Co-grafting of NCSCs with pancreatic islets improved insulin release in mixed transplants and enhanced beta cell proliferation, resulting in increased beta cell mass. This co-transplantation model offers an opportunity to restore neural-islet interactions and improve islet functions after transplantation.

Chlorambucil and prednisolone, two commonly used drugs in the treatment of chronic lymphocytic leukemia (CLL), induce apoptosis in CLL cells. We have investigated the involvement in this apoptotic cell death of caspases, which cleave critical cellular substrates thereby acting as the executioners of the apoptotic process. Induction of spontaneous or chlorambucil/ prednisolone-induced apoptosis of freshly isolated B-CLL cells in culture resulted in the activation of the 'effector' caspases, -3 and -7, but generally not of caspase-2. Activation of caspases-3 and -7 was accompanied by the proteolysis of the DNA repair enzyme, poly (ADP-ribose) polymerase. Induction of apoptosis was also accompanied by the processing of caspase-8, the extent of which varied between patients. Induction of apoptosis and processing of all the caspases was inhibited by the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.fmk). Our results demonstrate a key role for the activation and processing of caspases in the execution phase of apoptosis in CLL cells. Apoptosis of CLL cells resulted in the selective activation of some but not all caspases. Our results suggest that the dysregulation of apoptosis observed in CLL may be due to the signalling leading to the activation of caspases rather than a deletion of pro-caspases. High levels of caspase-8 in CLL cells in conjunction with low levels of CD95 receptor may offer new therapeutic opportunities for the treatment of CLL.

IntroductionRunx1, a Runt domain transcription factor, controls the differentiation of nociceptors that express the neurotrophin receptor Ret, regulates the expression of many ion channels and receptors, and controls the lamina-specific innervation pattern of nociceptive afferents in the spinal cord. Moreover, mice lacking Runx1 exhibit specific defects in thermal and neuropathic pain. We investigated whether conditional activation of Runx1 short isoform (Runx1a), which lacks a transcription activation domain, influences differentiation of neural crest stem cells (NCSCs) in vitro and in vivo during development and whether postnatal Runx1a activation affects the sensitivity to neuropathic pain.MethodsWe activated ectopic expression of Runx1a in cultured NCSCs using the Tet-ON gene regulatory system during the formation of neurospheres and analyzed the proportion of neurons and glial cells originating from NCSCs. In in vivo experiments we applied doxycycline (DOX) to pregnant mice (days 8–11), i.e. when NCSCs actively migrate, and examined the phenotype of offsprings. We also examined whether DOX-induced activation of Runx1a in adult mice affects their sensitivity to mechanical stimulation following a constriction injury of the sciatic nerve.ResultsEctopic Runx1a expression in cultured NCSCs resulted in predominantly glial differentiation. Offsprings in which Runx1a had been activated showed retarded growth and displayed megacolon, pigment defects, and dystrophic dorsal root ganglia. In the neuropathic pain model, the threshold for mechanical sensitivity was markedly increased following activation of Runx1a.ConclusionThese data suggest that Runx1a has a specific role in NCSC development and that modulation of Runx1a activity may reduce mechanical hypersensitivity associated with neuropathic pain.

