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Neutrophils might play an important role in the pathogenesis of autoimmune diseases, including type 1 diabetes (T1D), by contributing to immune dysregulation via a highly inflammatory program called neutrophil extracellular trap (NET) formation or NETosis, involving the extrusion of chromatin entangled with anti-microbial proteins. However, numerous studies reported contradictory data on NET formation in T1D. This might in part be due to the inherent heterogeneity of the disease and the influence of the disease developmental stage on neutrophil behavior. Moreover, there is a lack of a standardized method to measure NETosis in an unbiased and robust manner. In this study, we employed the Incucyte® ZOOM live-cell imaging platform to study NETosis levels in various subtypes of adult and pediatric T1D donors compared to healthy controls (HC) at baseline and in response to phorbol–myristate acetate (PMA) and ionomycin. Firstly, we determined that the technique allows for an operator-independent and automated quantification of NET formation across multiple time points, which showed that PMA and ionomycin induced NETosis with distinct kinetic characteristics, confirmed by high-resolution microscopy. NETosis levels also showed a clear dose-response curve to increasing concentrations of both stimuli. Overall, using Incucyte® ZOOM, no aberrant NET formation was observed over time in the different subtypes of T1D populations, irrespective of age, compared to HC. These data were corroborated by the levels of peripheral NET markers in all study participants. The current study showed that live-cell imaging allows for a robust and unbiased analysis and quantification of NET formation in real-time. Peripheral neutrophil measures should be complemented with dynamic quantification of NETing neutrophils to make robust conclusions on NET formation in health and disease.

Objective Activated synovial fibroblasts are key effector cells in RA. Selectively depleting these based upon their expression of fibroblast activation protein (FAP) is an attractive therapeutic approach. Here we introduce FAP imaging of inflamed joints using 68Ga-FAPI-04 in a RA patient, and aim to assess feasibility of anti-FAP targeted photodynamic therapy (FAP-tPDT) ex vivo using 28H1-IRDye700DX on RA synovial explants. Methods Remnant synovial tissue from RA patients was processed into 6 mm biopsies and, from several patients, into primary fibroblast cell cultures. Both were treated using FAP-tPDT. Cell viability was measured in fibroblast cultures and biopsies were evaluated for histological markers of cell damage. Selectivity of the effect of FAP-tPDT was assessed using flow cytometry on primary fibroblasts and co-cultured macrophages. Additionally, one RA patient intravenously received 68Ga-FAPI-04 and was scanned using PET/CT imaging. Results In the RA patient, FAPI-04 PET imaging showed high accumulation of the tracer in arthritic joints with very low background signal. In vitro, FAP-tPDT induced cell death in primary RA synovial fibroblasts in a light dose-dependent manner. An upregulation of cell damage markers was observed in the synovial biopsies after FAP-tPDT. No significant effects of FAP-tPDT were noted on macrophages after FAP-tPDT of neighbouring fibroblasts. Conclusion In this study the feasibility of selective FAP-tPDT in synovium of rheumatoid arthritis patients ex vivo is demonstrated. Furthermore, this study provides the first indication that FAP-targeted PET/CT can be used to image arthritic joints, an important step towards application of FAP-tPDT as a targeted locoregional therapy for RA.

Aims/hypothesis The role of beta cell mass in the balance of glucose control and hypoglycaemic burden in people with type 1 diabetes is unclear. We applied positron emission tomography (PET) imaging with radiolabelled exendin to compare beta cell mass among people with type 1 diabetes and either low glucose variability (LGV) or high glucose variability (HGV). Methods All participants with either LGV (n=9) or HGV (n=7) underwent a mixed-meal tolerance test to determine beta cell function and wore a blinded continuous glucose monitor for a week. After an i.v. injection with [68Ga]Ga-NODAGA-exendin-4, PET images were acquired for the quantification of pancreatic uptake of radiolabelled exendin. The mean standardised uptake value (SUVmean) of the pancreas was used to determine the amount of beta cell mass. Results Participants with LGV had lower HbA1c (46.0 mmol/mol [44.5–52.5] [6.4% (6.3–7)] vs 80 mmol/mol [69.0–110] [9.5% (8.5–12.2)], p=0.001) and higher time in range (TIR) (75.6% [73.5–90.3] vs 38.7% [25.1–48.5], p=0.002) than those with HGV. The SUVmean of the pancreas was higher for the LGV than for the HGV group (5.1 [3.6–5.6] vs 2.9 [2.1–3.4], p=0.008). The AUCC-peptide:AUCglucose ratio was numerically, but not statistically, higher in the LGV compared with the HGV group (2.7×10−2 [6.2×10−4–5.3×10−2] vs 9.3×10−4 [4.7×10−4–5.2×10−3], p=0.21). SUVmean correlated with the AUCC-peptide:AUCglucose ratio (Pearson r=0.64, p=0.01), as well as with the TIR (r=0.64, p=0.01) and the SD of interstitial glucose levels (r=−0.66, p=0.007). Conclusion/interpretation Our data show higher beta cell mass in people with type 1 diabetes and LGV than in those with HGV, independent of beta cell function. Graphical abstract

The role of beta cell mass in the balance of glucose control and hypoglycaemic burden in people with type 1 diabetes is unclear. We applied positron emission tomography (PET) imaging with radiolabelled exendin to compare beta cell mass among people with type 1 diabetes and either low glucose variability (LGV) or high glucose variability (HGV).All participants with either LGV (n=9) or HGV (n=7) underwent a mixed-meal tolerance test to determine beta cell function and wore a blinded continuous glucose monitor for a week. After an i.v. injection with [Participants with LGV had lower HbAOur data show higher beta cell mass in people with type 1 diabetes and LGV than in those with HGV, independent of beta cell function.

