Downregulation of ephrin-B1 is a critical event of podocyte injury

Proteinuria in several glomerular diseases results from dysfunction of the slit diaphragm, a cell-cell junction of glomerular epithelial cells (podocytes). Ephrin-B1 and its related molecule NHERF 2 are novel essential components of the slit diaphragm. Ephrin-B1 interacts with nephrin via the extra-cellular domain and interacts with NHERF2 via the cytoplasmic site. In the proteinuric state induced by the stimulation to nephrin, nephrin and ephrin-B1 are phosphorylated, and NHERF2 is de- phosphorylated and consequent disruption of the linkage and downregulation of nephrin, ephrin-B1 and NHERF2 are a critical pathogenic event of podocyte injury. Keywords: Podocyte; Slit Diaphragm; Ephrin-B1; Nephrin; NHERF2

Loss of Soluble (Pro)renin Receptor Attenuates Angiotensin-II Induced Hypertension and Renal Injury

Rationale: Cleavage of the extra-cellular domain of the (pro)renin receptor (PRR) yields a soluble fragment (sPRR). Although changes in plasma sPRR levels have been reported in hypertension, the causal role of sPRR in blood pressure (BP) regulation is unknown. Objective: Determine the role of sPRR in BP regulation at baseline and following Ang-II induced hypertension. Methods and Results: CRISPR-Cas9 was used to mutate the cleavage site of the PRR such that sPRR is not generated. Because the gene encoding PRR is on the X-chromosome and male mutant sPRR mice are infertile, only male mice were studied. Mutant sPRR mice had virtually undetectable plasma sPRR levels compared to littermate controls. Mutant sPRR mice had normal survival and development and no apparent histological abnormalities in the kidney, heart or aorta despite lower body weight. During normal Na+ intake, no differences in food or water intake, urinary water or Na+ excretion, or acid-base status were observed between control and mutant sPRR mice. Compared to controls, mutant sPRR mice had lower BP at baseline and an attenuated hypertensive response to 2 weeks of Ang-II infusion (400 ng/kg/min) which was partially reversed by infusion of mouse recombinant sPRR. Mutant sPRR mice also had lower albuminuria, renal tubular injury and oxidative stress relative to control mice post Ang-II infusion. Further, mesenteric arteries from mutant sPRR mice displayed reduced Ang-II-induced vasocontraction and greater acetylcholine, but not sodium nitroprusside, evoked vasorelaxation under baseline conditions. Conclusions: Loss of sPRR reduces BP at baseline and decreases Ang-II induced hypertension and renal injury. These effects of sPRR loss are associated with greater endothelium-dependent but not independent vasorelaxation of resistance-sized arteries.

89 First-in-human CAR T targets cleaved MUC1, the growth factor receptor form

BackgroundMinerva Biotechnologies has opened a Phase I 1st-in-human CAR T clinical trial, NCT-04020575, for metastatic breast cancers at the Fred Hutchinson Cancer Research Center. huMNC2-CAR44 targets the truncated extra cellular domain of MUC1* (muk 1 star), which is the transmembrane cleavage product that remains after MUC1 is cleaved and the tandem repeat domain is shed from the cancer cells. No therapeutic that targets MUC1* has ever been tested in humans. All previous, failed attempts to therapeutically target MUC1 have targeted the tandem repeat domains, which are cleaved and shed from the surface of cancer cells. MUC1 cleavage increases as tumor stage increases. Cleavage and shedding of the tandem repeat domain unmasks an ectopic binding site for onco-embryonic growth factor NME7AB. The antibody fragment that targets the CAR to the tumor competes with NME7AB for binding to this same ectopic site. MUC1* growth factor receptor is activated when onco-embryonic growth factor NME7AB dimerizes its truncated extra cellular domain.MethodsAutologous huMNC2-CAR44 T cells undergo a short 11-day manufacturing process, which includes an antigen stimulation step and preserves many of the cells in the naïve and central memory state. Patients are pre-treated with standard Cy-Flu lymphodepletion. Dose escalation phase is standard 3 × 3 with a starting dose 3.3 × 10e5 CAR T cells and going up to 1.0 × 10e7 cells. Patients are eligible if biopsy is greater than or equal to 30% reactive with MNC2 in a CLIA validated diagnostic assay.ResultsIn vitro, huMNC2-CAR44 T cells killed cancer cells, but not non-cancer cells even if they expressed MUC1 or a normal form of cleaved MUC1. In NSG mice (n>300), huMNC2-CAR44 T cells eliminated MUC1* positive tumors from implanted breast cancer cells. A single CAR T cell injection eliminated tumors for 100 days; control animals had to be sacrificed at Day 20. Further, huMNC2-CAR44 T cell mediated killing increased as MUC1* density increased. In tissue micro array studies, huMNC2-scFv recognized 95% of breast cancers, across all subtypes, 83% ovarian, 78% pancreatic and 71% of lung cancers. huMNC2-scFv showed almost no binding to normal tissues and no staining of critical organs. Although patient recruitment has been slowed by COVID-19, preliminary results indicate CAR T cell expansion and possible efficacy.ConclusionsPreliminary results show that patients experienced robust CAR T cell expansion with CAR-positive T cells persisting at Day 60 post huMNC2-CAR44 T cell treatment. Possible signs of efficacy were measured.Trial RegistrationNCT04020575

