Activation of dopamine D2 receptor promotes pepsinogen secretion by suppressing somatostatin release from the mouse gastric mucosa

In vivo administration dopamine (DA) receptor (DR)-related drugs modulates gastric pepsinogen secretion. However, DRs on gastric pepsinogen-secreting chief cells and DA D2 receptor (D2R) on somatostatin-secreting D cells were subsequently acquired. In this study, we aimed to further investigate the local effect of DA on gastric pepsinogen secretion through DRs expressed on chief cells or potential D2Rs expressed on D cells. To elucidate the modulation of DRs in gastric pepsinogen secretion, immunofluorescence staining, ex vivo incubation of gastric mucosa isolated from normal and D2R-/- mice were conducted, accompanied by measurements of pepsinogen or somatostatin levels using biochemical assays or enzyme-linked immunosorbent assays. D1R, D2R, and D5R-immunoreactivity (IR) were observed on chief cells in mouse gastric mucosa. D2R-IR was widely distributed on D cells from the corpus to the antrum. Ex vivo incubation results showed that DA and the D1-like receptor agonist SKF38393 increased pepsinogen secretion, which was blocked by the D1-like receptor antagonist SCH23390. However, D2-like receptor agonist quinpirole also significantly increased pepsinogen secretion, and D2-like receptor antagonist sulpiride blocked the promotion of DA. Besides, D2-like receptors exerted an inhibitory effect on somatostatin secretion, in contrast to their effect on pepsinogen secretion. Furthermore, D2R-/- mice showed much lower basal pepsinogen secretion but significantly increased somatostatin release and an increased number of D cells in gastric mucosa. Only SKF38393, not quinpirole, increased pepsinogen secretion in D2R-/- mice. DA promotes gastric pepsinogen secretion directly through D1-like receptors on chief cells and indirectly through D2R-mediated suppression of somatostatin release.

TriCCo v1.0.0 – a cubulation-based method for computing connected components on triangular grids

We present a new method to identify connected components on a triangular grid. Triangular grids are, for example, used in atmosphere and climate models to discretize the horizontal dimension. Because they are unstructured, neighbor relations are not self-evident and identifying connected components is challenging. Our method addresses this challenge by involving the mathematical tool of cubulation. We show that cubulation allows one to map the 2-d cells of the triangular grid onto the vertices of the 3-d cells of a cubic grid. The latter is structured and so connected components can be readily identified on the cubic grid by previously developed software packages. An advantage is that the cubulation, i.e., the mapping between the triangular and cubic grids, needs to be computed only once, which should be benifical for analysing many data fields for the same grid.We further implement our method in a python package that we name TriCCo and that is made available via pypi and gitlab. We document the package, demonstrate its application using cloud data from the ICON atmosphere model, and characterize its computational performance. This shows that TriCCo is ready for triangular grids with 100,000 cells, but that its speed and memory requirements need to be improved to analyse larger grids.

The Role of Cytokines Produced via the NLRP3 Inflammasome in Mouse Macrophages Stimulated with Dental Calculus in Osteoclastogenesis

Dental calculus (DC) is a common deposit in periodontitis patients. We have previously shown that DC contains both microbial components and calcium phosphate crystals that induce an osteoclastogenic cytokine IL-1β via the NLRP3 inflammasome in macrophages. In this study, we examined the effects of cytokines produced by mouse macrophages stimulated with DC on osteoclastogenesis. The culture supernatants from wild-type (WT) mouse macrophages stimulated with DC accelerated osteoclastogenesis in RANKL-primed mouse bone marrow macrophages (BMMs), but inhibited osteoclastogenesis in RANKL-primed RAW-D cells. WT, but not NLRP3-deficient, mouse macrophages stimulated with DC produced IL-1β and IL-18 in a dose-dependent manner, indicating the NLRP3 inflammasome-dependent production of IL-1β and IL-18. Both WT and NLRP3-deficient mouse macrophages stimulated with DC produced IL-10, indicating the NLRP3 inflammasome-independent production of IL-10. Recombinant IL-1β accelerated osteoclastogenesis in both RANKL-primed BMMs and RAW-D cells, whereas recombinant IL-18 and IL-10 inhibited osteoclastogenesis. These results indicate that DC induces osteoclastogenic IL-1β in an NLRP3 inflammasome-dependent manner and anti-osteogenic IL-18 and IL-10 dependently and independently of the NLRP3 inflammasome, respectively. DC may promote alveolar bone resorption via IL-1β induction in periodontitis patients, but suppress resorption via IL-18 and IL-10 induction in some circumstances.