Surgery with curative intent can be offered to congenital hyperinsulinism (CHI) patients, provided that the lesion is focal. Radiolabeled exendin-4 specifically binds the glucagonlike peptide 1 receptor on pancreatic β-cells. In this study, we compared the performance of 18F-DOPA PET/CT, the current standard imaging method for CHI, and PET/CT with the new tracer 68Ga-NODAGA-exendin-4 in the preoperative detection of focal CHI. Methods: Nineteen CHI patients underwent both 18F-DOPA PET/CT and 68Ga-NODAGA-exendin-4 PET/CT before surgery. The images were evaluated in 3 settings: a standard clinical reading, a masked expert reading, and a joint reading. The target (lesion)-to-nontarget (normal pancreas) ratio was determined using SUVmax Image quality was rated by pediatric surgeons in a questionnaire. Results: Fourteen of 19 patients having focal lesions underwent surgery. On the basis of clinical readings, the sensitivity of 68Ga-NODAGA-exendin-4 PET/CT (100%; 95% CI, 77%-100%) was higher than that of 18F-DOPA PET/CT (71%; 95% CI, 42%-92%). Interobserver agreement between readings was higher for 68Ga-NODAGA-exendin-4 than for 18F-DOPA PET/CT (Fleiss κ = 0.91 vs. 0.56). 68Ga-NODAGA-exendin-4 PET/CT provided significantly (P = 0.021) higher target-to-nontarget ratios (2.02 ± 0.65) than did 18F-DOPA PET/CT (1.40 ± 0.40). On a 5-point scale, pediatric surgeons rated 68Ga-NODAGA-exendin-4 PET/CT as superior to 18F-DOPA PET/CT. Conclusion: For the detection of focal CHI, 68Ga-NODAGA-exendin-4 PET/CT has higher clinical sensitivity and better interobserver correlation than 18F-DOPA PET/CT. Better contrast and image quality make 68Ga-NODAGA-exendin-4 PET/CT superior to 18F-DOPA PET/CT in surgeons' intraoperative quest for lesion localization. ispartof: JOURNAL OF NUCLEAR MEDICINE vol:63 issue:2 pages:310-315 ispartof: location:United States status: published

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In type 1 diabetes, autoimmune β-cell destruction may be favored by neo-antigens harboring post-translational modifications such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8+ T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8+ T cells circulated at similar frequencies in type 1 diabetic and healthy donors and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8+ T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidyl-arginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8+ T-cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, post-translational modifications may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Macrophages play a crucial role in the initiation and progression of rheumatoid arthritis (RA). Liposomes can be used to deliver therapeutics to macrophages by exploiting their phagocytic ability. However, since macrophages serve as the immune system’s first responders, it is inadvisable to systemically deplete these cells. By loading the liposomes with the photosensitizer IRDye700DX, we have developed and tested a novel way to perform photodynamic therapy (PDT) on macrophages in inflamed joints. PEGylated liposomes were created using the film method and post-inserted with micelles containing IRDye700DX. For radiolabeling, a chelator was also incorporated. RAW 264.7 cells were incubated with liposomes with or without IRDye700DX and exposed to 689 nm light. Viability was determined using CellTiterGlo. Subsequently, biodistribution and PDT studies were performed on mice with collagen-induced arthritis (CIA). PDT using IRDye700DX-loaded liposomes efficiently induced cell death in vitro, whilst no cell death was observed using the control liposomes. Biodistribution of the two compounds in CIA mice was comparable with excellent correlation of the uptake with macroscopic and microscopic arthritis scores. Treatment with 700DX-loaded liposomes significantly delayed arthritis development. Here we have shown the proof-of-principle of performing PDT in arthritic joints using IRDye700DX-loaded liposomes, allowing locoregional treatment of arthritis.

INTRODUCTION: Aberrant citrullination and excessive peptidylarginine deiminase (PAD) activity are detected in numerous challenging autoimmune diseases such as rheumatoid arthritis, inflammatory bowel diseases, systemic lupus erythematosus, multiple sclerosis, and type 1 diabetes. Because excessive PAD activity is a common denominator in these diseases, PADs are interesting potential therapeutic targets for future therapies. AREAS COVERED: This review summarizes the advances made in the design of PAD inhibitors, their utilization and therapeutic potential in preclinical mouse models of autoimmunity. Relevant literature encompasses studies from 1994 to 2021 that are available on PubMed.gov. EXPERT OPINION: Pan-PAD inhibition is a promising therapeutic strategy for autoimmune diseases. Drugs achieving pan-PAD inhibition were capable of ameliorating, reversing, and preventing clinical symptoms in preclinical mouse models. However, the implications for PADs in key biological processes potentially present a high risk for clinical complications and could hamper the translation of PAD inhibitors to the clinic. We envisage that PAD isozyme-specific inhibitors will improve the understanding the role of PAD isozymes in disease pathology, reduce the risk of side-effects and enhance prospects for future clinical translation. ispartof: EXPERT OPINION ON THERAPEUTIC TARGETS vol:25 issue:4 pages:269-281 ispartof: location:England status: published

In type 1 diabetes, autoimmune β-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8(+) T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8(+) T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8(+) T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8(+) T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Protein citrullination plays a role in several autoimmune diseases. Its involvement in murine and human type 1 diabetes has recently been recognized through the discovery of antibodies and T-cell reactivity against citrullinated peptides. In the current study, we demonstrate that systemic inhibition of peptidylarginine deiminases (PADs), the enzymes mediating citrullination, through BB-Cl-amidine treatment, prevents diabetes development in NOD mice. This prevention was associated with reduced levels of citrullination in the pancreas, decreased circulating autoantibody titers against citrullinated glucose-regulated protein 78, and reduced spontaneous neutrophil extracellular trap formation of bone marrow-derived neutrophils. Moreover, BB-Cl-amidine treatment induced a shift from Th1 to Th2 cytokines in the serum and an increase in the frequency of regulatory T cells in the blood and spleen. In the pancreas, BB-Cl-amidine treatment preserved insulin production and was associated with a less destructive immune infiltrate characterized by reduced frequencies of effector memory CD4+ T cells and a modest reduction in the frequency of interferon-γ-producing CD4+ and CD8+ T cells. Our results point to a role of citrullination in the pathogenesis of autoimmune diabetes, with PAD inhibition leading to disease prevention through modulation of immune pathways. These findings provide insight in the potential of PAD inhibition for treating autoimmune diseases like type 1 diabetes. ispartof: DIABETES vol:70 issue:2 pages:516-528 ispartof: location:United States status: published

