Reduced expression of central innate defense molecules in pancreatic biopsies from subjects with Type 1 diabetes.

AIMS/HYPOTHESIS: Defensins play a crucial role in the innate immune system's first defense against microbial threats. However, little is known about the defensin system in the pancreas, especially in relation to Type 1 diabetes. We explore the expression of defensins in different disease stages of Type 1 diabetes and correlated obtained findings to the degree of inflammation, providing new insights into the disease and the innate immune system. MATERIAL AND METHODS: Pancreases from non-diabetic human organ donors of different age groups and donors with Type 1 diabetes with different disease duration were examined. Sections from head, body and tail of the pancreas were stained for eight different defensins and for immune cells; CD3+, CD45+, CD68+ and NES+ (granulocytes). RESULTS: In non-diabetic adult controls the level of expression for defensins Beta-1,Alpha-1, Cathelicidin and REG3A correlated with the level of inflammation. In contrast, individuals with Type  1 diabetes exhibit a reduction or absence of several central defensins regardless of the level of inflammation in their pancreas. The expression of Cathelicidin is present in neutrophils and macrophages but not in T-cells in subjects with Type 1 diabetes. CONCLUSIONS: Obtained findings suggest a pancreatic dysfunction in the innate immune system and the bridging to the adaptive system in Type 1 diabetes. Further studies on the role of the local innate immune system in Type 1 diabetes is needed.

Non-invasive PET imaging of liver fibrogenesis using a RESCA-conjugated Affibody molecule.

Non-invasive assessment of fibrogenic activity, rather than fibrotic scars, could significantly improve the management of fibrotic diseases and the development of anti-fibrotic drugs. This study explores the potential of an Affibody molecule (Z09591) labeled with the Al(18)F-restrained complexing agent (RESCA) method as a tracer for the non-invasive detection of fibrogenic cells. Z09591 was functionalized with the RESCA chelator for direct labeling with [18F]AlF. In vivo positron emission tomography/magnetic resonance imaging scans on U-87 tumor-bearing mice exhibited high selectivity of the resulting radiotracer, [18F]AlF-RESCA-Z09591, for platelet-derived growth factor receptor ß (PDGFRß), with minimal non-specific background uptake. Evaluation in a mouse model with carbon tetrachloride-induced fibrotic liver followed by a disease regression phase, revealed the radiotracer's high affinity and specificity for fibrogenic cells in fibrotic livers (standardized uptake value [SUV] 0.43 ± 0.05), with uptake decreasing during recovery (SUV 0.29 ± 0.03) (p < 0.0001). [18F]AlF-RESCA-Z09591 accurately detects PDGFRß, offering non-invasive assessment of fibrogenic cells and promising applications in precise liver fibrogenesis diagnosis, potentially contributing significantly to anti-fibrotic drug development.

Illuminating the complete ß-cell mass of the human pancreas- signifying a new view on the islets of Langerhans.

Pancreatic islets of Langerhans play a pivotal role in regulating blood glucose homeostasis, but critical information regarding their mass, distribution and composition is lacking within a whole organ context. Here, we apply a 3D imaging pipeline to generate a complete account of the insulin-producing islets throughout the human pancreas at a microscopic resolution and within a maintained spatial 3D context. These data show that human islets are far more heterogenous than previously accounted for with regards to their size distribution and cellular make up. By deep tissue 3D imaging, this in-depth study demonstrates that 50% of the human insulin-expressing islets are virtually devoid of glucagon-producing α-cells, an observation with significant implications for both experimental and clinical research.

Assisting the implementation of screening for type 1 diabetes by using artificial intelligence on publicly available data.