Architecture of the Pancreatic Islets and Endocrine Cell Arrangement in the Embryonic Pancreas of the Grass Snake (Natrix natrix L.). Immunocytochemical Studies and 3D Reconstructions

During the early developmental stages of grass snakes, within the differentiating pancreas, cords of endocrine cells are formed. They differentiate into agglomerates of large islets flanked throughout subsequent developmental stages by small groups of endocrine cells forming islets. The islets are located within the cephalic part of the dorsal pancreas. At the end of the embryonic period, the pancreatic islet agglomerates branch off, and as a result of their remodeling, surround the splenic “bulb”. The stage of pancreatic endocrine ring formation is the first step in formation of intrasplenic islets characteristics for the adult specimens of the grass snake. The arrangement of endocrine cells within islets changes during pancreas differentiation. Initially, the core of islets formed from B and D cells is surrounded by a cluster of A cells. Subsequently, A, B, and D endocrine cells are mixed throughout the islets. Before grass snake hatching, A and B endocrine cells are intermingled within the islets, but D cells are arranged centrally. Moreover, the pancreatic polypeptide (PP) cells are not found within the embryonic pancreas of the grass snake. Variation in the proportions of different cell types, depending on the part of the pancreas, may affect the islet function—a higher proportion of glucagon cells is beneficial for insulin secretion.

The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.

Phase I Trial of MB-CART2019.1, a Novel CD20 and CD19 Targeting Tandem Chimeric Antigen Receptor, in Patients with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Background CD19 redirected chimeric antigen receptor (CAR) T-cell therapy has proven efficacy in relapsed or chemotherapy-refractory (r/r) aggressive B-cell non-Hodgkin lymphoma (B-NHL). However, targeting a single B-cell antigen leads to selective pressure with potential antigen-escape and subsequent relapse. A tandem CAR targeting CD20 and CD19 (pLTG1497) has been developed to overcome this limitation. Preclinical evaluation showed improved anti-lymphoma activity. Thus, we initiated a first-in-human, phase I clinical study of autologous pLTG1497-transduced CAR T-cells (MB-CART2019.1) in r/r B-NHL patients. Aims In this phase I prospective multi-center trial (NCT03870945) we aimed to evaluate the maximum tolerated dose (MTD) of MB-CART2019.1 in adult patients with CD20 and CD19 positive r/r B-NHL as determined by dose limiting toxicities (DLTs). Methods This was a 6+3 trial design with two predefined dose levels (DL1 1x106 and DL2 2.5x106 CAR T-cells/kg body weight, respectively). Secondary endpoints included adverse events (AEs) and best overall response rate (ORR). Pharmacodynamic assessments included maximum concentration (Cmax) of CAR T-cells, time to peak expansion (tmax), AUC (d0 to d28), and persistence. MB-CART2019.1 was produced by lentiviral transduction of autologous fresh leukapheresis in the closed automated CliniMACS Prodigy® System (Miltenyi Biotec, Bergisch Gladbach, Germany). Re-infusion (Day 0) of fresh MB-CART2019.1 was scheduled 14 days after leukapheresis. Fludarabine/cyclophosphamide lymphodepleting chemotherapy was administered from day -5 to -3. Results A total of 12 patients, 6 per dose level have been enrolled and treated between February and December 2019, 5 female and 7 male patients. Median age was 72 y (range 20, 78 y), with 10 patients >65 y and 8 >70 y. Histologies included aggressive B-NHL (11) and mantle cell lymphoma (1). Five (5) patients had refractory disease at study entry and IPI was ≥3 in 7 patients. Median time from leukapheresis to re-infusion was 14 d (range 13, 14 d). No DLT and no cytokine release syndrome (CRS) or neurotoxicity grade ≥3 were observed. One patient in dose level 1 experienced a grade 5 AE, which was due to disease progression. CRS grade 1 occurred in 3/6 patients on DL1 and DL2 each, and CRS grade 2 in 2 patients on DL2. Tocilizumab was given in 1 patient. Neurotoxicity grade 1 occurred in 1 patient on DL2. The above described CRS and neurotoxicity resolved completely. Mean Cmax of MB-CART2019.1 was 348.3 cells/µl (range 3.9, 830.4 cells/µl) on DL1 and 692 cells/µl (range 5.3, 3147.8 cells/µl) on DL2. Mean tmax was 15.8 d (range 9, 21 d) on DL1 and 11.5 d (range 9, 14 d) on DL2. Mean AUC was 3155 d*cells/µl (DL1) and 4339 d*cells/µl (DL2). Persistence of MB-CART2019.1 was observed in 12/12 patients until data cut-off. Altogether 9/12 patients (ORR 75%) responded to MB-CART2019.1 with 5/12 CRs. In DL1 3/6 patients responded (ORR 50%) and in DL2 6/6 patients (ORR 100%). The 3 patients without response to MB-CART2019.1 had a mean AUC0-28 of 870 d*cells/µl, whereas mean AUC0-28 in 9 responders was 4843 d*cells/µl reflecting the correlation between the pharmacodynamic parameters and the clinical response. Responses are ongoing in 5/9 patients, with a maximum duration of response of 330 days at data cut-off. Summary/Conclusions In this first-in-human dose finding study of MB-CART2019.1 no DLT and no severe (grade ≥3) CRS or neurotoxicity were observed. Feasibility and safety were very good in this cohort of elderly r/r B-NHL patients. The sustained expansion of tandem CAR T-cells was accompanied by efficacy: all patients (6/6) treated on DL2 responded and all 5 patients with CR (5/5) are in ongoing remission by the time of this report. Based on the promising risk-to-benefit ratio observed in our study, evaluation of MB-CART2019.1 at a dose of 2.5x106/kg body weight in clinical phase II and phase III trials for patients with relapsed aggressive B-NHL is underway. Disclosures Borchmann: Miltenyi Biotec B.V. & Co. KG: Honoraria. Balke-Want:Miltenyi Biotec B.V. & Co. KG: Honoraria. Ayuk:Celgene: Consultancy, Honoraria; Kite/Gilead: Honoraria; Therakos/Mallinckrodt: Honoraria, Research Funding; Neovii: Research Funding; Novartis: Honoraria. Holtkamp:Miltenyi Biomedicine GmbH: Current Employment. Preussner:Miltenyi Biomedicine GmbH: Ended employment in the past 24 months. Zadoyan:Miltenyi Biomedicine GmbH: Current Employment. Hanssens:Miltenyi Biomedicine GmbH: Current Employment. Kaiser:Miltenyi Biotec B.V. & Co. KG: Current Employment. Jurk:Miltenyi Biotec B.V. & Co. KG: Current Employment. Bürger:Miltenyi Biotec B.V. & Co. KG: Current Employment. Schneider:Lentigen Technology Inc., A Miltenyi Company: Current Employment, Patents & Royalties. Dropulic:Lentigen Technology Inc., A Miltenyi Company: Current Employment. Overstijns:Miltenyi Biomedicine GmbH: Current Employment, Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec B.V. & Co. KG: Current Employment, Membership on an entity's Board of Directors or advisory committees. Scheid:Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; BMS: Honoraria; Amgen: Honoraria; Takeda: Honoraria, Research Funding. Holtick:Miltenyi Biotec B.V. & Co. KG: Honoraria. Miltenyi:Miltenyi Biomedicine GmbH: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Lentigen Technology Inc., A Miltenyi Company: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Miltenyi Biotec B.V. & Co. KG: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