GLP-1R imaging with radiolabelled exendin has proven to be a powerful tool to quantify beta-cell mass (BCM) in vivo. As GLP-1R expression is thought to be influenced by glycemic control, we examined the effect of blood glucose levels on GLP-1R-mediated exendin uptake in both murine and human islets and its implications for BCM quantification. Periods of hyperglycemia significantly reduced exendin uptake in murine and human islets, which was paralleled by a reduction in GLP-1R expression. Detailed mapping of the tracer uptake and insulin and GLP-1R expression conclusively demonstrated that the observed reduction in tracer uptake directly correlates to GLP-1R expression levels. Importantly, the linear correlation between tracer uptake and beta-cell area was maintained in spite of the reduced GLP-1R expression levels. Subsequent normalization of blood glucose levels restored absolute tracer uptake and GLP-1R expression in beta-cells and the observed loss in islet volume was halted. This manuscript emphasizes the potency of nuclear imaging techniques to monitor receptor regulation non-invasively. Our findings have significant implications for clinical practice, indicating that blood glucose levels should be near-normalized for at least three weeks prior to GLP-1R agonist treatment or quantitative radiolabeled exendin imaging for BCM analysis.

The treatment of choice for insulinomas and focal lesions in congenital hyperinsulinism (CHI) is surgery. However, intraoperative detection can be challenging. This challenge could be overcome with intraoperative fluorescence imaging, which provides real-time lesion detection with a high spatial resolution. Here, a novel method for targeted near-infrared (NIR) fluorescence imaging of glucagonlike peptide 1 receptor (GLP-1R)-positive lesions, using the GLP-1 agonist exendin-4 labeled with IRDye 800CW, was examined in vitro and in vivo. Methods: A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with GLP-1R. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. In vivo NIR fluorescence imaging of CHL-GLP-1R xenografts was performed. Localization of the tracer in the pancreatic islets of BALB/c nude mice was examined using fluorescence microscopy. Laparoscopic imaging was performed to detect the fluorescent signal of the tracer in the pancreas of mini pigs. Results: Exendin-4-IRDye 800CW binds GLP-1R with a half-maximal inhibitory concentration of 3.96 nM. The tracer accumulates in CHL-GLP-1R xenografts. Subcutaneous CHL-GLP-1R xenografts were visualized using in vivo NIR fluorescence imaging. The tracer accumulates specifically in the pancreatic islets of mice, and a clear fluorescent signal was detected in the pancreas of mini pigs. Conclusion: These data provide the first in vivo evidence of the feasibility of targeted fluorescence imaging of GLP-1R-positive lesions. Intraoperative lesion delineation using exendin-4-IRDye 800CW could benefit open as well as laparoscopic surgical procedures for removal of insulinomas and focal lesions in CHI.

Glucagon-like peptide 1 receptor (GLP-1R) imaging with radiolabeled exendin has proven to be a powerful tool to quantify β-cell mass (BCM) in vivo. As GLP-1R expression is thought to be influenced by glycemic control, we examined the effect of blood glucose (BG) levels on GLP-1R–mediated exendin uptake in both murine and human islets and its implications for BCM quantification. Periods of hyperglycemia significantly reduced exendin uptake in murine and human islets, which was paralleled by a reduction in GLP-1R expression. Detailed mapping of the tracer uptake and insulin and GLP-1R expression conclusively demonstrated that the observed reduction in tracer uptake directly correlates to GLP-1R expression levels. Importantly, the linear correlation between tracer uptake and β-cell area was maintained in spite of the reduced GLP-1R expression levels. Subsequent normalization of BG levels restored absolute tracer uptake and GLP-1R expression in β-cells and the observed loss in islet volume was halted. This manuscript emphasizes the potency of nuclear imaging techniques to monitor receptor regulation noninvasively. Our findings have significant implications for clinical practice, indicating that BG levels should be near-normalized for at least 3 weeks prior to GLP-1R agonist treatment or quantitative radiolabeled exendin imaging for BCM analysis.

Objective In RA, synovial fibroblasts become activated. These cells express fibroblast activation protein (FAP) and contribute to the pathogenesis by producing cytokines, chemokines and proteases. Selective depletion in inflamed joints could therefore constitute a viable treatment option. To this end, we developed and tested a new therapeutic strategy based on the selective destruction of FAP-positive cells by targeted photodynamic therapy (tPDT) using the anti-FAP antibody 28H1 coupled to the photosensitizer IRDye700DX. Methods After conjugation of IRDye700DX to 28H1, the immunoreactive binding and specificity of the conjugate were determined. Subsequently, tPDT efficiency was established in vitro using a 3T3 cell line stably transfected with FAP. The biodistribution of [111In]In-DTPA-28H1 with and without IRDye700DX was assessed in healthy C57BL/6N mice and in C57BL/6N mice with antigen-induced arthritis. The potential of FAP-tPDT to induce targeted damage was determined ex vivo by treating knee joints from C57BL/6N mice with antigen-induced arthritis 24 h after injection of the conjugate. Finally, the effect of FAP-tPDT on arthritis development was determined in mice with collagen-induced arthritis. Results 28H1-700DX was able to efficiently induce FAP-specific cell death in vitro. Accumulation of the anti-FAP antibody in arthritic knee joints was not affected by conjugation with the photosensitizer. Arthritis development was moderately delayed in mice with collagen-induced arthritis after FAP-tPDT. Conclusion Here we demonstrate the feasibility of tPDT to selectively target and kill FAP-positive fibroblasts in vitro and modulate arthritis in vivo using a mouse model of RA. This approach may have therapeutic potential in (refractory) arthritis.

