Oncolytic DNX-2401 Virus for Pediatric Diffuse Intrinsic Pontine Glioma.

BACKGROUND: Pediatric patients with diffuse intrinsic pontine glioma (DIPG) have a poor prognosis, with a median survival of less than 1 year. Oncolytic viral therapy has been evaluated in patients with pediatric gliomas elsewhere in the brain, but data regarding oncolytic viral therapy in patients with DIPG are lacking. METHODS: We conducted a single-center, dose-escalation study of DNX-2401, an oncolytic adenovirus that selectively replicates in tumor cells, in patients with newly diagnosed DIPG. The patients received a single virus infusion through a catheter placed in the cerebellar peduncle, followed by radiotherapy. The primary objective was to assess the safety and adverse-event profile of DNX-2401. The secondary objectives were to evaluate the effect of DNX-2401 on overall survival and quality of life, to determine the percentage of patients who have an objective response, and to collect tumor-biopsy and peripheral-blood samples for correlative studies of the molecular features of DIPG and antitumor immune responses. RESULTS: A total of 12 patients, 3 to 18 years of age, with newly diagnosed DIPG received 1×1010 (the first 4 patients) or 5×1010 (the subsequent 8 patients) viral particles of DNX-2401, and 11 received subsequent radiotherapy. Adverse events among the patients included headache, nausea, vomiting, and fatigue. Hemiparesis and tetraparesis developed in 1 patient each. Over a median follow-up of 17.8 months (range, 5.9 to 33.5), a reduction in tumor size, as assessed on magnetic resonance imaging, was reported in 9 patients, a partial response in 3 patients, and stable disease in 8 patients. The median survival was 17.8 months. Two patients were alive at the time of preparation of the current report, 1 of whom was free of tumor progression at 38 months. Examination of a tumor sample obtained during autopsy from 1 patient and peripheral-blood studies revealed alteration of the tumor microenvironment and T-cell repertoire. CONCLUSIONS: Intratumoral infusion of oncolytic virus DNX-2401 followed by radiotherapy in pediatric patients with DIPG resulted in changes in T-cell activity and a reduction in or stabilization of tumor size in some patients but was associated with adverse events. (Funded by the European Research Council under the European Union's Horizon 2020 Research and Innovation Program and others; EudraCT number, 2016-001577-33; ClinicalTrials.gov number, NCT03178032.).

Cell Type Purification by Single-Cell Transcriptome-Trained Sorting.

Much of current molecular and cell biology research relies on the ability to purify cell types by fluorescence-activated cell sorting (FACS). FACS typically relies on the ability to label cell types of interest with antibodies or fluorescent transgenic constructs. However, antibody availability is often limited, and genetic manipulation is labor intensive or impossible in the case of primary human tissue. To date, no systematic method exists to enrich for cell types without a priori knowledge of cell-type markers. Here, we propose GateID, a computational method that combines single-cell transcriptomics with FACS index sorting to purify cell types of choice using only native cellular properties such as cell size, granularity, and mitochondrial content. We validate GateID by purifying various cell types from zebrafish kidney marrow and the human pancreas to high purity without resorting to specific antibodies or transgenes.

A transcriptomic roadmap to α- and ß-cell differentiation in the embryonic pancreas.

During pancreatic development, endocrine cells appear from the pancreatic epithelium when Neurog3-positive cells delaminate and differentiate into α-, ß-, γ- and δ-cells. The mechanisms involved in this process are still incompletely understood. We characterized the temporal, lineage-specific developmental programs during pancreatic development by sequencing the transcriptome of thousands of individual pancreatic cells from E12.5 to E18.5 in mice, and identified all known cell types that are present in the embryonic pancreas, but focused specifically on α- and ß-cell differentiation by enrichment of a MIP-GFP reporter. We characterized transcriptomic heterogeneity in the tip domain based on proliferation, and characterized two endocrine precursor clusters marked by expression of Neurog3 and Fev Pseudotime analysis revealed specific branches for developing α- and ß-cells, which allowed identification of specific gene regulation patterns. These include some known and many previously unreported genes that appear to define pancreatic cell fate transitions. This resource allows dynamic profiling of embryonic pancreas development at single cell resolution and reveals novel gene signatures during pancreatic differentiation into α- and ß-cells.

Siglec-7 restores ß-cell function and survival and reduces inflammation in pancreatic islets from patients with diabetes.

Chronic inflammation plays a key role in both type 1 and type 2 diabetes. Cytokine and chemokine production within the islets in a diabetic milieu results in ß-cell failure and diabetes progression. Identification of targets, which both prevent macrophage activation and infiltration into islets and restore ß-cell functionality is essential for effective diabetes therapy. We report that certain Sialic-acid-binding immunoglobulin-like-lectins (siglecs) are expressed in human pancreatic islets in a cell-type specific manner. Siglec-7 was expressed on ß-cells and down-regulated in type 1 and type 2 diabetes and in infiltrating activated immune cells. Over-expression of Siglec-7 in diabetic islets reduced cytokines, prevented ß-cell dysfunction and apoptosis and reduced recruiting of migrating monocytes. Our data suggest that restoration of human Siglec-7 expression may be a novel therapeutic strategy targeted to both inhibition of immune activation and preservation of ß-cell function and survival.

