Delivery of Soluble Heme Oxygenase 1 Cell-Penetrating Peptide into Liver Cells in in vitro and ex vivo Models of Cold Ischemia.

BACKGROUND/PURPOSE: Liver transplantation is the treatment of choice in patients with end-stage liver disease. During liver transplantation, ischemia-reperfusion injury (IRI) occurs, which is an inevitable consequence of the transplantation process. To reduce the extent of cellular injury, one of the proteins that have been extensively investigated is heme oxygenase 1 (HO-1), which plays an important role in protecting the organs against IRI. The aim of this study was to introduce an active and functional HO-1 protein conjugated to a cell-penetrating peptide (CPP) in vitro and ex vivo into liver cells in hypothermic and anoxic conditions and to assert its cytoprotective effects. METHODS: We generated an enzymatically active soluble (s)HO-1-CPP recombinant protein. The ability of the sHO-1-CPP protein to penetrate McA-RH7777, Clone 9, and Hep G2 cells, primary hepatocytes, and Kupffer and human umbilical vein endothelial cells in vitro, as well as its ability to penetrate a whole liver ex vivo under hypothermic and anoxic conditions, was assessed. An in vitro hypoxia-reoxygenation (HR) model was used to determine the cytoprotective effect of the sHO-1-CPP protein. RESULTS: We showed that our recombinant protein sHO-1-CPP can cross cell membranes into rodent and human liver cells in vitro, and the results were further validated ex vivo, where rodent livers were perfused with an organ preservation solution supplemented with sHO-1-CPP under anoxic and hypothermic conditions. Immunohistochemistry revealed an intracellular localization of sHO-1-CPP in zones 1-3 of the perfused livers. The CPP did not exert any significant toxicity on the cells. Treating cells with sHO-1-CPP showed significant cytoprotection in the in vitro HR model. CONCLUSIONS: Our findings show that the recombinant protein sHO-1-CPP can be successfully delivered to cells of a whole organ in an ex vivo hypothermic and anoxic perfusion model and that it provides cytoprotection to hepatocytes in an in vitro HR model. These results hold great potential for future repair and protection of donor organs. Future experiments are planned to confirm these data in in vivo models of IRI.

Cux2 activity defines a subpopulation of perinatal neurogenic progenitors in the hippocampus.

The hippocampus arises from the medial region of the subventricular (SVZ) within the telencephalon. It is one of two regions in the postnatal brain that harbors neural progenitors (NPs) capable of giving rise to new neurons. Neurogenesis in the hippocampus is restricted to the subgranular zone (SGZ) of the dentate gyrus (DG) where it contributes to the generation of granule cell layer (gcl) neurons. It is thought that SGZ progenitors are heterogeneous, differing in their morphology, expression profiles, and developmental potential, however it is currently unknown whether they display differences in their developmental origins and cell fate-restriction in the DG. Here we demonstrate that Cux2 is a marker for SGZ progenitors and nascent granule cell neurons in the perinatal brain. Cux2 was expressed in the presumptive hippocampal forming region of the embryonic forebrain from E14.5 onwards. At fetal stages, Cux2 was expressed in early-forming Prox1(+) granule cell neurons as well as the SVZ of the DG germinal matrix. In the postnatal brain, Cux2 was expressed in several types of progenitors in the SGZ of the DG, including Nestin/Sox2 double-positive radial glia, Sox2(+) cells that lacked a radial glial process, DCX(+) neuroblasts, and Calretinin-expressing nascent neurons. Another domain characterized by a low level of Cux2 expression emerged in Calbindin(+) neurons of the developing DG blades. We used Cux2-Cre mice in genetic fate-mapping studies and showed almost exclusive labeling of Calbindin-positive gcl neurons, but not in any progenitor cell types or astroglia. This suggests that Cux2(+) progenitors directly differentiate into gcl neurons and do not self-renew. Interestingly, developmental profiling of cell fate revealed an outside-in formation of gcl neurons in the DG, likely reflecting the activity of Cux2 in the germinative matrices during DG formation and maturation. However, DG morphogenesis proceeded largely normally in hypomorphic Cux2 mutants lacking Cux2 expression. Taken together we conclude that Cux2 expression reflects hippocampal neurogenesis and identifies non-self-renewing NPs in the SGZ.

Immunohistochemical validation study of 15-gene biomarker panel predictive of benefit from adjuvant chemotherapy in resected non-small-cell lung cancer: analysis of JBR.10.

OBJECTIVE: There are no validated approaches to predict benefit from adjuvant chemotherapy for resected patients with non-small-cell lung cancer (NSCLC). The aim of this study was to translate a 15-gene mRNA expression profile published by Zhu et al, shown to be prognostic and predictive of benefit, into a readily applicable immunohistochemistry (IHC) panel. METHODS: For seven of the genes in the gene expression profile (GEP) for which suitable commercial antibodies were available, we semiquantitatively assessed the IHC expression and prognostic significance for 173 patients treated at the Saint John Regional Hospital (SJRH). Cut-offs for high and low expression were defined for each marker and applied to IHC scores from 291 of the 482 patients in JBR.10, including patients on both the adjuvant chemotherapy and observation arms. The prognostic and predictive value of these markers on overall survival (OS) or recurrence-free survival (RFS) was assessed by Cox regression models. RESULTS: In the SJRH cohort, in 62 patients with resected stage II-III NSCLC, the prognostic significance of IHC assays for four proteins were concordant with Zhu's GEP results. Low FOSL2 (OS, HR=0.15; p=0.0001; RFS, HR=0.14; p<0.0001) and high STMN2 (RFS, HR=2.501; p=0.0197) were adverse prognostic factors. Low ATP1B1 and low TRIM14 expression trended toward worse OS and RFS. Validation of these markers with JBR.10 patients failed to show prognostic significance either individually or in combined risk classifications. Additionally, the interaction between these markers and chemotherapy treatment in predicting OS (FOSL2, p=0.52; STMN2 p=0.14; ATP1B1, p=0.33; TRIM14, p=0.81) or RFS (FOSL2, p=0.63; STMN2, p=0.12; ATP1B1, p=0.66; TRIM14, p=0.57) did not reach significance, individually or in combination panels. CONCLUSIONS: Zhu's GEP could not be translated into an IHC panel predictive of benefit from adjuvant chemotherapy. Future predictive biomarker analysis in the adjuvant NSCLC setting may need to focus on novel therapies.