The type 1 diabetes community is coalescing around the benefits and advantages of early screening for disease risk. To be accepted by healthcare providers, regulatory authorities and payers, screening programmes need to show that the testing variables allow accurate risk prediction and that individualised risk-informed monitoring plans are established, as well as operational feasibility, cost-effectiveness and acceptance at population level. Artificial intelligence (AI) has the potential to contribute to solving these issues, starting with the identification and stratification of at-risk individuals. ASSET (AI for Sustainable Prevention of Autoimmunity in the Society; www.asset.healthcare ) is a public/private consortium that was established to contribute to research around screening for type 1 diabetes and particularly to how AI can drive the implementation of a precision medicine approach to disease prevention. ASSET will additionally focus on issues pertaining to operational implementation of screening. The authors of this article, researchers and clinicians active in the field of type 1 diabetes, met in an open forum to independently debate key issues around screening for type 1 diabetes and to advise ASSET. The potential use of AI in the analysis of longitudinal data from observational cohort studies to inform the design of improved, more individualised screening programmes was also discussed. A key issue was whether AI would allow the research community and industry to capitalise on large publicly available data repositories to design screening programmes that allow the early detection of individuals at high risk and enable clinical evaluation of preventive therapies. Overall, AI has the potential to revolutionise type 1 diabetes screening, in particular to help identify individuals who are at increased risk of disease and aid in the design of appropriate follow-up plans. We hope that this initiative will stimulate further research on this very timely topic.

Noninvasive PET Detection of CD69-Positive Immune Cells Before Signs of Clinical Disease in Inflammatory Arthritis.

Rheumatoid arthritis (RA) is the most common inflammatory joint disease, and early diagnosis is key for effective disease management. CD69 is one of the earliest cell surface markers seen at the surface of activated immune cells, and CD69 is upregulated in synovial tissue in patients with active RA. In this study, we evaluated the performance of a CD69-targeting PET agent, [68Ga]Ga-DOTA-ZCAM241, for early disease detection in a model of inflammatory arthritis. Methods: A model of inflammatory arthritis was induced by transferring splenocytes from KRN T-cell receptor transgenic B6 mice into T-cell-deficient I-Ag7 major histocompatibility complex class II-expressing recipient mice. The mice were examined longitudinally by [68Ga]Ga-DOTA-ZCAM241 PET/CT before and 3, 7, and 12 d after induction of arthritis. Disease progression was monitored by clinical parameters, including measuring body weight and scoring the swelling of the paws. The uptake of [68Ga]Ga-DOTA-ZCAM241 in the paws was analyzed and expressed as SUVmean Tissue biopsy samples were analyzed for CD69 expression by flow cytometry or immunostaining for a histologic correlate. A second group of mice was examined by a nonbinding, size-matched Affibody molecule as the control. Results: Clinical symptoms appeared 5-7 d after induction of arthritis. The uptake of [68Ga]Ga-DOTA-ZCAM241 in the joints was negligible at baseline but increased gradually after disease induction. An elevated PET signal was found on day 3, before the appearance of clinical symptoms. The uptake of [68Ga]Ga-DOTA-ZCAM241 correlated with the clinical score and disease severity. The presence of CD69-positive cells in the joints and lymph nodes was confirmed by flow cytometry and immunostaining. The uptake of the nonbinding tracer that was the negative control also increased gradually with disease progression, although to a lesser extent than with [68Ga]Ga-DOTA-ZCAM241 Conclusion: The uptake of [68Ga]Ga-DOTA-ZCAM241 in the inflamed joints preceded the clinical symptoms in the KRN T-cell transfer model of inflammatory arthritis, in accordance with immunostaining for CD69. [68Ga]Ga-DOTA-ZCAM241 is thus a promising PET imaging marker of activated immune cells in tissue during RA onset.

Outcome after total pancreatectomy with islet autotransplantation: A European single-center study.

BACKGROUND AND AIMS: Chronic pancreatitis may cause intractable abdominal pain, with total pancreatectomy sometimes being the last resort. To mitigate the subsequent diabetes, total pancreatectomy can be followed by islet autotransplantation (TP-IAT). The primary aim of this study was to assess the outcomes in patients undergoing TP-IAT at Karolinska University Hospital with respect to safety, postoperative complications, and islet graft function. A secondary aim was to compare liver to skeletal muscle as autotransplantation sites. METHODS: Single-center observational cohort study on patients undergoing TP-IAT. Islets were transplanted either into the liver or skeletal muscle. Data on baseline characteristics and pretransplantory conditions were collected. Outcome measures included mortality and major postoperative complications as well as the glycemic measures: insulin use, fasting C-peptide, and HbA1c. RESULTS: Between 2004 and 2020, 24 patients underwent TP-IAT. Islets were transplanted into the liver in 9 patients and into skeletal muscle in 15 patients. There was no 90-day mortality, and major complications (Clavien-Dindo ⩾IIIa) occurred in 26.7%, all related to the procedure of total pancreatectomy. Fasting C-peptide could be detected postoperatively, with higher levels in patients receiving islet autotransplantation into the liver (p = 0.006). Insulin independence was not achieved, although insulin doses at last follow-up were significantly lower in patients receiving islet autotransplantation into the liver compared to skeletal muscle (p = 0.036). CONCLUSION: TP-IAT is safe and associated with tolerable risk, the component of islet autotransplantation being seemingly harmless. Although islet grafts maintain some endocrine function, insulin independence should not be expected. Regarding islet autotransplantation sites, the liver seems superior to skeletal muscle. CLINICAL TRIAL REGISTRATION: Not applicable.