Despite the clinical success of intrahepatic islet transplantation in treating type 1 diabetes, factors specific to this transplantation site hinder long-term insulin independence. The adoption of alternative, extravascular sites likely improve islet survival and function, but few locations are able to sufficiently confine islets in order to facilitate engraftment. This work describes a porous microwell scaffold with a well-defined pore size and spacing designed to guarantee islet retention at an extrahepatic transplantation site and facilitate islet revascularization. Three techniques to introduce pores were characterized: particulate leaching; solvent casting on pillared wafers; and laser drilling. Our criteria of a maximum pore diameter of 40 mm were best achieved via laser drilling. Transplantation studies in the epididymal fat of diabetic mice elucidated the potential of this porous scaffold platform to restore blood glucose levels and facilitate islet engraftment. Six out of eight mice reverted to stable normoglycemia with a mean time to remission of 6.2 +/- 3.2 days, which was comparable to that of the gold standard of renal subcapsular islet grafts. In contrast, when islets were transplanted in the epididymal fat pad without a microwell scaffold, only two out of seven mice reverted to stable normoglycemia. Detailed histological evaluation four weeks after transplantation found a comparable vascular density in scaffold-seeded islets, renal subcapsular islets and native pancreatic islets. However, the vascularization pattern in scaffold-seeded islets was more inhomogeneous compared to native pancreatic islets with a higher vascular density in the outer shell of the islets compared to the inner core. We also observed a corresponding decrease in the beta-cell density in the islet core. Despite this, our data indicated that islets transplanted in the microwell scaffold platform were able to maintain a viable beta-cell population and restore glycemic control. Furthermore, we demonstrated that the microwell scaffold platform facilitated detailed analysis at a subcellular level to correlate design parameters with functional physiological observations. (C) 2017 Elsevier Ltd. All rights reserved.

Insulinomas, neuroendocrine tumors arising from pancreatic beta cells, often show overexpression of the glucagon-like peptide-1 receptor. Therefore, imaging with glucagon-like peptide analog exendin-4 can be used for diagnosis and preoperative localization. This review presents an overview of the development and clinical implementation of exendin-based tracers for nuclear imaging, and the potential use of exendin-4 based tracers for optical imaging and therapeutic applications such as peptide receptor radionuclide therapy or targeted photodynamic therapy.Insulinomas are neuroendocrine tumors arising from the pancreatic beta cells. Currently, surgical resection is the therapy of choice, and therefore, preoperative localization of insulinomas is essential. Nearly all insulinomas show overexpression of the glucagon-like peptide-1 receptor (GLP-1R), and therefore, radiolabeled GLP-1 peptide analog exendin-4 can be used for diagnosis and preoperative localization with nuclear imaging. Here, we present an overview of the development and clinical implementation of exendin-4-based tracers for single-photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging of insulinomas, and we address the potential use of this molecule for optical imaging. At last, we discuss the possibilities and pitfalls of the use of exendin-4-based tracers for therapeutic applications such as peptide receptor radionuclide therapy (PRRT) or targeted photodynamic therapy (tPDT), giving a future outlook on the use of exendin-4 in insulinoma theranostics.

Background: Activated synovial fibroblasts (SF) contribute to rheumatoid arthritis (RA) by producing a multitude of cytokines, chemokines and proteases thus aggravating disease. Activated SF can be distinguished from quiescent fibroblasts by their expression of fibroblast activation protein (FAP). Selective depletion of FAP+ SF in inflamed joints could decrease their contribution to the arthritis process and thus constitute a viable treatment option. Further focussing of the treatment to only those areas affected by the disease can be accomplished by applying targeted photodynamic therapy (tPDT). In tPDT a light sensitive molecule, a photosensitizer (PS), is conjugated to a targeting moiety. Upon activation by light this construct produces reactive oxygen species, killing the targeted cells. Objectives: To this end we developed and tested a therapy that selectively depletes activated SF by targeting FAP on these cells with an antibody, 28H1, to which the PS, IRDye700DX, for tPDT is attached. Here we investigated the feasibility of using FAP-tPDT to induce cell death in murine arthritic synovium ex vivo. Methods: After conjugation of the IDRye700DX to 28H1 (28H1-700DX), binding and specificity of the conjugate was determined. Subsequently, tPDT efficiency in vitro was established using a 3T3 fibroblast cell line stably transfected with FAP. Biodistribution using an [111In] In-DTPA-28H1 conjugate with and without IRDye700DX was performed in healthy C57BL/6N mice as well as in C57BL/6N mice with antigen induced arthritis (AIA). Finally, the potential of FAP-tPDT to induce targeted cell death in the synovial lining was determined by treating knee joints from mice with AIA ex vivo. Results: Conjugation of IRDye700DX to the antibody did not negatively influence the immunoreactive fraction or binding capacity of the conjugate (94.7% for 28H1-700DX). 28H1-700DX was able to efficiently induce FAP-specific cell death in vitro. At 17.6 J/cm2 radiant exposure, 89.24% ± 3.67% of fibroblasts died in the group incubated with antibody compared to control incubated with buffer only (p Conclusion: Here we demonstrated the feasibility of conjugating a PS to an antibody targeting FAP on activated SF without negatively impacting the binding capacity thereof. Furthermore we showed that this construct can then be used to deliver cell specific cytotoxicity through tPDT both in vitro and ex vivo in a mouse model of arthritis. This approach may have therapeutic potential in the treatment of RA. Disclosure of Interests: Daphne Dorst: None declared, Mark Rijpkema: None declared, Mijke Buitinga: None declared, Peter Laverman: None declared, Marti Boss: None declared, Christian Klein Employee of: Roche pharmaceutics, Anne Freimoser-Grundschober Employee of: Roche pharmaceutics, Birgitte Walgreen: None declared, Peter van der Kraan: None declared, Martin Gotthardt: None declared, Marije Koenders: None declared