Meprins are metalloproteases that are expressed abundantly in the proximal kidney tubules. The level of expression and the localization of meprins are associated ischemia/reperfusion (IR)‐induced acute kidney injury (AKI). Previous studies have shown that meprin β deficiency protect mice from IR‐induced kidney injury, resulting in better overall preservation of kidney function. In vitro studies have shown that meprins are capable of proteolytically processing proteins associated with the hypoxia response (e.g OS‐9) and inflammation (e.g IL‐β1, IL‐6, IL‐18). Previous work from our lab showed that proteolytic processing of PKA C by meprins occurs in vivo (in IR‐induced AKI). In vitro studies with purified PKA C proteins further demonstrated that cleaving of PKA C by meprin B is isoform‐specific and significantly reduces the PKA C kinase activity; which could potentially affect downstream targets of the protein kinase A (PKA) signaling pathway and hypoxia response genes. The goal of the current study was to determine how meprin β expression affects downstream mediators of the PKA signaling pathway under hypoxia conditions. To this end, Madin‐Darby canine kidney (MDCK) cells stably transfected with meprin β cDNA and sham‐transfected control cells cultured to 90–95% confluence then subjected to hypoxia (1% O2) for 2h followed by 2h of recovery (under normoxia conditions). Proteins were extracted from the cells and fractionated into cytosolic‐, nuclear‐, and membrane‐enriched fractions. Nuclear levels of hypoxia inducible factor (HIF‐1α) were evaluated to confirm induction of hypoxia. Western blot analysis was used to determine the levels of PKA C, PKA Cα and PKA Cβ isoforms, Phospho‐PKA C, phospho‐extracellular signal‐regulated kinase (P‐ERK), and heat shock protein (Hsp27) in the cytosolic‐ and nuclear‐enriched fractions. Statistical analysis utilized 2‐way ANOVA. Our data showed that the levels of PKA C in cytosolic‐enriched fraction decreased in meprin β transfected cells subjected to hypoxia. No change was observed in the levels of PAK Cα in protein fractions of both cell genotypes. In contrast, the levels PKA Cβ in nuclear and cytosolic‐enriched fractions of meprin β transfected cells subjected to hypoxia decreased when compared to non‐transfected cells. P‐PKA C levels in nuclear and cytosolic‐enriched fractions decreased in meprin β expressing cells subjected to hypoxia when compared to non‐transfected control cells. The level of P‐ERK in meprin β‐transfected cells increased significantly compared to non‐transfected control cells, which showed a significant reduction after hypoxia induction. There was no change in the levels of HSP27 in cytosolic‐enriched fraction for either genotype of cells. These results suggest that meprin B expression has a direct impact on the levels of ERK and could thus affect downstream hypoxia response genes (e.g. ANKRD37, RCOR2, and NDRG1) which may impact IR‐induced AKI via modulation of the PKA pathway.Support or Funding InformationNIH/National Institutes of General Medical Sciences (NIGMS) Grant #s SC3GM102049 and SC1GM118271 to E. Moige OngeriThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Meprins are zinc dependent metalloproteases that are abundantly expressed in the brush border membranes (BBM) of proximal kidney tubules. Previous studies have implicated meprins in Ischemic/Reperfusion (IR) induced acute kidney injury (AKI), and diabetic nephropathy (DN). However, the mechanisms by which meprins modulate kidney injury are not fully understood. Known meprin substrates include extracellular matrix (ECM) proteins (e.g. Collagen, Fibronectin, Laminin, and Nidogen‐1) and modulators of inflammation such as interleukin 1β (IL‐1β), IL‐6, pro‐IL‐18, monocyte chemoattractant protein‐1 (MCP‐1), and thymosin β4 – leading to release of the anti‐inflammatory peptide Ac‐SDKP. Furthermore, meprins are expressed in leukocytes (monocytes, macrophages), and play a role in leukocyte infiltration to sites of inflammation in the small intestines. Inflammation is an underlying cause of renal injury in both IR‐induced AKI and DN. The objective of the current study was to determine how meprin deficiency impacts extracellular matrix protein build up and leukocyte infiltration in unilateral ureteral obstruction (UUO)‐induced inflammation. Surgical procedures were used to ligate one ureter in twelve‐week‐old wild‐type (WT) and meprin β knockout male mice. The contralateral kidney served as the control for each mouse. At 7 days post‐UUO, the kidneys were perfused with saline to remove leukocytes present in kidney blood vessels and kidneys were harvested. Sections of the kidney tissue were snap frozen for protein extraction and some were paraffin embedded and sectioned for microscopy. For immunofluorescence F4/80, was used as a macrophage maker. The images were captured and the number of cells that stained positive for F4/80 counted. Western blot analysis was used to evaluate the levels of ECM proteins (fibronectin, laminin, and nidogen‐1) in the kidney lysates. Statistical analysis utilized 2‐way ANOVA (Graphpad Prism 7 software). Our data demonstrates increases in fibronectin protein levels in both WT and βKO kidneys subjected to UUO. However, UUO‐induced fibronectin increases were higher in βKO than in WT (p=0.0016 vs p=0.004). The baseline levels of nidogen‐1 were higher in WT mice (p=0.01) when compared to βKOs. At 7 days post‐UUO, the levels of nidogen‐1 increased significantly in the kidneys of βKO subjected to UUO (p=0.001), but not in WT kidneys. There were no significant differences in the baseline or post‐UUO levels of laminin in either genotype. Results from immunofluorescence showed that WT and meprin βKO kidneys subjected to UUO had significant increases in the number of macrophages in tubulo‐interstitial tissue when compared to control counterparts (≤0.007 and ≤0.001 respectively). Taken together, our data suggest that meprin expression/activity reduces the buildup of specific ECM proteins (fibronectin and nidogen‐1), thus reducing the fibrosis observed in inflammation. Furthermore, meprins modulate inflammation via enhanced leukocyte infiltration of (monocytes/macrophages).Support or Funding InformationNIH/National Institutes of General Medical Sciences (NIGMS) Grant #s SC3GM102049 and #SC1GM118271 to Elimelda Moige OngeriThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.