a50cc825091ea0e313078f19.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Distribution of glutathione peroxidase-1 immunoreactive cells in pancreatic islets from type 1 diabetic donors and non-diabetic donors with and without islet cell autoantibodies is variable and independent of disease ","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDuring type 1 diabetes (T1D), beta cells in pancreatic islets are selectively and progressively destroyed by immune-mediated processes, culminating in an abrupt clinical onset (Atkinson et al, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, 2014). Its diagnosis is preceded by an asymptomatic protracted phase lasting several months to years when subjects manifest variable degrees of beta cell dysfunction (Atkinson and Mirmira, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Evans-Molina, et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Following onset, daily administration of insulin is mandatory to suppress hyperglycaemia. The immune destructive process is beta cell specific since the closely-located non-beta endocrine cells within the islet escape destruction, although glucagon cells display functional impairment (Atkinson and Mirmira, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Doliba, et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Yosten, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The exquisite immune specificity for beta cells suggests that such cells may already harbour molecular signatures of vulnerability which may act not only as initiators, or conduits for the early immune response but also amplifiers of ongoing beta cell-directed immune destruction (Kracht, et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Mallone and Eizirik, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Mallone, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Peters, et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Roep, et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Thompson, et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, a subset of beta cells in some subjects remain detectable many years after diagnosis (Keenan, et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Reddy, et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eAlthough low levels of reactive oxygen species (ROS) mediate critical physiological signalling pathways, accumulation of raised levels, if not cleared rapidly, can lead to major deleterious consequences affecting several organs, such as the heart, kidney, eye and brain, culminating in a variety of human disorders (Forman and Zhang, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Elevated ROS injure cells by promoting degradation of cellular proteins, lipids and DNA. In addition they affect several adverse cell signalling pathways involving mammalian target of rapamycin complex 1 (mTORC1), c-jun N-terminal kinase (JNK) and also invoke inflammation by activating nuclear factor-kB (nF-κB) (Kulkarni, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeta cells exhibit sustained high respiratory, metabolic, secretory and protein synthetic rates. High respiratory activity results in raised levels of ROS originating from the electron transport chain. However, little is known on how human beta cells eliminate excess levels of ROS efficiently. During T1D, early environmental agents, such as certain viruses and chemicals may also provoke deleterious events in beta cells leading to oxidative, nitrosative and endoplasmic reticulum (ER) stress (Amirruddin, et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Craig, et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Hasnain, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Hober and Sauter, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Leenders, et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Mallone and Eizirik, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Roep, Thomaidou, van Tienhoven and Zaldumbide, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Such stressors result in the formation of misfolded proteins, and neoantigens which trigger a clinically silent phase of beta cell dysfunction and self-directed immune reactivity prior to clinical diagnosis (Atkinson, et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Sims, et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Sims, et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, recent studies have shown that ER stress within beta cells leads to translational errors in the reading frame of human insulin mRNA, resulting in the generation of defective polypeptides and formation of neo-epitopes, capable of recognition by circulating T cell populations which have escaped prior thymic deletion (Kracht, van Lummel, Nikolic, Joosten, Laban, van der Slik, van Veelen, Carlotti, de Koning, Hoeben, Zaldumbide and Roep, 2017, Roep, Thomaidou, van Tienhoven and Zaldumbide, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, neo-epitope formation from degraded citrullinated proteins such as glucose-regulated protein 78 following exposure of human islets to inflammatory molecules, can lead to immune responses against beta cells (Buitinga, et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eHigh levels of ROS, such as superoxide anions (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), originate from the electron transport chain and to a lesser extent following catalysis by cytosolic and plasma membrane oxidoreductases (Nolfi-Donegan, et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Excess levels of superoxide can dismutate either spontaneously to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or following catalysis by superoxide dismutase (SOD). Hydrogen peroxide is converted to water and oxygen by glutathione peroxidases, catalase and peroxiredoxins (Bast, et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Handy and Loscalzo, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Stancill, et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Stancill and Corbett, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Wolf, et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Mouse islets express lower levels of SOD, glutathione peroxidase (GPX) and catalase at the mRNA and protein level than other tissues such as liver and kidneys (Grankvist, et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1981\u003c/span\u003e, Lenzen, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Tiedge, et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Tiedge, et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). During the prodromal phase of T1D and preceding early insulitis, islet cell autoantibodies against several antigens such as insulin, glutamic acid decarboxylase and zinc transporter-8 are detectable in sera of most subjects (Bogun, et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Campbell-Thompson, et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Krischer, et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Whether excess uncleared ROS originating from beta cell mitochondrial respiration contributes to the ontogeny of islet cell autoantibodies and insulitis remains unclear.