The 78-kDa glucose-regulated protein (GRP78) is an ubiquitously expressed endoplasmic reticulum chaperone, with a central role in maintaining protein homeostasis. Recently, an alternative role for GRP78 under stress conditions has been proposed, with stress-induced extracellular secretion and translocation of GRP78 to the cell surface where it acts as a multifunctional signaling receptor. Here we demonstrate translocation of GRP78 to the surface of human EndoC-βH1 cells and primary human islets upon cytokine exposure, in analogy to observations in rodent INS-1E and MIN6 beta cell lines. We show that GRP78 is shuttled via the anterograde secretory pathway, through the Golgi complex and secretory granules, and identify the DNAJ homolog subfamily C member 3 (DNAJC3) as a GRP78-interacting protein that facilitates its membrane translocation. Evaluation of downstream signaling pathways, using N- and C-terminal anti-GRP78 blocking antibodies, demonstrates that both GRP78 signaling domains initiate pro-apoptotic signaling cascades in beta cells. Extracellular GRP78 itself is identified as a ligand for cell surface GRP78 (sGRP78), increasing caspase 3/7 activity and cell death upon binding, which is accompanied by enhanced Chop and Bax mRNA expression. These results suggest that inflammatory cytokines induce a self-destructive pro-apoptotic feedback loop through the secretion and membrane translocation of GRP78. This proapoptotic function distinguishes the role of sGRP78 in beta cells from its reported anti-apoptotic and proliferative role in cancer cells, opening the road for the use of compounds that block sGRP78 as potential beta cell-preserving therapies in type 1 diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

68Ga-NODAGA-exendin-4 is a promising tracer for β-cell imaging using PET/CT. Possible applications include preoperative visualization of insulinomas and discrimination between focal and diffuse forms of congenital hyperinsulinism. There is also a significant role for this tracer in extending our knowledge on the role of β-cell mass in the pathophysiology of type 1 and type 2 diabetes by enabling noninvasive quantification of tracer uptake as a measure for β-cell mass. Calculating radiation doses from this tracer is important to assess its safety for use in patients (including young children) with benign diseases and healthy individuals. Methods: Six patients with hyperinsulinemic hypoglycemia were included. After intravenous injection of 100 MBq of the tracer, 4 successive PET/CT scans were obtained at 30, 60, 120, and 240 min after injection. Tracer activity in the pancreas, kidneys, duodenum, and remainder of the body were determined, and time-integrated activity coefficients for the measured organs were calculated. OLINDA/EXM software, version 1.1, was applied to calculate radiation doses using the reference adult male and female models and to estimate radiation doses to children. Results: The mean total effective dose for adults was very low (0.71 ± 0.07 mSv for a standard injected dose of 100 MBq). The organ with the highest absorbed dose was the kidney (47.3 ± 10.2 mGy/100 MBq). The estimated effective dose was 2.32 ± 0.32 mSv for an injected dose of 20 MBq in newborns. This dose decreased to 0.77 ± 0.11 mSv/20 MBq for 1-y-old children and 0.59 ± 0.05 mSv for an injected dose of 30 MBq in 5-y-old children. Conclusion: Our human PET/CT-based dosimetric calculations show that the effective radiation doses from the novel tracer 68Ga-NODAGA-exendin-4 are very low for adults and children. The doses are lower than reported for other polypeptide tracers such as somatostatin analogs (2.1–2.6 mSv/100 MBq) and are beneficial for application as a research tool, especially when repeated examinations are needed.

Career situation of first and presenting author Student for a master or a PhD. Introduction Activated synovial fibroblasts (ASF) play an important role in the pathogenesis of rheumatoid arthritis (RA). They contribute to the pro-inflammatory environment in the joint and degradation of cartilage and bone. Depleting ASF could ameliorate both these hallmarks of RA. ASF are characterized by the expression of fibroblast activation protein (FAP). Objectives Here, we investigated the potential of FAP-targeted photodynamic therapy (tPDT) using the photosensitizer IRDye700DX conjugated to the anti-FAP antibody 28 H1 (28H1–700DX) to selectively kill these cells. Methods To demonstrate the proof-of-concept of FAP-based tPDT in ex vivo human tissue, RA synovial tissue obtained during joint replacement surgery was trypsin digested and fibroblasts were cultured for at least 5 passages. Cells were incubated with or without 1 ug/well 28H1–700DX for 4 hour, washed with PBS and either exposed to 690 nm light or not exposed. A luminescent cell viability assay (CellTiter-Glo) was used to measure cell viability. In parallel, 6 mm biopsies of synovial tissue were taken and used for FAP-based tPDT (n=8 patients). They were subjected to tPDT as described above and formalin fixed after 1 hour. To measure cell death, 5 µm paraffin slices were stained for FAP, gamma-H2AX and caspase-3 expression for activated fibroblasts, DNA double-strand breaks and early apoptosis, respectively. Results FAP-tPDT performed on the cultured fibroblasts showed a light dose dependent increase in cell death when incubated with 28H1–700DX for 4 hour. Cell viability was not affected when cells were incubated with 28H1-700DX without illumination (101.37%±3.9% remaining compared to 100%±5.07% in the normalized control). Radiant exposure of 17.6 J/cm2 did not significantly decrease cell viability (6.4%±9.8% decrease, ns). Radiant exposures of 52.8, 105.6 and 158.4 J/cm2 significantly decreased cell viability (38.6%±6.9%, 67.5%±10.9% and 80.6%±5.8% respectively, p Conclusions FAP-tPDT induces cell death of FAP-positive activated fibroblasts in synovial tissue from RA patients. This is a first indication that FAP-targeted PDT can be a feasible new treatment strategy in RA. Disclosure of Interest D. Dorst: None declared, M. Rijpkema: None declared, M. Buitinga: None declared, P. Laverman: None declared, M. Brom: None declared, D. Bos: None declared, A. Freimoser-Grundschober Grant/research support from: support in kind, C. Klein Grant/research support from: support in kind, B. Walgreen: None declared, P. Van der Kraan: None declared, M. Gotthardt: None declared, M. Koenders: None declared.