Preclinical evaluation of Affibody molecule for PET imaging of human pancreatic islets derived from stem cells.

BACKGROUND: Beta-cell replacement methods such as transplantation of isolated donor islets have been proposed as a curative treatment of type 1 diabetes, but widespread application is challenging due to shortages of donor tissue and the need for continuous immunosuppressive treatments. Stem-cell-derived islets have been suggested as an alternative source of beta cells, but face transplantation protocols optimization difficulties, mainly due to a lack of available methods and markers to directly monitor grafts survival, as well as their localization and function. Molecular imaging techniques and particularly positron emission tomography has been suggested as a tool for monitoring the fate of islets after clinical transplantation. The integral membrane protein DGCR2 has been demonstrated to be a potential pancreatic islet biomarker, with specific expression on insulin-positive human embryonic stem-cell-derived pancreatic progenitor cells. The candidate Affibody molecule ZDGCR2:AM106 was radiolabeled with fluorine-18 using a novel click chemistry-based approach. The resulting positron emission tomography tracer [18F]ZDGCR2:AM106 was evaluated for binding to recombinant human DGCR2 and cryosections of stem-cell-derived islets, as well as in vivo using an immune-deficient mouse model transplanted with stem-cell-derived islets. Biodistribution of the [18F]ZDGCR2:AM106 was also assessed in healthy rats and pigs. RESULTS: [18F]ZDGCR2:AM106 was successfully synthesized with high radiochemical purity and yield via a pretargeting approach. [18F]ZDGCR2:AM106 retained binding to recombinant human DCGR2 as well as to cryosectioned stem-cell-derived islets, but in vivo binding to native pancreatic tissue in both rat and pig was low. However, in vivo uptake of [18F]ZDGCR2:AM106 in stem-cell-derived islets transplanted in the immunodeficient mice was observed, albeit only within the early imaging frames after injection of the radiotracer. CONCLUSION: Targeting of DGCR2 is a promising approach for in vivo detection of stem-cell-derived islets grafts by molecular imaging. The synthesis of [18F]ZDGCR2:AM106 was successfully performed via a pretargeting method to label a site-specific covalently bonded fluorine-18 to the Affibody molecule. However, the rapid washout of [18F]ZDGCR2:AM106 from the stem-cell-derived islets graft indicates that dissociation kinetics can be improved. Further studies using alternative binders of similar classes with improved binding potential are warranted.

Imaging of fibrogenesis in the liver by [18F]TZ-Z09591, an Affibody molecule targeting platelet derived growth factor receptor ß.

BACKGROUND: Platelet-derived growth factor receptor beta (PDGFRß) is a receptor overexpressed on activated hepatic stellate cells (aHSCs). Positron emission tomography (PET) imaging of PDGFRß could potentially allow the quantification of fibrogenesis in fibrotic livers. This study aims to evaluate a fluorine-18 radiolabeled Affibody molecule ([18F]TZ-Z09591) as a PET tracer for imaging liver fibrogenesis. RESULTS: In vitro specificity studies demonstrated that the trans-Cyclooctenes (TCO) conjugated Z09591 Affibody molecule had a picomolar affinity for human PDGFRß. Biodistribution performed on healthy rats showed rapid clearance of [18F]TZ-Z09591 through the kidneys and low liver background uptake. Autoradiography (ARG) studies on fibrotic livers from mice or humans correlated with histopathology results. Ex vivo biodistribution and ARG revealed that [18F]TZ-Z09591 binding in the liver was increased in fibrotic livers (p = 0.02) and corresponded to binding in fibrotic scars. CONCLUSIONS: Our study highlights [18F]TZ-Z09591 as a specific tracer for fibrogenic cells in the fibrotic liver, thus offering the potential to assess fibrogenesis clearly.