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eGlutathione peroxidase (GPX) is present in six isoforms. Isoforms 1 to 4 are homotetramers, containing selenium bound to cysteine in their active site and are referred as selenoproteins (Handy and Loscalzo, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). They play a key role in detoxifying H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and lipid peroxides in many tissues (Robertson and Harmon, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Tanaka, et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Glutathione peroxidase catalysis occurs in the presence of reduced glutathione, a tripeptide, in a paired reaction involving glutathione reductase which regenerates reduced glutathione from oxidized glutathione (Handy and Loscalzo, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, whether the previously reported lower levels glutathione peroxidase-1 (GPX1) in mouse and human islets by Western blotting and enzyme assays are also an intrinsic feature of human beta cells in situ are unclear (Grankvist, Marklund and T\u0026auml;ljedal, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1981\u003c/span\u003e, Tonooka, et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLittle is known on the cellular expression of GPX1 in human pancreatic islets in donors with increasing duration of T1D and whether the expression patterns differ in non-diabetic cases with and without islet cell autoantibodies. In a brief study, immunohistochemical analysis of pancreatic sections from deceased non-diabetic human donors showed expression of GPX1 and catalase predominantly in selective glucagon cells and in some beta cells, with an absence in the remaining islet cell-types (Miki, et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, this study did not fully analyze the possible changing expression pattern of GPX1 in glucagon and beta cells from donors with variable duration of T1D, including new-onset cases. In addition, similar analysis was not undertaken by the same authors in non-diabetic donors with and without islet cell autoantibodies. Thus, there is an urgent need to investigate and compare the expression of GPX1 in persisting beta cells and other islet cell-types at and after diagnosis of T1D with non-diabetic cases with and without islet cell autoantibodies.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe recent availability of rare pancreatic samples from cases with variable but precisely defined duration of T1D and non-diabetic donors with and without serum islet cell autoantibodies has provided us an impetus and rationale to undertake detailed studies on the expression of GPX1 in islets. In the present study, we have, therefore, utilized such unique samples and applied an optimized triple-label immunohistochemical protocol for comparing the localization of GPX1 in beta and alpha cells and estimate its level and relative expression in the two cell-types in high quality human pancreatic sections from diabetic and non-diabetic deceased donors, supplied by the Network for Pancreatic Organ Donors with Diabetes (nPOD) and the Exeter Archival Diabetes Biobank (EADB) (Morgan and Richardson, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Pugliese, et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). We have also analyzed sections of well-preserved surgically-retrieved rare pancreatic biopsies from four living volunteers with recent-onset T1D, supplied by the Diabetes Virus Detection (DiViD) Study (Krogvold, et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eSubjects and pancreatic sections\u003c/h2\u003e\n \u003cp\u003eParaffin-embedded pancreatic sections (5 \u0026micro;m) from formalin-fixed biopsy specimens from 4 living individuals with newly-diagnosed type 1 diabetes from DiViD, 22 formalin-fixed cadaveric pancreases supplied by nPOD, and 1 pancreas at post-mortem from EADB, United Kingdom were analysed (Krogvold, Edwin, Buanes, Ludvigsson, Korsgren, Hy\u0026ouml;ty, Frisk, Hanssen and Dahl-J\u0026oslash;rgensen, 2014, Morgan and Richardson, \u003cspan\u003e2018\u003c/span\u003e, Pugliese, Yang, Kusmarteva, Heiple, Vendrame, Wasserfall, Rowe, Moraski, Ball, Jebson, Schatz, Gianani, Burke, Nierras, Staeva, Kaddis, Campbell-Thompson and Atkinson, 2014). In addition, sections prepared from formalin-fixed liver (University of Harbin, Harbin, China), breast (Auckland Tissue Biobank, University of Auckland) and lung (Department of Anatomy and Medical Imaging, University of Auckland) carcinoma were tested as positive controls for GPX1 immunostaining. Pancreatic sections from human cases were triple-immunostained for GPX1, insulin and glucagon and analysed. Please refer to \u0026ldquo;Acknowledgements\u0026rdquo; for ethical approval.\u003c/p\u003e\n \u003cp\u003eThe main study groups are summarised in Table\u0026nbsp;1 and donor demographics are detailed in the electronic supplementary material (ESM Table\u0026nbsp;1).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eTriple-label immunohistochemical protocol\u003c/h3\u003e\n\u003cp\u003eWe employed a commercially available affinity-purified rabbit polyclonal antibody monospecific for human GPX1 (Abcam ab22604, supplied as 1 mg/ml IgG). This antibody has been validated by the suppliers as suitable for immunohistochemistry and Western blotting (refer below for further details on immunohistochemical protocols and to ESM Table \u003cspan\u003e2\u003c/span\u003e). It has also been validated and employed by previous investigators for immunohistochemical localization of GPX1 in formalin-fixed human lung sections and Bouin\u0026rsquo;s fixed human pancreatic sections (Basnet, et al., \u003cspan\u003e2019\u003c/span\u003e, Miki, Ricordi, Sakuma, Yamamoto, Misawa, Mita, Molano, Vaziri, Pileggi and Ichii, 2018). Anti-GPX1 was titrated to a working dilution of 1:120 (final concentration: 8.33 \u0026micro;g/ml IgG), prior to its application to sections from formalin-fixed human pancreas. As negative controls, anti-GPX1 was replaced with diluent or normal rabbit IgG in the immunohistochemical procedure. In initial studies, immunohistochemical staining for GPX1 in pancreatic sections with species-specific secondary antibodies linked to anti-rabbit IgG-horseradish peroxidase polymer and subsequent reaction with diaminobenzidine hydrochloride (adopted in this study) was compared with donkey anti-rabbit IgG-Alexa 568 as secondary antibody. The two protocols yielded concordant cellular staining (see below under \u0026ldquo;Methods\u0026rdquo;). In addition, we stained pancreatic sections with a mixture rabbit anti-glucagon (same immunizing species as GPX1 antibody), guinea pig anti-insulin and mouse anti-CD45. Immunohistochemical specificity of anti-GPX1 was also evaluated in formalin-fixed paraffin sections from liver, breast and lung cancer, known to express GPX1 (Basnet, Tian, Ganesh, Huang, Macalinao, Brogi, Finley and Massagu\u0026eacute;, 2019, Woolston, et al., \u003cspan\u003e2011\u003c/span\u003e). Please see ESM Fig. \u003cspan\u003e1\u003c/span\u003e and ESM Fig. \u003cspan\u003e2\u003c/span\u003e under \u0026ldquo;Results\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe immunohistochemical protocol for GPX1 applied in this study was slightly modified from our previous procedures for interleukin-1\u0026beta; and inducible nitric oxide synthase (Reddy, et al., \u003cspan\u003e2018\u003c/span\u003e, Reddy, Krogvold, Martin, Sun, Martin, Al-Ani and Dahl-J\u0026oslash;rgensen, 2021). Relevant immunohistochemical steps, reagents and incubation times are summarised in ESM