Being highly expressed in insulinomas, the glucagon-like peptide-1 receptor (GLP-1R) is a potential target for diagnosis, localization and treatment with the radiolabeled GLP-1R agonist exendin. Tracer accumulation in the kidneys, however, hampers accurate diagnostic visualization of pancreatic tissue and prohibits the therapeutic application of radiolabeled exendin for beta-cell-derived tumors. Therefore, we evaluated the ability of succinylated gelatin (Gelofusine) to reduce the renal accumulation of radiolabeled exendin in humans and we performed dosimetric calculations to estimate the maximum absorbed insulinoma dose that could be achieved when exendin would be used for peptide receptor radionuclide therapy. Methods: Ten healthy volunteers received 50 MBq 111In-exendin-4, in combination with Gelofusine or saline in a crossover design. SPECT/CT images were obtained after 24 hours. The procedure was repeated three weeks later. Uptake of 111In-exendin was determined by drawing regions of interest around the kidneys and in the pancreas. Planar scintigraphic 111In-exendin images of five insulinoma patients were used for dosimetry studies estimating the maximum insulinoma absorbed dose that could be achieved without causing radiotoxicity to other organs. Results: Gelofusine reduced the renal accumulation of 111In-exendin-4 with 18.1%, whereas the pancreatic uptake remained unchanged. In 3 out of 10 subjects, the kidney uptake was reduced to such an extent that the pancreatic tail could be better discriminated from the kidney signal. Dosimetric estimations suggested that the insulinoma absorbed dose ranges from 30.3-127.8 Gy. This dose could be further increased to maximally 156.1 Gy when Gelofusine would be used. Conclusion: We have shown that Gelofusine can reduce the renal accumulation of 111In-exendin-4 in humans. This reduction does not only allow more accurate qualitative and quantitative analyses of radiolabeled exendin uptake in the tail region of the pancreas, but it also potentiates the safe delivery of a higher radiation dose to GLP-1R positive tumors for therapy. ispartof: JOURNAL OF NUCLEAR MEDICINE vol:60 issue:6 pages:812-816 ispartof: location:United States status: Published online

The β-cell has become recognized as a central player in the pathogenesis of type 1 diabetes with the generation of neoantigens as potential triggers for breaking immune tolerance. We report that posttranslationally modified glucose-regulated protein 78 (GRP78) is a novel autoantigen in human type 1 diabetes. When human islets were exposed to inflammatory stress induced by interleukin-1β, tumor necrosis factor-α, and interferon-γ, arginine residue R510 within GRP78 was converted into citrulline, as evidenced by liquid chromatography-tandem mass spectrometry. This conversion, known as citrullination, led to the generation of neoepitopes, which effectively could be presented by HLA-DRB1*04:01 molecules. With the use of HLA-DRB1*04:01 tetramers and ELISA techniques, we demonstrate enhanced antigenicity of citrullinated GRP78 with significantly increased CD4+ T-cell responses and autoantibody titers in patients with type 1 diabetes compared with healthy control subjects. Of note, patients with type 1 diabetes had a predominantly higher percentage of central memory cells and a lower percentage of effector memory cells directed against citrullinated GRP78 compared with the native epitope. These results strongly suggest that citrullination of β-cell proteins, exemplified here by the citrullination of GRP78, contributes to loss of self-tolerance toward β-cells in human type 1 diabetes, indicating that β-cells actively participate in their own demise. ispartof: DIABETES vol:67 issue:11 pages:2337-2348 ispartof: location:United States status: published

Background Activated synovial fibroblasts (SF) play an important role in the pathogenesis of rheumatoid arthritis (RA). They contribute to the pro-inflammatory environment in the joint as well as to the degradation of cartilage. Depleting SF could ameliorate both the symptoms of joint inflammation and degradation in RA. SF are characterised by the expression of Fibroblast Activation Protein (FAP). Here, we investigated the potential of photodynamic therapy (PDT) targeting FAP to selectively induce cell death in these cells as well as in synovial tissue from RA patients. In PDT, a light-sensitive molecule is delivered to a target cell and activated with light of a specific wavelength. This causes cell death through the production of reactive oxygen species. Methods The anti-FAP antibody 28 H1 was conjugated with the photosensitizer IRDye700DX (28 H1–700DX). In vitro PDT assays were performed with 3 T3 fibroblasts stably transfected with FAP. 3T3-FAP cells were incubated with 28 H1–700DX or a control conjugate for 4 hours, and exposed to varying 690 nm light exposures. Subsequently, cell viability was measured using the CellTiter-Glo assay. For ex vivo evaluation of PDT efficiency, human RA synovial tissue obtained after joint replacement surgery was processed into standardised 6 mm biopsies and used for FAP-based PDT. The biopsies were incubated with 28 H1–700DX for 4 hours, subjected to 52 J/cm2 light exposure and fixed in formalin after 1 hour. Tissue was then embedded in paraffin and stained for the presence of gH2AX and caspase 3 as indicators of DNA double-strand breaks and early apoptosis on sequential slides. The presence of FAP was also determined on subsequent slides. Results The effect of PDT was optimal at 13.7 J/cm2 light exposure to 3T3-FAP cells incubated with 6.67 pM 28 H1–700DX, which dramatically reduced cell viability with 89.27%±2.48 compared to control (p In the PDT experiment on human RA synovial biopsies, the groups incubated with 28 H1–700DX and exposed to light showed apparent cell death in the synovial tissue as evidenced by the positive staining of both the gH2AX and caspase 3 markers (figure J and K). Staining of these markers co-localised with areas of high FAP staining (figure L). This was not the case in the control samples that were not exposed to either 28 H1–700DX and/or light (figure A – I). All biopsies did show FAP staining indicating that the cell death was only achieved when the biopsies were exposed to both the antibody and the light. Conclusions We have demonstrated fibroblast-specific cell death by targeted PDT using 700DX-conjugated 28 H1. Furthermore, we demonstrated that PDT also induces cell death of FAP-positive cells in synovial tissue from RA patients, suggesting FAP-targeted PDT as a promising new tool in treating RA. Disclosure of Interest None declared