[18F]MK-7246 for Positron Emission Tomography Imaging of the Beta-Cell Surface Marker GPR44.

The progressive loss of beta-cell mass is a hallmark of diabetes and has been suggested as a complementary approach to studying the progression of diabetes in contrast to the beta-cell function. Non-invasive nuclear medicinal imaging techniques such as Positron Emission Tomography using radiation emitting tracers have thus been suggested as more viable methodologies to visualize and quantify the beta-cell mass with sufficient sensitivity. The transmembrane G protein-coupled receptor GPR44 has been identified as a biomarker for monitoring beta-cell mass. MK-7246 is a GPR44 antagonist that selectively binds to GPR44 with high affinity and good pharmacokinetic properties. Here, we present the synthesis of MK-7246, radiolabeled with the positron emitter fluorine-18 for preclinical evaluation using cell lines, mice, rats and human pancreatic cells. Here, we have described a synthesis and radiolabeling method for producing [18F]MK-7246 and its precursor compound. Preclinical assessments demonstrated the strong affinity and selectivity of [18F]MK-7246 towards GPR44. Additionally, [18F]MK-7246 exhibited excellent metabolic stability, a fast clearance profile from blood and tissues, qualifying it as a promising radioactive probe for GPR44-directed PET imaging.

PET Imaging of Neutrophil Elastase with 11C-GW457427 in Acute Respiratory Distress Syndrome in Pigs.

Today, there is a lack of clinically available imaging techniques to detect and quantify specific immune cell populations. Neutrophils are one of the first immune cells at the site of inflammation, and they secrete the serine protease neutrophil elastase (NE), which is crucial in the fight against pathogens. However, the prolonged lifespan of neutrophils increases the risk that patients will develop severe complications, such as acute respiratory distress syndrome (ARDS). Here, we evaluated the novel radiolabeled NE inhibitor 11C-GW457427 in a pig model of ARDS, for detection and quantification of neutrophil activity in the lungs. Methods: ARDS was induced by intravenous administration of oleic acid to 5 farm pigs, and 4 were considered healthy controls. The severity of ARDS was monitored by clinical parameters of lung function and plasma biomarkers. Each pig was studied with 11C-GW457427 and PET/CT, before and after pretreatment with the NE inhibitor GW311616 to determine in vivo binding specificity. PET image data were analyzed as SUVs and correlated with immunohistochemical staining for NE in biopsies. Results: The binding of 11C-GW457427 was increased in pig lungs with induced ARDS (median SUVmean, 1.91; interquartile range [IQR], 1.67-2.55) compared with healthy control pigs (P < 0.05 and P = 0.03, respectively; median SUVmean, 1.04; IQR, 0.66-1.47). The binding was especially strong in lung regions with high levels of NE and ongoing inflammation, as verified by immunohistochemistry. The binding was successfully blocked by pretreatment of an NE inhibitor drug, which demonstrated the in vivo specificity of 11C-GW457427 (P < 0.05 and P = 0.04, respectively; median SUVmean, 0.60; IQR, 0.58-0.77). The binding in neutrophil-rich tissues such as bone marrow (P < 0.05 and P = 0.04, respectively; baseline median SUVmean, 5.01; IQR, 4.48-5.49; block median SUVmean, 1.57; IQR, 0.95-1.85) and spleen (median SUVmean, 2.14; IQR, 1.19-2.36) was also high in all pigs. Conclusion: 11C-GW457427 binds to NE in a porcine model of oleic acid-induced lung inflammation in vivo, with a specific increase in regional lung, bone marrow, and spleen SUV. 11C-GW457427 is a promising tool for localizing, tracking, and quantifying neutrophil-facilitated inflammation in clinical diagnostics and drug development.

Organ-specific metabolic pathways distinguish prediabetes, type 2 diabetes, and normal tissues.