Table \u003cspan\u003e2\u003c/span\u003e. Thus, sections were deparaffinized, rehydrated and subjected to antigen retrieval with citrate buffer, pH 6, containing 0.05% (vol/vol) Tween-20 (Sigma-Aldrich, Darmstadt, Germany), cooled in distilled water and permeabilized in 0.4% (vol/vol) Triton-X-100 in phosphate-buffered saline (PBS), pH 7.4, on ice for 30 min. After washing sections in PBS for 10 min, they were exposed to 3% (vol/vol) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in PBS for 15 min. Following two consecutive 5- minute washing steps in PBS, sections were blocked with 10% (vol./vol.) normal goat serum constituted in low protein blocking solution (EBioSciences, San Diego, CA, USA) for 1 h at 37\u003csup\u003eᵒ\u003c/sup\u003eC, followed by incubation with rabbit anti-GPX1 (1:120, diluted in antibody diluent from Cell Signaling Technology, Danvers, MA, USA) for 16\u0026ndash;18 h at 4\u003csup\u003eᵒ\u003c/sup\u003eC. After a wash step, sections were incubated with anti-rabbit IgG horseradish peroxidase polymer (Cell Signaling Technology), diluted 1:1 with 0.1% vol/vol Tween-20 in PBS (wash buffer). After further washing, sections were reacted with 3,3\u0026apos;-diaminobenzidine (DAB) substrate (freshly-prepared in accordance with the manufacturer\u0026rsquo;s instructions; catalogue number: 8059; Cell Signaling Technology) for 5\u0026ndash;6 minutes. Following monitoring of peroxidase-catalysed DAB-product, by bright field microscopy, reaction was terminated in excess distilled water. Sections were then briefly immersed in wash buffer and incubated with a mixture of guinea pig anti-insulin and mouse anti-glucagon (both at 1:600 dilution in antibody diluent from Cell Signaling) and incubated for 90 minutes at 37\u003csup\u003eᵒ\u003c/sup\u003eC. Sections were washed in wash buffer and reacted with a mixture of highly cross-absorbed species-specific donkey anti-guinea pig IgG-Alexa 488 (1:300) and donkey anti-mouse IgG-Alexa 568 (1:600), diluted in 0.2% vol/vol Tween-20 in PBS for 1 h at 37\u003csup\u003eᵒ\u003c/sup\u003eC. Following washing, sections were mounted with CitiFluor mountant for microscopic analysis.\u003c/p\u003e\n\u003ch3\u003eImage acquisition\u003c/h3\u003e\n\u003cp\u003eSections were examined with a Nikon Eclipse-epifluorescence-bright field microscope and digital images were recorded and saved as Tiffs. Groups 2 and 3 were blinded to the investigator, but not Groups 1 and 4, where many islets from all cases were either insulin-negative or showed reduced number of residual beta cells. Such distinct pathologies displayed by all cases from the two diabetic groups did not permit sample blinding. However, case characteristics such as duration of T1D and clinical information was blinded to the investigator. From each section, all islets with approximately\u0026thinsp;\u0026ge;\u0026thinsp;20 endocrine cells were imaged for the presence of GPX1 by bright field microscopy. Uniform gain and exposure settings in the Nikon NIS Imaging Software with identical objective lens magnification of 40x with a numerical aperture of 0.75 were applied to all sections for ensuring consistency in image intensities and magnification. Following image acquisition by bright field, the same islets were co-imaged for insulin and glucagon staining by fluorescence microscopy equipped with appropriate narrow band filters. Each of the specific image sets from multiple acquisitions (GPX1, \u0026plusmn;insulin and glucagon) were merged using Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA). For image display in combination with insulin and glucagon staining by fluorescence, GPX-1 immunostained cells (initially brown) were converted to a blue fluorescent pseudocolour for photographic display.\u003c/p\u003e\n\u003ch3\u003eImage analysis\u003c/h3\u003e\n\u003cp\u003eInsulin-positive and -negative islets in all donors were enumerated.\u003c/p\u003e\n\u003cp\u003eFollowing triple staining (GPX1\u0026thinsp;\u0026plusmn;\u0026thinsp;insulin\u0026thinsp;+\u0026thinsp;glucagon), the overall cellular intensity for GPX1 (brown) and its distribution pattern in all islets with more than 20 estimated cells were graded visually following display of acquired images by Photoshop and magnification for determining distribution of cells as follows. Islets with very weak staining in almost all islet cells were assigned grade 1, those with overall weak staining as grade 2 and those with moderate to strong staining in most islet cells were assigned grade 3 (Pattern 1). In addition, islets with strong GPX1 staining in only a few selective islet cells with negative staining in a majority of islet cells were assigned grade 4, while those with strong staining in a minority of islet cells accompanied by weak staining in a majority of islet cells were assigned grade 5. Islets showing strong staining in a minority of cells coupled with moderate staining in a majority of islet cells were assigned \u0026ldquo;grade 6\u0026rdquo;. Grades 4\u0026ndash;6 were defined as \u0026ldquo;Pattern 2\u0026rdquo;. The grading system for the two patterns was based on a visual guide shown in Fig. \u003cspan\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eWe also graded the intensities of GPX1 staining in each insulin and glucagon cell located in 10 randomly selected islets per case by viewing, magnifying and merging image sets in Photoshop, to ascertain the precise identity of the two cell-types, GPX-1 positivity, grading and enumeration. Staining for GPX1 in each insulin and glucagon cell from 10 islets per case was scored as either negative, weak, moderate or strong in accordance with a visual template guide depicted in Fig. \u003cspan\u003e2\u003c/span\u003e. In insulin-negative islets from diabetic cases the various intensity grades for GPX1 in glucagon cells.\u003c/p\u003e\n\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003eData Analysis\u003c/h2\u003e\n \u003cp\u003eResults were tabulated and represented as bar graphs for each donor and analysed for:\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003col\u003e\n \u003cli\u003e\u003cspan\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eNumber of islets imaged per section\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eNumber of islets per case positive and negative for insulin\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003ePercentage of all islets including insulin-positive and negative islets from each donor with GPX-1 positive cells and their intensities, graded on a scale from 1\u0026ndash;3 (Pattern 1; Fig. \u003cspan\u003e1\u003c/span\u003e) 4\u0026ndash;6 (Pattern 2; Fig. \u003cspan\u003e1\u003c/span\u003e)\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003ePercentage of insulin and glucagon cells in 10 randomly selected islets from each case with various intensities of GPX-1 staining, graded on a scale of negative, weak, moderate and strong, indicated in Fig. \u003cspan\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eTotal percentage of insulin and glucagon cells per group with various grades of GPX1 staining as specified under (d). For each group, the total percentages were calculated from the number of insulin- and glucagon-positive cells in 10 islets per case\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003c/span\u003e\u003c/li\u003e\n \u003c/ol\u003e\n \u003cdiv\u003e\n \u003cp\u003eFollowing image acquisition and analysis, sections were counterstained with haematoxylin to reveal islet histology and its surrounding area.