Mechanism of cell polarisation and first lineage segregation in the human embryo

The formation of differential cell lineages in the mammalian blastocyst from the totipotent zygote is crucial for implantation and the success of the whole pregnancy. The first lineage segregation generates the polarised trophectoderm (TE) tissue, which forms the placenta, and the apolar inner cell mass (ICM), which mainly gives rise to all foetal tissues and also the yolk sac1–3. The mechanism underlying this cell fate segregation has been extensively studied in the mouse embryo4,5. However, when and how it takes place in the human embryo remains unclear. Here, using time-lapse imaging and 325 surplus human embryos, we provide a detailed characterisation of morphological events and transcription factor expression and localisation to understand how they lead to the first lineage segregation in human embryogenesis. We show that the first lineage segregation of the human embryo is triggered by cell polarisation that occurs at the 8-cell stage in two sequential steps. In the first step, F-actin becomes apically polarised concomitantly with embryo compaction. In the second step, the Par complex becomes polarised to form the apical cellular domain. Mechanistically, we show that activation of Phospholipase C (PLC) triggers actin polarisation and is therefore essential for apical domain formation, as is the case in mouse embryos6. Finally, we show that, in contrast to the mouse embryo, the key extra-embryonic determinant GATA37,8 is expressed not only in extra-embryonic lineage precursors upon blastocyst formation. However, the cell polarity machinery enhances the expression and nuclear accumulation of GATA3. In summary, our results demonstrate for the first time that cell polarisation reinforces the first lineage segregation in the human embryo.

Abstract P020: LOSS OF SOLUBLE (PRO)RENIN RECEPTOR ATTENUATES DOCA-SALT HYPERTENSION

Cleavage of the extra-cellular domain of the (pro)renin receptor (PRR) yields a soluble fragment (sPRR), which maybe involved in mediating hypertension. We recently developed a novel mouse model with mutation in the cleavage site of the PRR using CRISPR/Cas9 such that sPRR is not generated and showed that absence of sPRR attenuated angiotensin-II induced hypertension and kidney damage. In this study, we examined if sPRR alters blood pressure (BP) in angiotensin-II independent hypertension using deoxycorticosterone acetate (DOCA)-salt treatment. Mutant sPRR mice and littermate controls were treated with DOCA (50 mg/kg) and high Na + diet for 3 weeks. BP was monitored by radio-telemetry and metabolic balance studies performed at the end of the study (Day 17-18). Compared to controls, male mutant sPRR mice had markedly lower plasma sPRR levels (control: 21.5 ± 2.5 vs mutant 0.2 ± 0.03 ng/ml) and baseline BP (systolic control: 122 ± 3 vs mutant 114 ± 3; diastolic control: 94 ±5 vs mutant 82 ± 3 mm Hg). BP remained low in mutant sPRR mice relative to controls following 12 days of DOCA-salt treatment (systolic control: 141 ± 2 vs mutant 132 ± 5; diastolic control: 110 ± 4 vs mutant 95 ± 5 mm Hg). Mutant sPRR mice had lower body weight but similar food intake and urinary albumin excretion compared to controls (Table 1). Mutant sPRR mice had lower urine volume, water intake and urinary K + but not Na + excretion. No differences in renal histology were noted between control and mutant sPRR mice. Loss of sPRR attenuates DOCA-salt mediated hypertension. The mechanisms by which sPRR might regulate BP and water/Na + homeostasis in DOCA-salt hypertension are currently being investigated.

Surface LSP1 is a phenotypic marker distinguishing human classical versus monocyte-derived dendritic cells

Human mononuclear phagocytes comprise several subsets of dendritic cells (DCs), monocytes and macrophages. Distinguishing one population from another is challenging, especially in inflammed tissues, due to the promiscuous expression of phenotypic markers. Using a synthetic library of humanized llama single domain antibodies, we identified a novel surface marker for human naturally-occuring monocyte-derived DCs. Our antibody targets an extra-cellular domain of LSP-1, specifically on monocyte-derived DCs, but not on monocytes, macrophages or classical DCs. Using this antibody, we provide evidence that the recently described blood DC3 population does not correspond to circulating monocyte-derived DCs. Our findings will pave the way for a better characterization of human mononuclear phagocytes in pathological settings.

Dissecting the Repertoire of DNA-Binding Transcription Factors of the Archaeon Pyrococcus furiosus DSM 3638

In recent years, there has been a large increase in the amount of experimental evidence for diverse archaeal organisms, and these findings allow for a comprehensive analysis of archaeal genetic organization. However, studies about regulatory mechanisms in this cellular domain are still limited. In this context, we identified a repertoire of 86 DNA-binding transcription factors (TFs) in the archaeon Pyrococcus furiosus DSM 3638, that are clustered into 32 evolutionary families. In structural terms, 45% of these proteins are composed of one structural domain, 41% have two domains, and 14% have three structural domains. The most abundant DNA-binding domain corresponds to the winged helix-turn-helix domain; with few alternative DNA-binding domains. We also identified seven regulons, which represent 13.5% (279 genes) of the total genes in this archaeon. These analyses increase our knowledge about gene regulation in P. furiosus DSM 3638 and provide additional clues for comprehensive modeling of transcriptional regulatory networks in the Archaea cellular domain.