Environmental and genetic factors cause defects in pancreatic islets driving type 2 diabetes (T2D) together with the progression of multi-tissue insulin resistance. Mass spectrometry proteomics on samples from five key metabolic tissues of a cross-sectional cohort of 43 multi-organ donors provides deep coverage of their proteomes. Enrichment analysis of Gene Ontology terms provides a tissue-specific map of altered biological processes across healthy, prediabetes (PD), and T2D subjects. We find widespread alterations in several relevant biological pathways, including increase in hemostasis in pancreatic islets of PD, increase in the complement cascade in liver and pancreatic islets of PD, and elevation in cholesterol biosynthesis in liver of T2D. Our findings point to inflammatory, immune, and vascular alterations in pancreatic islets in PD that are hypotheses to be tested for potential contributions to hormonal perturbations such as impaired insulin and increased glucagon production. This multi-tissue proteomic map suggests tissue-specific metabolic dysregulations in T2D.

Altered microvasculature in pancreatic islets from subjects with type 1 diabetes.

AIMS: The transcriptome of different dissociated pancreatic islet cells has been described in enzymatically isolated islets in both health and disease. However, the isolation, culturing, and dissociation procedures likely affect the transcriptome profiles, distorting the biological conclusions. The aim of the current study was to characterize the cells of the islets of Langerhans from subjects with and without type 1 diabetes in a way that reflects the in vivo situation to the highest possible extent. METHODS: Islets were excised using laser capture microdissection directly from frozen pancreatic tissue sections obtained from organ donors with (n = 7) and without (n = 8) type 1 diabetes. Transcriptome analysis of excised samples was performed using AmpliSeq. Consecutive pancreatic sections were used to estimate the proportion of beta-, alpha-, and delta cells using immunofluorescence and to examine the presence of CD31 positive endothelial regions using immunohistochemistry. RESULTS: The proportion of beta cells in islets from subjects with type 1 diabetes was reduced to 0% according to both the histological and transcriptome data, and several alterations in the transcriptome were derived from the loss of beta cells. In total, 473 differentially expressed genes were found in the islets from subjects with type 1 diabetes. Functional enrichment analysis showed that several of the most upregulated gene sets were related to vasculature and angiogenesis, and histologically, vascular density was increased in subjects with type 1 diabetes. Downregulated in type 1 diabetes islets was the gene set epithelial mesenchymal transition. CONCLUSION: A number of transcriptional alterations are present in islets from subjects with type 1 diabetes. In particular, several gene sets related to vasculature and angiogenesis are upregulated and there is an increased vascular density, suggesting an altered microvasculature in islets from subjects with type 1 diabetes. By studying pancreatic islets extracted directly from snap-frozen pancreatic tissue, this study reflects the in vivo situation to a high degree and gives important insights into islet pathophysiology in type 1 diabetes.

Islet Gene View-a tool to facilitate islet research.

Characterization of gene expression in pancreatic islets and its alteration in type 2 diabetes (T2D) are vital in understanding islet function and T2D pathogenesis. We leveraged RNA sequencing and genome-wide genotyping in islets from 188 donors to create the Islet Gene View (IGW) platform to make this information easily accessible to the scientific community. Expression data were related to islet phenotypes, diabetes status, other islet-expressed genes, islet hormone-encoding genes and for expression in insulin target tissues. The IGW web application produces output graphs for a particular gene of interest. In IGW, 284 differentially expressed genes (DEGs) were identified in T2D donor islets compared with controls. Forty percent of DEGs showed cell-type enrichment and a large proportion significantly co-expressed with islet hormone-encoding genes; glucagon (<i>GCG</i>, 56%), amylin (<i>IAPP</i>, 52%), insulin (<i>INS</i>, 44%), and somatostatin (<i>SST</i>, 24%). Inhibition of two DEGs, <i>UNC5D</i> and <i>SERPINE2</i>, impaired glucose-stimulated insulin secretion and impacted cell survival in a human ß-cell model. The exploratory use of IGW could help designing more comprehensive functional follow-up studies and serve to identify therapeutic targets in T2D.

Alternative splicing encodes functional intracellular CD59 isoforms that mediate insulin secretion and are down-regulated in diabetic islets.

Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of ß-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse ß-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed non­GPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.

PET-CT imaging of pulmonary inflammation using [68Ga]Ga-DOTA-TATE.