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv\u003e\n \u003cp\u003eA student t-test in the excel of Microsoft Office 2016 was used for statistical analysis. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical specificity of rabbit anti-GPX-1\u003c/h2\u003e \u003cp\u003eIn the immunohistochemical procedure for GPX-1, substitution of anti-rabbit GPX-1 with antibody diluent or normal rabbit IgG did not show staining of islets cells and the surrounding region (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-f). Incubation of a pancreatic section with rabbit anti-glucagon (also a rabbit antibody as for GPX1) in combination with guinea pig anti-insulin and mouse anti-CD45 (antibody of mouse origin as for glucagon) showed the expected staining of alpha cells, beta cells and CD45-positive cells, respectively (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-i). Addition of anti-GPX1 to separate pancreatic sections showed staining of several islet cells following incubation with anti-rabbit IgG horseradish peroxidase polymer and reaction with 3,3\u0026rsquo;diaminobenzidine (DAB) substrate (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej-l) or donkey anti-rabbit IgG-Alexa 568 (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003em-o).\u003c/p\u003e \u003cp\u003ePrevious reports demonstrated that anti-GPX-1 (Abcam, ab22604; employed in this study) stained distinct cells in formalin-fixed tissue sections from lung carcinoma (Basnet, Tian, Ganesh, Huang, Macalinao, Brogi, Finley and Massagu\u0026eacute;, 2019). The corresponding transcript was also demonstrated in multiple tissues including breast cancer (Chu, et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, Woolston, Al-Attar, Storr, Ellis, Morgan and Martin, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Here we confirm that anti-GPX1 stained cells in sections of formalin-fixed human breast, liver, lung and pancreas (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePercentage of insulin-positive islets\u003c/h2\u003e \u003cp\u003ePlease refer to the demographic data for characteristics of each donor in ESM Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eThe percentage of insulin-positive islets per donor from the 4 study groups is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d (values per case are itemized in the second column of ESM Tables\u0026nbsp;3\u0026ndash;6). The number of islets analysed per case is indicated above each bar. In the non-diabetic autoantibody-negative and positive groups, all islets were insulin-positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c), but were highly variable in diabetic donors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,d). Islets from DiViD donor 1 and nPOD 6088, 6045 and 6220 (long-standing diabetes), were insulin-negative. In DiViD donors 2, 3 and 4, the percentages were 34.15%, 58.44% and 21.43%, respectively. Among the long-term diabetic donors (Group 4), the highest percentages were in case E560 (55.7%), nPOD 6211 (21.25%) and nPOD 6070 (29.76%). There was a marked reduction in the number of insulin-positive islets in nPOD 6245 and nPOD 6262, being absent in cases 6088 and 6220 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDistribution and localization of GPX1 in islet cells\u003c/h2\u003e \u003cp\u003eWe observed inter-islet variability in the distribution and staining intensities of GPX1-positive islet cells in each donor and among different donors. Additional immunoreactivity was observed in selective pancreatic ductal cells, some of which were glucagon-positive (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Some cells negative for insulin and glucagon within the intra-islet vascular space and external to the islet showed GPX1 positivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003ed-f). It was only weakly expressed in pancreatic acinar cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn pancreatic islets, two major patterns of GPX1 expression were observed and defined as pattern 1 or 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-f).\u003c/p\u003e \u003cp\u003eExamples of the overall distribution of GPX1-positive cells in selected islets from various donors are depicted as assembled micrographs, where the first column shows the presence of GPX1 cells alone within islets and the surrounding regions, the second column displays merged images of cells positive for GPX1 (brown GPX1 cells converted to a blue pseudocolour), insulin (green) and glucagon (red), while the third column shows corresponding fields after counterstaining with haematoxylin.\u003c/p\u003e \u003cp\u003eGroup 1 (newly-diagnosed cases from DiViD): In DiViD case 1, all islets were devoid of beta cells, with strong expression of GPX1 in a majority of glucagon cells which occupied almost the entire islet area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-f). In DiViD case 2, GPX1 was weakly expressed in glucagon cells and in the remaining beta cells; islets devoid of beta cells showed GPX1 immunoreactivity in selective glucagon cells and in some cells negative for insulin and glucagon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-l). In DiViD cases 3 and 4, GPX1 was strongly as well as weakly expressed in glucagon cells, in association with positivity in selective insulin cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-l). In insulin-negative islets, it was also observed in almost all islet cells, with many corresponding to glucagon cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-f, j-l). It was also seen in some islet cells negative for insulin and glucagon (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ej-l).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGroup 2 (Non-diabetic autoantibody-negative cases): In nPOD 6289, 6234 and 6160, weak to moderate GPX1 intensities were present in almost all islet cells, while only some islets showed slightly stronger expression in selective insulin and glucagon cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-l; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-f). However, in nPOD 6178, a majority of islets showed strong expression of GPX1 in only a minority of glucagon cells, with either an absence or weak to moderate staining in the remaining glucagon cells; only weak or negative expression was present in selective insulin cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eg-l). A similar pattern was observed in nPOD 6229 (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-l). In nPOD 6401, 6055 and 6048, moderate to strong staining for GPX1 was present in entire islet cells and was colocalized in glucagon and insulin cells (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-f; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-l) while in nPOD 6369 strong expression was observed in selective glucagon cells with moderate expression in most glucagon and insulin cells (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGroup 3 (Non-diabetic autoantibody-positive cases): In nPOD 6424 and 6267 (with 2 autoantibodies), an overall weak GPX staining was present in most islet cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea-l). Occasional strong GPX1-expressing cells within the intra-islet space were also present (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eg-l). In nPOD 6301 and 6310 (single autoantibody-positive), selective insulin and glucagon cells showed moderate to strong GPX1 staining, while a majority showed weaker expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea-l). An overall weak to moderate expression was present in most islet cells in nPOD 6167 and 6158 (with two autoantibodies; ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-l).