Introduction Activated synovial fibroblasts (SF) are characterised by the expression of Fibroblast Activation Protein (FAP). Here, we investigated the potential of photodynamic therapy (PDT) targeting FAP to selectively induce cell death in these cells. In PDT, a light-sensitive molecule is delivered to a target cell and activated with light of a specific wavelength. This causes cell death through the production of reactive oxygen species. Methods The anti-FAP antibody 28 H1 was conjugated with the photosensitizer IRDye700DX (28H1-700DX). In vitro PDT assays were performed with 3 T3 fibroblasts stably transfected with FAP. 3T3-FAP cells were incubated with 28H1-700DX or a control conjugate for 4 hours, and exposed to varying 690 nm light doses. Subsequently, cell viability was measured using the CellTiter-Glo assay. For in vivo biodistribution, 5 days after onset of antigen-induced arthritis (AIA) C57Bl6 mice were injected with 28H1±700 DX labelled with radioactive Indium for quantification purposes (expressed as% injected dose per gram tissue (%ID/g)). For the PDT treatment experiment, arthritic mice were injected at day 5 of AIA with 28H1-700DX or PBS and exposed to 50 or 90 J/cm2 light 24 hours post injection. Joints were isolated at day 10 for histological analysis. Results To assess PDT efficacy, we applied 13.7 J/cm2 light exposure to 3T3-FAP cells incubated with 6.67 pM 28H1-700DX, which significantly reduced cell viability (89.27%±2.48 compared to control (p Conjugating the anti-FAP antibody to 700DX changed the in vivo biodistribution of the antibody, with a higher accumulation in the liver (27,06±0,95%ID/g vs. 6,08±0,42%ID/g with control (p Conclusions We have demonstrated fibroblast-specific cell death using 700DX-conjugated 28 H1 PDT, indicating FAP-based PDT as a promising new tool in treating RA. Furthermore, we demonstrated that adding 700DX results in faster liver clearance of the antibody, but does not affect uptake in the inflamed knee joint. Future research will further elucidate the applicability of our conjugate for PDT in animal models of RA. Disclosure of interest None declared

In this issue of Diabetologia, Alavi and Werner (https://doi.org/10.1007/s00125-018-4676-1) criticise the attempts to use positron emission tomography (PET) for in vivo imaging of pancreatic beta cells, which they consider as ‘futile’. In support of this strong statement, they point out the limitations of PET imaging, which they believe render beta cell mass impossible to estimate using this method. In our view, the Alavi and Werner presentation of the technical limitations of PET imaging does not reflect the current state of the art, which leads them to questionable conclusions towards the feasibility of beta cell imaging using this approach. Here, we put forward arguments in favour of continuing the development of innovative technologies enabling in vivo imaging of pancreatic beta cells and concisely present the current state of the art regarding putative technical limitations of PET imaging. Indeed, far from being a ‘futile’ effort, we demonstrate that beta cell imaging is now closer than ever to becoming a long-awaited clinical reality., SCOPUS: no.j, info:eu-repo/semantics/published

Contains fulltext : 177862.pdf (Publisher’s version ) (Open Access) Allogeneic islet transplantation into the liver in combination with immune suppressive drug therapy is widely regarded as a potential cure for type 1 diabetes. However, the intrahepatic system is suboptimal as the concentration of drugs and nutrients there is higher compared to pancreas, which negatively affects islet function. Islet encapsulation within semipermeable membranes is a promising strategy that allows for the islet transplantation outside the suboptimal liver portal system and provides environment, where islets can perform their endocrine function. In this study, we develop a macroencapsulation device based on thin microwell membranes. The islets are seeded in separate microwells to avoid aggregation, whereas the membrane porosity is tailored to achieve sufficient transport of nutrients, glucose and insulin. The non-degradable, microwell membranes are composed of poly (ether sulfone)/polyvinylpyrrolidone and manufactured via phase separation micro molding. Our results show that the device prevents aggregation and preserves the islet's native morphology. Moreover, the encapsulated islets maintain their glucose responsiveness and function after 7 days of culture (stimulation index above 2 for high glucose stimulation), demonstrating the potential of this novel device for islet transplantation.

Pancreatic islet transplantation is a promising therapy for patients with type 1 diabetes. However, the duration of long-term graft survival is limited due to inflammatory as well as non-inflammatory processes and routine clinical tests are not suitable to monitor islet survival. 111In-exendin-SPECT (single photon emission computed tomography) is a promising method to non-invasively image islets after transplantation and has the potential to help improve the clinical outcome. Whether 111In-exendin-SPECT allows detecting small differences in beta-cell mass (BCM) and measuring the actual volume of islets that were successfully engrafted has yet to be demonstrated. Here, we evaluated the performance of 111In-exendin-SPECT using an intramuscular islet transplantation model in C3H mice. In vivo imaging of animals transplanted with 50, 100, 200, 400 and 800 islets revealed an excellent linear correlation between SPECT quantification of 111In-exendin uptake and insulin-positive area of islet transplants, demonstrating that 111In-exendin-SPECT specifically and accurately measures BCM. The high sensitivity of the method allowed measuring small differences in graft volumes, including grafts that contained less than 50 islets. The presented method is reliable, convenient and holds great potential for non-invasive monitoring of BCM after islet transplantation in humans.