PURPOSE: In the characterization of severe lung diseases, early detection of specific inflammatory cells could help to monitor patients' response to therapy and increase chances of survival. Macrophages contribute to regulating the resolution and termination of inflammation and have increasingly been of interest for targeted therapies. [68Ga]Ga-DOTA-TATE is an established clinical radiopharmaceutical targeting somatostatin receptor subtype 2 (SSTR 2). Since activated macrophages (M1) overexpress SSTR 2, the aim of this study was to investigate the applicability of [68Ga]Ga-DOTA-TATE for positron emission tomography (PET) imaging of M1 macrophages in pulmonary inflammation. METHODS: Inflammation in the pig lungs was induced by warm saline lavage followed by injurious ventilation in farm pigs (n = 7). Healthy pigs (n = 3) were used as control. A 60-min dynamic PET scan over the lungs was performed after [68Ga]Ga-DOTA-TATE injection and [18F]FDG scan was executed afterward for comparison. The uptake of both tracers was assessed as mean standardized uptake values (SUVmean) 30-60-min post-injection. The PET scans were followed by computed tomography (CT) scans, and the Hounsfield units (HU) were quantified of the coronal segments. Basal and apical segments of the lungs were harvested for histology staining. A rat lung inflammation model was also studied for tracer specificity using lipopolysaccharides (LPS) by oropharyngeal aspiration. Organ biodistribution, ex vivo autoradiography (ARG) and histology samples were conducted on LPS treated, octreotide induced blocking and control healthy rats. RESULTS: The accumulation of [68Ga]Ga-DOTA-TATE on pig lavage model was prominent in the more severely injured dorsal segments of the lungs (SUVmean = 0.91 ± 0.56), compared with control animals (SUVmean = 0.27 ± 0.16, p < 0.05). The tracer uptake corresponded to the damaged areas assessed by CT and histology and were in line with HU quantification. The [68Ga]Ga-DOTA-TATE uptake in LPS treated rat lungs could be blocked and was significantly higher compared with control group. CONCLUSION: The feasibility of the noninvasive assessment of tissue macrophages using [68Ga]Ga-DOTA-TATE/PET was demonstrated in both porcine and rat lung inflammation models. [68Ga]Ga-DOTA-TATE has a great potential to be used to study the role and presence of macrophages in humans in fight against severe lung diseases.

Autologous regulatory T cells in clinical intraportal allogenic pancreatic islet transplantation.

Allogeneic islet transplantation in type 1 diabetes requires lifelong immunosuppression to prevent graft rejection. This medication can cause adverse effects and increases the susceptibility for infections and malignancies. Adoptive therapies with regulatory T cells (Tregs) have shown promise in reducing the need for immunosuppression in human transplantation settings but have previously not been evaluated in islet transplantation. In this study, five patients with type 1 diabetes undergoing intraportal allogeneic islet transplantation were co-infused with polyclonal autologous Tregs under a standard immunosuppressive regimen. Patients underwent leaukapheresis from which Tregs were purified by magnetic-activated cell sorting (MACS) and cryopreserved until transplantation. Dose ranges of 0.14-1.27 × 106 T cells per kilo bodyweight were transplanted. No negative effects were seen related to the Treg infusion, regardless of cell dose. Only minor complications related to the immunosuppressive drugs were reported. This first-in-man study of autologous Treg infusion in allogenic pancreatic islet transplantation shows that the treatment is safe and feasible. Based on these results, future efficacy studies will be developed under the label of advanced therapeutic medical products (ATMP), using modified or expanded Tregs with the aim of minimizing the need for chronic immunosuppressive medication in islet transplantation.

Discovery, optimization and biodistribution of an Affibody molecule for imaging of CD69.

Due to the wide scale of inflammatory processes in different types of disease, more sensitive and specific biomarkers are required to improve prevention and treatment. Cluster of differentiation 69 (CD69) is one of the earliest cell surface proteins expressed by activated leukocytes. Here we characterize and optimize potential new imaging probes, Affibody molecules targeting CD69 for imaging of activated immune cells. Analysis of candidates isolated in a previously performed selection from a Z variant E. coli library to the recombinant extracellular domain of human CD69, identified one cross-reactive Z variant with affinity to murine and human CD69. Affinity maturation was performed by randomization of the primary Z variant, followed by selections from the library. The resulting Z variants were evaluated for affinity towards human and murine CD69 and thermal stability. The in vivo biodistribution was assessed by SPECT/CT in rats following conjugation of the Z variants by a DOTA chelator and radiolabeling with Indium-111. A primary Z variant with a Kd of approximately 50 nM affinity to human and murine CD69 was identified. Affinity maturation generated 5 additional Z variants with improved or similar affinity. All clones exhibited suitable stability. Radiolabeling and in vivo biodistribution in rat demonstrated rapid renal clearance for all variants, while the background uptake and washout varied. The variant ZCD69:4 had the highest affinity for human and murine CD69 (34 nM) as well as the lowest in vivo background binding. In summary, we describe the discovery, optimization and evaluation of novel Affibody molecules with affinity for CD69. Affibody molecule ZCD69:4 is suitable for further development for imaging of activated immune cells.