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGroup 4 (Long-term diabetic cases): In E560 (1.5 years of T1D), moderate to strong expression of GPX1 was present in selective glucagon and some remaining insulin cells and in cells negative for both cell-types (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ea-f). In nPOD 6211 (4 years of T1D), islet cells showed either weak or stronger expression in selective glucagon and remaining insulin cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eg-l). A similar pattern was also present in nPOD 6088 (4 years of T1D; Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003ea-f) and nPOD 6070 (7 years of T1D; Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eg-l). In nPOD 6245 (7 years of T1D), most islet cells showed an overall weak expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ea-f). A similar cellular pattern was observed in some islets from nPOD 6045 (8 years of T1D). However, in this donor, several cells in specific islets negative for insulin and glucagon, showed unusually strong staining intensities for GPX1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ej-l). In nPOD 6262 (8 years of T1D) and nPOD 6220 (11 years of T1D), islets were negative for insulin, but showed strong expression of GPX1 in selective glucagon cells, with moderate expression in the remaining islet cells (ESM Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-l).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of overall GPX1 staining patterns in islets in the 4 study groups\u003c/h2\u003e \u003cp\u003eThe overall cellular pattern of GPX1 expression in islets and its staining intensities in islet cells and in insulin-negative and positive islets, were quantified based on a visual grading system depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Pattern 1 (grades 1\u0026ndash;3) and Pattern 2 (grades 4\u0026ndash;6)). The various grades are represented graphically in Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003ea, b and values listed in ESM Tables\u0026nbsp;3\u0026ndash;6. The percentage of islets with combined grades of 1, 2 and 3 (pattern 1) versus 4, 5 and 6 (pattern 2) are summarised in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In groups 1, 2 and 3, differences between the two patterns reached statistical significance (p\u0026thinsp;=\u0026thinsp;0.035, 0.006 and 0.008, respectively but not in group 4 (p\u0026thinsp;=\u0026thinsp;0.071)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGradings for GPX1 in insulin-positive and negative islets (Groups 1 and 4) are represented as segmented bar graphs in Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eb and tabulated in ESM Tables\u0026nbsp;7\u0026ndash;10. The variable percentages of islets with the two patterns probably reflect re-distribution of islet cells during beta cell loss. In group 1 (newly-diagnosed diabetic group), there was no significant difference between the percentage of insulin-positive islets with pattern 1 and pattern 2 (p\u0026thinsp;=\u0026thinsp;0.09) and in insulin-negative islets (p\u0026thinsp;=\u0026thinsp;0.80). However, in Group 4 (long-term diabetic group) there was a significant difference between the two patterns in insulin-positive islets (p\u0026thinsp;=\u0026thinsp;0.04) but not in insulin-negative islets (p\u0026thinsp;=\u0026thinsp;0.06).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of GPX1 staining intensities in individual insulin and glucagon cells in 10 randomly selected islets per case from all study groups (refer to\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003e \u003cb\u003eand ESM Tables\u0026nbsp;11\u0026ndash;15)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGroup 1 (newly-diagnosed cases): Islets from case 1 were insulin-negative, with strong GPX1 staining in approximately 90% of glucagon cells, while in case 2, 80% of beta cells had weak staining and almost 55% of glucagon cells had moderate to strong staining. In case 3, 77% of insulin cells had negative GPX1 staining, while almost 90% glucagon cells had moderate to strong staining for GPX1. In case 4, almost all insulin cells and glucagon cells showed moderate GPX1 staining intensities.\u003c/p\u003e \u003cp\u003eGroup 2 (non-diabetic autoantibody-negative cases): In cases 6289, 6160, 6055 and 6229, many insulin cells showed weak GPX1 staining while in case 6178, 97% of such cells were negative with only 24% of glucagon cells with moderate to strong staining. In case 6401, \u0026gt;\u0026thinsp;78% of insulin cells had moderate GPX1 intensities. More than 80% of glucagon cells in cases 6401 and 6369 had moderate GPX1 intensity while in the remaining cases the intensities in the same cell-types were more variable.\u003c/p\u003e \u003cp\u003eGroup 3 (non-diabetic autoantibody-positive cases): In cases 6424, 6301 and 6158, more than 65% of insulin cells showed moderate GPX1 staining intensities while in cases 6267, 6310 and 6167, 99%, 62% and 56%, respectively showed weak intensities. In cases 6424, 6301, 6310 and 6158, moderate intensities were present in 68%, 88%, 64% and 94%, of glucagon cells, respectively. In cases 6267 and 6167, weak intensities were present in 73% and 56% of glucagon cells, respectively. In all 6 cases, strong intensities were observed in a minority of glucagon cells (\u0026lt;\u0026thinsp;15% of glucagon cells), with staining intensities being independent of the presence of single or double autoantibodies.\u003c/p\u003e \u003cp\u003eIn groups 1, 2 and 3, differences between the percentages of insulin cells versus glucagon cells with negative, weak and moderate GPX1 staining intensities did not reach statistical difference.\u003c/p\u003e \u003cp\u003eHowever, there was a significant difference in Group 2 between insulin and glucagon cells with strong GPX1 staining (p\u0026thinsp;=\u0026thinsp;0.03).\u003c/p\u003e \u003cp\u003eGroup 4 (long-term diabetic cases): Four of 8 cases from this group, showed an absence of insulin cells in 10 randomly selected islets per case. In cases 6211, 6070, and 6245 more than 50% of insulin cells showed weak staining intensities for GPX1 while in case E560, approximately 75% of insulin cells displayed moderate staining intensities. Weak intensities were displayed in a majority of the remaining insulin cells (6211: 50.55%; 6070: 76.6% and 6245: 83.5%). In 7/8 cases from the same group, more than 50% of glucagon cells per case showed moderate staining intensities while in the remaining case (E560: 1.5 years of T1D), 75% glucagon cells showed moderate-strong intensities. In this group, there was a significant difference in the percentages of insulin cells versus glucagon cells with moderate to strong GPX1 staining intensities (p\u0026thinsp;=\u0026thinsp;0.003).