A Single-Cell Transcriptome Atlas of the Human Pancreas.

To understand organ function, it is important to have an inventory of its cell types and of their corresponding marker genes. This is a particularly challenging task for human tissues like the pancreas, because reliable markers are limited. Hence, transcriptome-wide studies are typically done on pooled islets of Langerhans, obscuring contributions from rare cell types and of potential subpopulations. To overcome this challenge, we developed an automated platform that uses FACS, robotics, and the CEL-Seq2 protocol to obtain the transcriptomes of thousands of single pancreatic cells from deceased organ donors, allowing in silico purification of all main pancreatic cell types. We identify cell type-specific transcription factors and a subpopulation of REG3A-positive acinar cells. We also show that CD24 and TM4SF4 expression can be used to sort live alpha and beta cells with high purity. This resource will be useful for developing a deeper understanding of pancreatic biology and pathophysiology of diabetes mellitus.

Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies.

AIMS: Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. METHODS: The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, ß-ACTIN and TUBULIN) and exemplary of SIGLEC-7. RESULTS: Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible. CONCLUSIONS: This study confirms that it is not possible to mix or change sampling or extraction strategies during the same study because of large variations of RNA yield and expression levels.

Sequential intravital imaging reveals in vivo dynamics of pancreatic tissue transplanted under the kidney capsule in mice.

AIMS/HYPOTHESIS: Dynamic processes in pancreatic tissue are difficult to study. We aimed to develop an intravital imaging method to longitudinally examine engraftment, vascularisation, expansion and differentiation in mature islets or embryonic pancreases transplanted under the kidney capsule. METHODS: Isolated pancreatic islets from adult mice and murine embryonic day (E)12.5 pancreases containing fluorescent biomarkers were transplanted under the kidney capsule of immunodeficient recipient mice. Human islet cells were dispersed, transduced with a lentivirus expressing a fluorescent label and reaggregated before transplantation. Graft-containing kidneys were positioned subcutaneously and an imaging window was fitted into the skin on top of the kidney. Intravital imaging using multiphoton microscopy was performed for up to 2 weeks. Volumes of fluorescently labelled cells were determined as a measure of development and survival. RESULTS: Transplanted islets and embryonic pancreases showed good engraftment and remained viable. Engraftment and vascularisation could be longitudinally examined in murine and human islet cells. Murine islet beta cell volume was unchanged over time. Transplanted embryonic pancreases increased to up to 6.1 times of their original volume and beta cell volume increased 90 times during 2 weeks. CONCLUSIONS/INTERPRETATION: This method allows for repeated intravital imaging of grafts containing various sources of pancreatic tissue transplanted under the kidney capsule. Using fluorescent markers, dynamic information concerning engraftment or differentiation can be visualised and measured.

De Novo Prediction of Stem Cell Identity using Single-Cell Transcriptome Data.

Adult mitotic tissues like the intestine, skin, and blood undergo constant turnover throughout the life of an organism. Knowing the identity of the stem cell is crucial to understanding tissue homeostasis and its aberrations upon disease. Here we present a computational method for the derivation of a lineage tree from single-cell transcriptome data. By exploiting the tree topology and the transcriptome composition, we establish StemID, an algorithm for identifying stem cells among all detectable cell types within a population. We demonstrate that StemID recovers two known adult stem cell populations, Lgr5+ cells in the small intestine and hematopoietic stem cells in the bone marrow. We apply StemID to predict candidate multipotent cell populations in the human pancreas, a tissue with largely uncharacterized turnover dynamics. We hope that StemID will accelerate the search for novel stem cells by providing concrete markers for biological follow-up and validation.

Possible role of interleukin-1ß in type 2 diabetes onset and implications for anti-inflammatory therapy strategies.

Increasing evidence of a role of chronic inflammation in type 2 diabetes progression has led to the development of therapies targeting the immune system. We develop a model of interleukin-1ß dynamics in order to explain principles of disease onset. The parameters in the model are derived from in vitro experiments and patient data. In the framework of this model, an IL-1ß switch is sufficient and necessary to account for type 2 diabetes onset. The model suggests that treatments targeting glucose bear the potential of stopping progression from pre-diabetes to overt type 2 diabetes. However, once in overt type 2 diabetes, these treatments have to be complemented by adjuvant anti-inflammatory therapies in order to stop or decelerate disease progression. Moreover, the model suggests that while glucose-lowering therapy needs to be continued all the way, dose and duration of the anti-inflammatory therapy needs to be specifically controlled. The model proposes a framework for the discussion of clinical trial outcomes.

The DPP-4 inhibitor linagliptin restores ß-cell function and survival in human isolated islets through GLP-1 stabilization.