3D imaging of human organs with micrometer resolution - applied to the endocrine pancreas.

The possibility to quantitatively study specific molecular/cellular features of complete human organs with preserved spatial 3D context would have widespread implications for pre-clinical and clinical medicine. Whereas optical 3D imaging approaches have experienced a formidable revolution, they have remained limited due to current incapacities in obtaining specific labelling within large tissue volumes. We present a simple approach enabling reconstruction of antibody labeled cells within entire human organs with preserved organ context. We demonstrate the utility of the approach by providing volumetric data and 3D distribution of hundreds of thousands of islets of Langerhans within the human pancreas. By assessments of pancreata from non-diabetic and type 2 diabetic individuals, we display previously unrecognized features of the human islet mass distribution and pathology. As such, this method may contribute not only in unraveling new information of the pancreatic anatomy/pathophysiology, but it may be translated to essentially any antibody marker or organ system.

Adenoviral CD40 Ligand Immunotherapy in 32 Canine Malignant Melanomas-Long-Term Follow Up.

Malignant melanoma is a serious disease in both humans and dogs, and the high metastatic potential results in poor prognosis for many patients. Its similarities with human melanoma make spontaneous canine melanoma an excellent model for comparative studies of novel therapies and tumor biology. Gene therapy using adenoviruses encoding the immunostimulatory gene CD40L (AdCD40L) has shown promise in initial clinical trials enrolling human patients with various malignancies including melanoma. We report a study of local AdCD40L treatment in 32 cases of canine melanoma (23 oral, 5 cutaneous, 3 ungual and 1 conjunctival). Eight patients were World Health Organization (WHO) stage I, 9 were stage II, 12 stage III, and 3 stage IV. One to six intratumoral injections of AdCD40L were given every seven days, combined with cytoreductive surgery in 20 cases and only immunotherapy in 12 cases. Tumor tissue was infiltrated with T and B lymphocytes after treatment, suggesting immune stimulation. The best overall response based on result of immunotherapy included 7 complete responses, 5 partial responses, 5 stable and 2 progressive disease statuses according to the World Health Organization response criteria. Median survival was 285 days (range 20-3435 d). Our results suggest that local AdCD40L therapy is safe and could have beneficial effects in dogs, supporting further treatment development. Clinical translation to human patients is ongoing.

Characterisation of enterovirus RNA detected in the pancreas and other specimens of live patients with newly diagnosed type 1 diabetes in the DiViD study.

AIMS/HYPOTHESIS: The Diabetes Virus Detection (DiViD) study is the first study to laparoscopically collect pancreatic tissue and purified pancreatic islets together with duodenal mucosa, serum, peripheral blood mononuclear cells (PBMCs) and stools from six live adult patients (age 24-35 years) with newly diagnosed type 1 diabetes. The presence of enterovirus (EV) in the pancreatic islets of these patients has previously been reported. METHODS: In the present study we used reverse transcription quantitative real-time PCR (RT-qPCR) and sequencing to characterise EV genomes present in different tissues to understand the nature of infection in these individuals. RESULTS: All six patients were found to be EV-positive by RT-qPCR in at least one of the tested sample types. Four patients were EV-positive in purified islet culture medium, three in PBMCs, one in duodenal biopsy and two in stool, while serum was EV-negative in all individuals. Sequencing the 5' untranslated region of these EVs suggested that all but one belonged to enterovirus B species. One patient was EV-positive in all these sample types except for serum. Sequence analysis revealed that the virus strain present in the isolated islets of this patient was different from the strain found in other sample types. None of the islet-resident viruses could be isolated using EV-permissive cell lines. CONCLUSIONS/INTERPRETATION: EV RNA can be frequently detected in various tissues of patients with type 1 diabetes. At least in some patients, the EV strain in the pancreatic islets may represent a slowly replicating persisting virus.