\u003c/p\u003e \u003cp\u003eCumulative GPX1 staining intensities per study group (refer to ESM Table\u0026nbsp;15 and Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e16\u003c/span\u003e): GPX1 staining in Group 1 was absent in a higher percentage of insulin cells (30.34%) than glucagon cells (0.2%). A similar difference was seen in groups 2 and 3 while in group 4 (long-term diabetic group), almost all insulin and glucagon cells showed weak to strong GPX1 staining intensities. In all 4 groups moderate to strong intensities were observed in higher percentage of glucagon cells than beta cells (refer to ESM Table\u0026nbsp;15). In Group 4, there was a significant difference between the percentage of beta cells and glucagon cells with moderate GPX1 staining intensity (p\u0026thinsp;=\u0026thinsp;0.005).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEarlier results from experimental mice show an intrinsic absence or lower expression levels of the three important ROS clearing enzymes, namely GPX1, catalase and SOD in islets (Grankvist, Marklund and T\u0026auml;ljedal, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1981\u003c/span\u003e, Harmon, et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Lenzen, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Tiedge, Lortz, Drinkgern and Lenzen, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Tiedge, Lortz, Munday and Lenzen, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). If such findings hold true for human islets, one may conclude that human beta cells may also fail to clear excess ROS. However, low levels of expression of the three enzymes selectively in beta cells of mice have not been studied in detail. If such deficits are present in human beta cells, the resulting redox imbalance may activate critical immune signals in these cells prior to and during human T1D (Gerber and Rutter, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Leenders, Groen, de Graaf, Engelse, Rabelink, de Koning and Carlotti, 2021, Mallone, Halliez, Rui and Herold, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Newsholme, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Robertson and Harmon, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Robertson and Harmon, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Despite such void, animal findings have led to the evaluation of several therapeutic approaches targeted at boosting levels of SOD and/or GPX1 or the use of low molecular weight mimetics to suppress excess ROS in beta cell lines and in experimental animals afflicted with various pathologies (Bertera, et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Forman and Zhang, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Harmon, Bogdani, Parazzoli, Mak, Oseid, Berghmans, Leboeuf and Robertson, 2009, Mallone, Halliez, Rui and Herold, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Tiedge, Lortz, Munday and Lenzen, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Although such efforts have yielded little success, more recent ploys incorporating specific and targeted payload delivery systems and techniques for up-regulating transcription factors that activate specific genes encoding anti-oxidant enzymes are in progress (Poprac, et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe availability of rare pancreatic sections from clinically well-defined diabetic and non-diabetic donors has permitted us to develop and apply a validated immunohistochemical protocol for the analysis of GPX1 expression in combination with insulin and glucagon and compare its distribution and staining intensities in donors with T1D and in autoantibody-positive and -negative non-diabetic donors.\u003c/p\u003e \u003cp\u003eThe specificity of anti-GPX1 was confirmed in this study by demonstrating positive staining in human liver, lung and breast carcinoma, omission of anti-GPX1 in the immunohistochemical procedure and the use of GPX1 antibody from the same supplier by others (Basnet, Tian, Ganesh, Huang, Macalinao, Brogi, Finley and Massagu\u0026eacute;, 2019, Chu, Esworthy, Doroshow, Doan and Liu, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, Miki, Ricordi, Sakuma, Yamamoto, Misawa, Mita, Molano, Vaziri, Pileggi and Ichii, 2018). We also validated the expression of GPX1 in human pancreatic sections by employing an immunofluorescence technique. Staining of the enzyme in some ductal cells, a proportion of which co-localized with glucagon is novel. Glutathione peroxidase-1 immunoreactivity in insulin- and glucagon-negative islet cells, shown here, likely represents somatostatin and/or pancreatic polypeptide hormone and ghrelin cells or non-endocrine cells within the intra-islet vascular region, such as endothelial cells or immune cells. Thus, we show that the distribution of GPX1 in the human pancreatic islet cells is more widespread than previously reported by others (Miki, Ricordi, Sakuma, Yamamoto, Misawa, Mita, Molano, Vaziri, Pileggi and Ichii, 2018).\u003c/p\u003e \u003cp\u003eOur observation of two major cellular patterns of GPX1 immunoreactivity in islet cells (Pattern 1 and Pattern 2) has not been observed previously. Different percentages of islets with the two staining patterns, with varying intensities were observed in all donors, irrespective of diabetes status or the presence or absence of beta cells. In the 2 non-diabetic autoantibody-positive and negative groups (Groups 2 and 3), Pattern 1 was present in a majority of cases. Differences in the frequency of the 2 distribution patterns in newly-diagnosed and long-term diabetic groups (Groups 1 and 4) may have been due to the presence or absence of residual beta cells in the islets, variability in the expression of the GPX1 in non-beta cells, including glucagon cells or spatial re-distribution of endocrine cells in beta cell-negative pseudoatrophic islets.\u003c/p\u003e \u003cp\u003eWe highlight that various percentages of beta cells do express GPX1 in all study groups, with moderate to strong staining intensities in a higher percentage of glucagon cells than beta cells, except in nPOD case 6178, where a majority of insulin and glucagon cells were negative for GPX1. These observations suggest that in most cases many beta cells express weaker or negative staining intensities for GPX1 than glucagon cells and may, therefore, be ill-equipped to clear excess H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, conferring selective vulnerability. Heterogeneity of intra-islet staining intensities in insulin and glucagon cells with islet-to-islet variability within the same donor may suggest differing rates of intracellular storage of GPX1 and its turnover within the same donor pancreas. Previous findings which show intra-islet functional heterogeneity, such as asynchronous secretion of insulin in response to glucose and senescence lend support to our current findings (Farack, et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Thompson, Pipella, Rutter, Gaisano and Santamaria, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Van Schravendijk, et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1992\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMore recent studies in mice suggest that beta cells are equipped with other anti-oxidant systems to scavenge excess ROS, and in particular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. These include various isoforms of peroxiredoxin, thioredoxin and thioredoxin reductase (Stancill, Broniowska, Oleson, Naatz and