Currently, radionuclide imaging after injection of radiolabeled tracers seems to be the most promising approach to noninvasively determine the beta cell mass (BCM) in vivo. Despite the limited resolution of positron emission tomography (PET) and single photon emission computed tomography (SPECT), the very high sensitivity of these imaging modalities allows detection of very low radiotracer concentrations. Since the noninvasive imaging modalities that offer the highest spatial resolution, i.e., computed tomography (CT) and magnetic resonance imaging (MRI), which are not able to resolve single islets in vivo, radionuclide imaging would be the method of choice for beta cell imaging. However, noninvasive visualization of the pancreatic beta cells is a highly challenging endeavor. The beta cells are located in the islets of Langerhans in the pancreas. The islets are small (usually between 50 and 400 μm), only account for 1–2 % of the total pancreatic mass, and are spread throughout the pancreas (Bonner-Weir 1994; Weir et al. 1990). Targeting of these small structures spread through an organ is highly challenging when compared to tumor imaging, where a focal solid mass containing the target cells is visualized. Moreover, the islets consist of several cell types (beta cells, alpha cells, delta cells, and pp cells) with the beta cells representing around 60–80 % of the islet mass, making specific visualization of beta cells even more difficult. Since PET and SPECT cannot resolve single islets, let alone beta cells, a highly beta cell-specific radiotracer is required. Only when the radiotracer specifically accumulates in beta cells and not in other cell types of the endocrine or exocrine pancreas, the accumulation of the radiotracer in the pancreas could be used as a surrogate measure of the beta cell mass. To enable this, a target specifically expressed on beta cells and an optimal respective ligand should be selected for the development of beta cell targeting radiotracers. In contrast to tumor imaging, where several targets are overexpressed, beta cells express endogenous levels of receptors leading to generally lower accumulation of radiotracers when compared to tumor cells. In this book chapter, the challenges regarding tracer development and preclinical and clinical characterization will be discussed in detail.

While subcutaneous tissue has been proposed as a clinically relevant site for pancreatic islet transplantation, a major issue of concern remains, which is its poor vascular state. In an effort to overcome this limitation, we present an efficient and reproducible method to form human composite islets (CIs) with proangiogenic cell types in a controlled manner using nonadherent agarose microwell templates. In this study, we assessed the three-dimensional structure, function, and angiogenic potential of human CIs with human mesenchymal stromal cells (hMSCs), with or without human umbilical vein endothelial cells (HUVECs), and preconditioned hMSCs (PC-hMSCs) in EGM-2 under shear stress. Distinct cellular rearrangements could be observed in CIs, but islet functionality was maintained. In vitro angiogenesis assays found significantly enhanced sprout formation in case of CIs. In particular, the number of sprouts emanating from CIs with PC-hMSCs was significantly increased compared to other conditions. Subsequent in vivo assessment confirmed the proangiogenic potential of CIs. However, in contrast to our in vitro angiogenesis assays, CIs with hMSCs and HUVECs exhibited a higher in vivo angiogenic potential compared to control islets or islets combined with hMSCs or PC-hMSCs. These findings highlight the importance and necessity of verifying in vitro studies with in vivo models to reliably predict, in this case, revascularization outcomes. Regardless, we demonstrate here the therapeutic potential of CIs with proangiogenic support cells to enhance islet revascularization at a clinically relevant, although poorly vascularized, transplantation site.

Allogeneic islet transplantation into the liver has the potential to restore normoglycemia in patients with type 1 diabetes. However, the suboptimal microenvironment for islets in the liver is likely to be involved in the progressive islet dysfunction that is often observed post-transplantation. This study validates a novel microwell scaffold platform to be used for the extrahepatic transplantation of islet of Langerhans. Scaffolds were fabricated from either a thin polymer film or an electrospun mesh of poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) block copolymer (composition: 4000PEOT30PBT70) and were imprinted with microwells, ∼400 µm in diameter and ∼350 µm in depth. The water contact angle and water uptake were 39±2° and 52.1±4.0 wt%, respectively. The glucose flux through electrospun scaffolds was three times higher than for thin film scaffolds, indicating enhanced nutrient diffusion. Human islets cultured in microwell scaffolds for seven days showed insulin release and insulin content comparable to those of free-floating control islets. Islet morphology and insulin and glucagon expression were maintained during culture in the microwell scaffolds. Our results indicate that the microwell scaffold platform prevents islet aggregation by confinement of individual islets in separate microwells, preserves the islet's native rounded morphology, and provides a protective environment without impairing islet functionality, making it a promising platform for use in extrahepatic islet transplantation. ispartof: PLOS ONE vol:8 issue:5 ispartof: location:United States status: published

68Ga-NODAGA-exendin-4 is a promising tracer for β-cell imaging using PET/CT. Possible applications include preoperative visualization of insulinomas and discrimination between focal and diffuse forms of congenital hyperinsulinism. There is also a significant role for this tracer in extending our knowledge on the role of β-cell mass in the pathophysiology of type 1 and type 2 diabetes by enabling noninvasive quantification of tracer uptake as a measure for β-cell mass. Calculating radiation doses from this tracer is important to assess its safety for use in patients (including young children) with benign diseases and healthy individuals. Methods: Six patients with hyperinsulinemic hypoglycemia were included. After intravenous injection of 100 MBq of the tracer, 4 successive PET/CT scans were obtained at 30, 60, 120, and 240 min after injection. Tracer activity in the pancreas, kidneys, duodenum, and remainder of the body were determined, and time-integrated activity coefficients for the measured organs were calculated. OLINDA/EXM software, version 1.1, was applied to calculate radiation doses using the reference adult male and female models and to estimate radiation doses to children. Results: The mean total effective dose for adults was very low (0.71 ± 0.07 mSv for a standard injected dose of 100 MBq). The organ with the highest absorbed dose was the kidney (47.3 ± 10.2 mGy/100 MBq). The estimated effective dose was 2.32 ± 0.32 mSv for an injected dose of 20 MBq in newborns. This dose decreased to 0.77 ± 0.11 mSv/20 MBq for 1-y-old children and 0.59 ± 0.05 mSv for an injected dose of 30 MBq in 5-y-old children. Conclusion: Our human PET/CT-based dosimetric calculations show that the effective radiation doses from the novel tracer 68Ga-NODAGA-exendin-4 are very low for adults and children. The doses are lower than reported for other polypeptide tracers such as somatostatin analogs (2.1-2.6 mSv/100 MBq) and are beneficial for application as a research tool, especially when repeated examinations are needed.