ET-06 Suppression of glioblastoma through novel drug based on “Gene Switch Technology”

Glioblastoma (GBM) is the most common and aggressive malignancy primarily affecting adults. Despite intensive multimodal therapies, the prognosis of GBM is dismal and a novel therapy is needed. Here, we focused on RUNX, a transcription factor involved in the malignant transformation of GBM, and developed a novel Chlorambucil-conjugated PI-polyamides (Chb-M’), which “switches off” RUNX family. Chb-M’ specifically recognizes the consensus RUNX-binding sequences (TGTGGT) and alkylates it to inhibit transcription of the downstream gene of RUNX family. Chb-M’ has been shown to induce apoptosis and suppress proliferation in a variety of cancers including leukemia, and in this study, similar results were found for glioblastoma cells in vitro. Specific inhibition of RUNX1 led to a marked inhibition of tumor growth through cell cycle arrest and apoptosis. By using apoptosis array, we isolated several candidate genes which regulated by RUNX1. And some types of glioblastoma cell lines treated with Chb-M’ showed elevated expression of p21 and decreased survivin. From in silico analysis using glioma patient cohorts, survivin expression was significantly higher in GBM and it was possibly involved in maintaining the malignancy of GBM. Mechanistically survivin was found to be directly transcriptionally regulated by RUNX1 through ChIP assay and reporter assay. In addition, survivin K/D cells upregulated p21 expression and accelerated apoptosis. Taken together, we hypothesized that the RUNX1-survivin-p21 pathway can potentially be exploited in the management of this malignancy. Chb-M’ mediated regulation of RUNX1 can be a novel therapeutic strategy against GBM.

Single-cell mass cytometry reveals cross-talk between inflammation-dampening and inflammation-amplifying cells in osteoarthritic cartilage

Aging or injury leads to degradation of the cartilage matrix and the development of osteoarthritis (OA). Because of a paucity of single-cell studies of OA cartilage, little is known about the interpatient variability in its cellular composition and, more importantly, about the cell subpopulations that drive the disease. Here, we profiled healthy and OA cartilage samples using mass cytometry to establish a single-cell atlas, revealing distinct chondrocyte progenitor and inflammation-modulating subpopulations. These rare populations include an inflammation-amplifying (Inf-A) population, marked by interleukin-1 receptor 1 and tumor necrosis factor receptor II, whose inhibition decreased inflammation, and an inflammation-dampening (Inf-D) population, marked by CD24, which is resistant to inflammation. We devised a pharmacological strategy targeting Inf-A and Inf-D cells that significantly decreased inflammation in OA chondrocytes. Using our atlas, we stratified patients with OA in three groups that are distinguished by the relative proportions of inflammatory to regenerative cells, making it possible to devise precision therapeutic approaches.