Corbett, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Stancill and Corbett, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, whether such findings hold true for human islets remains to be proven. In support, earlier studies have shown that human islets are more resistant to oxidant-generating beta cell toxins such as alloxan, streptozotocin, nitroprusside or cytokine-induced injury than mouse islets (Welsh, et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). In the same studies, enzyme activities of catalase and SOD were significantly lower in islets from mice than humans. More recent work suggests that high levels of thioredoxin-interacting protein (TXNIP) in beta cells may inhibit the ability of thioredoxin in the peroxiredoxin-thioredoxin system to detoxify H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Ovalle, et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The latter findings have led to an ongoing clinical trial in adults with recent-onset T1D, involving the use of verapamil, a known anti-hypertensive calcium channel blocker, in suppressing TXNIP levels and improving beta cell function (Ovalle, Grimes, Xu, Patel, Grayson, Thielen, Li and Shalev, 2018) (clinical trial reg. no. NCT0455151, ClinicalTrials.gov).\u003c/p\u003e \u003cp\u003eWe are cognisant of some of the limitations of the present study. For example, the use of immunohistochemistry as the sole experimental approach may have some caveats. However, the availability of pancreatic sections only precluded us from undertaking additional confirmatory studies such as Western blotting or proteomic analysis of isolated human islets for assessing GPX1 levels. However, Western blotting has inherent limitations since it involves disruption of islet architecture and subsequent loss of spatial and cell-type distribution and identification of proteins under study, and we propose that immunohistochemical approach remains compatible with the aims of the present study. We recognize that this study involved analysis of a smaller sample size due to the limited availability of rare human samples with inter-individual heterogeneity and variations in sample acquisition. Therefore, generalizations of our findings to all individuals with new-onset and long-term T1D require some caution. We recognize that while DiViD donors were in better diabetic control during and prior to biopsy, deceased donors from cadaveric and autopsy pancreas may have experienced variable degrees of unavoidable metabolic fluctuations, hyperglycaemia-driven oxidative and nitrosative stress and cold ischaemia before pancreas sampling. Prior medications during hospitalisation, patient stress and variable but unavoidable delays between death and tissue procurement may have also influenced the dynamics, expression pattern and staining intensities of GPX1. Since our immunohistochemical approach does not discriminate between enzymatically active and inactive forms of GPX1, monomeric and tetrameric forms and gene variants, caution also needs to be exercised in ascribing a physiological role of GPX1 solely based on immunohistochemical approaches (Mohammedi, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). We also recognize that although our studies are cross-sectional and reflect a static snapshot of GPX1 expressing cells in human pancreatic sections, they are nevertheless informative and provide clues on beta cell stress. Other limitations include the possibility of regional differences in the frequency, distribution and intensity of GPX1-positive cells within various islets from the same donor. However, in this study, we report our results following careful analysis of a large number of separate islets with various cross-sectional areas per donor, providing scientific rigour. Future non-invasive deep tissue multiplex imaging of a panel of anti-oxidant enzymes in islets of diabetic and non-diabetic cases may provide more precise and mechanistic information on the distribution and vulnerability of human beta cells to excess levels of ROS, how they impact on the early origins of beta cell destruction and whether they show adaptive protective mechanisms when exposed to raised levels of ROS.\u003c/p\u003e \u003cp\u003eThe present study conducted in rare human pancreatic tissues, unveils scientific perspectives and offers new and previously unreported findings on the cellular patterns of GPX1 expression. Contrary to recent findings, we report that the expression of the enzyme is seen with varying intensities in not only in selective glucagon cells but also in a lower percentage of beta cells in both non-diabetic and diabetic human donors. Differences in intensities of GPX1 expression in intra- and inter-islet beta cells among the study groups, implies different rates GPX1 biosynthesis and turnover in individual beta cells and their selective vulnerability to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026minus;\u003c/sub\u003emediated oxidative injury. Contrary to previous reports in experimental mice, our current findings demonstrating GPX1 positivity in beta cells, albeit in lower numbers, are novel and suggest that a proportion of human beta cells have the capacity to clear GPX1-mediated excess H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Of note, the distribution of GPX1 immunoreactivity in islet cells showed two distinct patterns of staining, namely whole islet cell staining and selective islet cell staining in a minority of islet cells. The heterogeneity of GPX1 staining in beta cells is consistent with recent observations that demonstrate their asynchronous destruction during T1D (Thompson, Pipella, Rutter, Gaisano and Santamaria, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Targeted delivery of GPX1 or its mimetics to beta cells at the early stages of T1D or pharmacological attempts to boost GPX-1 via the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), and other beta cell protective candidates may mitigate further destruction of beta cells (Forman and Zhang, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e The online version contains supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eSR conceived and designed the experimental studies, carried out a considerable portion of them, acquired and analysed the data, wrote, and revised the manuscript critically for publication. He also led and directed the study. KP also carried out considerable portion of the experimental studies and compiled numerous microscopic images into plates and prepared all tables and edited the manuscript. KS, KD-J and LK made a significant contribution towards editing and revising the manuscript for intellectual content. KS also carried out statistical analysis and prepared all bar graphs and edited the manuscript. All authors have given their final approval of the version to be published. SR is the guarantor of the work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e Electronic supplementary material is available online.