CONTEXT: Inhibition of dipeptidyl peptidase-4 (DPP-4) is a potent strategy to increase glucose-dependent insulinotropic polypeptide and glucagon like peptide 1 (GLP-1) induced insulin secretion in diabetes. It is important to know whether new drugs approved for the treatment of type 2 diabetes have direct effects on the ß-cell. OBJECTIVE: Herein we investigated the effect of linagliptin, a novel DPP-4 inhibitor, on ß-cell function and survival. DESIGN: Human islets were exposed to a diabetic milieu (11.1-33.3 mM glucose, 0.5 mM palmitate, the mixture of 2 ng/mL IL-1ß+1000 U/mL interferon-γ, or 50 µM H2O2) with or without 500 ng/mL IL-1 receptor antagonist (IL-1Ra) or 30-50 nM linagliptin. RESULTS: Linagliptin restored ß-cell function and turnover, which was impaired when islets were exposed to elevated glucose, palmitate, cytokines, or H2O2. Pretreatment with IL-1Ra was similarly effective, except against H2O2 treatment. Nitrotyrosine concentrations in islet lysates, an indicator of oxidative stress, were highly elevated under diabetic conditions but not in islets treated with linagliptin or IL-1Ra. Linagliptin also reduced cytokine secretion and stabilized GLP-1 in islet supernatants. CONCLUSIONS: We show that the novel DPP-4 inhibitor linagliptin protected from gluco-, lipo-, and cytokine-toxicity and stabilized active GLP-1 secreted from human islets. This provides a direct GLP-1 mediated protective effect of linagliptin on ß-cell function and survival.

Interleukin-targeted therapy for metabolic syndrome and type 2 diabetes.

Interleukin-1ß Interleukin-1ß (IL-1ß) is a key regulator of the body's inflammatory response and is produced after infection, injury, and an antigenic challenge. Cloned in 1984, the single polypeptide IL-1ß has been shown to exert numerous biological effects. It plays a role in various diseases, including autoimmune diseases such as rheumatoid arthritis, inflammatory bowel diseases, and Type 1 diabetes, as well as in diseases associated with metabolic syndrome such as atherosclerosis, chronic heart failure, and Type 2 diabetes. The macrophage is the primary source of IL-1ß, but epidermal, epithelial, lymphoid, and vascular tissues also synthesize IL-1. Recently, IL-1ß production and secretion have also been reported from pancreatic islets. Insulin-producing ß-cells ß-cells within the pancreatic islets are specifically prone to IL-ß-induced destruction and loss of function. Macrophage-derived IL-1ß production in insulin-sensitive organs leads to the progression of inflammation inflammation and induction of insulin resistance in obesity. This chapter explains the mechanisms involved in the inflammatory response during diabetes progression with specific attention to the IL-1ß signal effects influencing insulin action and insulin secretion insulin secretion . We highlight recent clinical studies, rodent and in vitro experiments with isolated islets using IL-1ß as a potential target for the therapy of Type 2 diabetes.

Neutralizing interleukin-1beta (IL-1beta) induces beta-cell survival by maintaining PDX1 protein nuclear localization.

The transcription factor PDX1 plays a critical role during ß-cell development and in glucose-induced insulin gene transcription in adult ß-cells. Acute glucose exposure leads to translocalization of PDX1 to the nucleoplasm, whereas under conditions of oxidative stress, PDX1 shuttles from the nucleus to the cytosol. Here we show that cytosolic PDX1 expression correlated with ß-cell failure in diabetes. In isolated islets from patients with type 2 diabetes and from diabetic mice, we found opposite regulation of insulin and PDX1 mRNA; insulin was decreased in diabetes, but PDX1 was increased. This suggests that elevated PDX1 mRNA levels may be insufficient to regulate insulin. In diabetic islets, PDX1 protein was localized in the cytosol, whereas in non-diabetic controls, PDX1 was in the nucleus. In contrast, overexpression of either IL-1 receptor antagonist or shuttling-deficient PDX1 restored ß-cell survival and function and PDX1 nuclear localization. Our results show that nuclear localization of PDX1 is essential for a functional ß-cell and provides a novel mechanism of the protective effect of IL-1 receptor antagonist on ß-cell survival and function.

Interleukin-1 beta targeted therapy for type 2 diabetes.

Since having been cloned in 1984, IL-1beta has been the subject of over 22,000 citations in Pubmed, among them over 800 reviews. This is because of its numerous effects. IL-1beta is a regulator of the body's inflammatory response and is produced after infection, injury, and antigenic challenge. It plays a role in various diseases, including autoimmune diseases such as rheumatoid arthritis, inflammatory bowel diseases and type 1 diabetes, as well as in diseases associated with metabolic syndrome such as atherosclerosis, chronic heart failure and type 2 diabetes. Macrophage are the primary source of IL-1, but epidermal, epithelial, lymphoid and vascular tissues also synthesize IL-1. IL-1beta production and secretion have also been reported from pancreatic islets. Insulin-producing beta-cells within pancreatic islets are specifically prone to IL-beta-induced destruction and loss of function. Macrophage-derived IL-1beta production in insulin-sensitive organs, leads to progression of inflammation and induction of insulin resistance in obesity. We summarize the mechanisms involved in inflammation and specifically the IL-1beta signals that lead to the progression of insulin resistance and diabetes. We highlight recent clinical studies and experiments in animals and isolated islets using IL-1beta as a potential target for the therapy of type 2 diabetes.