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u0026nbsp;\u003c/strong\u003eInformed consent for donation and analysis of pancreatic biopsies from DiViD donors were obtained by the DiViD Study Group from the Norwegian Government\u0026rsquo;s Regional Ethics Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eEthical approval for conducting this study was granted by the New Zealand Ministry of Health and Disability Ethics Committee (Ethics reference: 2023 PR 2121). Harbin Medical University (China), Auckland Tissue Bank and the Department of Anatomy of this faculty provided control tissue sections from the breast, liver and lung and were also covered by the above New Zealand ethics committee approval. \u0026nbsp;Approval for biopsy procurement from new-onset type 1 diabetes donors was granted by the Norwegian Government\u0026rsquo;s Regional Ethics Committee following informed consent. Ethical approval for the analysis of pancreatic sections from deceased nPOD donors was granted to nPOD. Ethical approval for the analysis of sections from post-mortem pancreas for EADB (http://foulis.vub.ac.be) was granted by the West of Scotland Research Committee 4 ((WoSREC4; 15//WS/0258) to Professors S Richardson and N Morgan, University of Exeter. Therefore, all procedures performed involving human tissue donors were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclaimer\u003c/strong\u003e The content and views expressed in this publication are the responsibility of the authors and do not reflect an official view of nPOD, DiViD and EADB.\u003c/p\u003e\n\u003cp\u003eThis research is dedicated to the Late Sir Robert B Elliott, Former Professor of Paediatrics and Child Health Research, University of Auckland, Auckland, New Zealand\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmirruddin NS, Tan WX, Tan YS, Gardner DS, Bee YM, Verma CS, Hoon S, Lee KO, Teo AKK (2021) Progressive endoplasmic reticulum stress over time due to human insulin gene mutation contributes to pancreatic beta cell dysfunction. 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Peptides 100:54\u0026ndash;60\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable 2.\u0026nbsp;Comparison of mean percentages of islets with GPX1 staining per study group\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eStudy groups\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNo. of cases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePattern 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePattern 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eP values\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGroup 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e24.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e75.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.035*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGroup 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e65.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.006*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGroup 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e68.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e31.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.008*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGroup 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e64.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e35.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.1071\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: The percentages of islets with GPX1 staining were combined grades of 1, 2 and 3 (pattern 1) versus 4, 5 and 6 (pattern 2). The mean values were compared by using a student t-test per study group. \u0026ldquo;*\u0026rdquo; indicates a significant difference.\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Type 1 diabetes, Glutathione peroxidase-1, Reactive oxygen species, Beta cell damage, Immunohistochemistry","lastPublishedDoi":"10.21203/rs.3.rs-5369709/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5369709/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDuring type 1 diabetes (T1D), oxidative stress in beta cells may cause early dysfunction and initiate autoimmunity. Mouse islets express lower levels of reactive oxygen species (ROS) clearing enzymes, glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase in comparison with several other tissues. It remains unclear if human beta cells show a similar deficiency, particularly during T1D or show a higher degree oxidative resistance. We compared islet cell distributions and determined graded intensities of glutathione peroxidase1 (GPX1), a key enzyme which detoxifies hydrogen peroxide, by applying combined immunohistochemistry for GPX1, insulin and glucagon, in pancreatic sections from new-onset T1D (group 1), non-diabetic autoantibody-negative (group 2), non-diabetic autoantibody-positive (group 3) and long-term diabetic (group 4) donors. Islets from all study groups demonstrated either uniform but graded staining intensities for GPX1 in almost all islet cells or strong staining in selective islet cells with weaker intensities in the remaining cells. GPX1 was present in selective glucagon and in insulin cells also and in cells negative for both hormones, with stronger intensities in a higher percentage of glucagon than insulin cells. The enzyme was absent in a higher percentage of beta cells than glucagon cells independent of disease or autoantibody positivity. We conclude that a proportion of human beta cells and glucagon cells express GPX1 but show heterogeneity in its distribution and intensities, independent of disease or autoantibody status. Pharmacologic attempts to boost GPX1 level in human beta cells during early T1D or in isolated islets for transplantation may offer clinical benefits.\u003c/p\u003e","manuscriptTitle":"Distribution of glutathione peroxidase-1 immunoreactive cells in pancreatic islets from type 1 diabetic donors and non-diabetic donors with and without islet cell autoantibodies is variable and independent of disease ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-15 14:11:40","doi":"10.21203/rs.3.rs-5369709/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-18T06:22:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-17T19:55:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305579582841864106790093069528516929175","date":"2024-12-13T19:11:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-14T00:15:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110424889679076642572791396608812306433","date":"2024-11-05T10:21:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60425095254167634667441665675482304477","date":"2024-11-05T06:51:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-05T06:46:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-04T05:05:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-04T05:04:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell and Tissue Research","date":"2024-11-01T01:21:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dd2dd3cb-edd7-4f8b-ad7b-effd4ead366d","owner":[],"postedDate":"November 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-17T16:00:49+00:00","versionOfRecord":{"articleIdentity":"rs-5369709","link":"https://doi.org/10.1007/s00441-025-03955-5","journal":{"identity":"cell-and-tissue-research","isVorOnly":false,"title":"Cell and Tissue Research"},"publishedOn":"2025-03-10 15:57:25","publishedOnDateReadable":"March 10th, 2025"},"versionCreatedAt":"2024-11-15 14:11:40","video":"","vorDoi":"10.1007/s00441-025-03955-5","vorDoiUrl":"https://doi.org/10.1007/s00441-025-03955-5","workflowStages":[]},"version":"v1","identity":"rs-5369709","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5369709","identity":"rs-5369709","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}