Delivery of Heme Oxygenase-1-Cell Penetrating Peptide (HO-1-CPP) Into Hepatocytes in In Vitro and Ex Vivo Model of Cold Ischemia.
Bibliographic record
Abstract
In an attempt to increase the number of donor organs, one of the major strategies being employed by transplant surgeons is the use of extended criteria donor (ECD) organs. Unfortunately, ECD organs are more susceptible to cold ischemia. Several methods have been described that may reduce the cellular injury in experimental models of cold ischemia. One of the proteins that have gained a lot of traction is the heme-oxygenase-1 (HO-1). Induction of HO-1 expression under conditions of cellular stress has targeted it as a key gene for protection against injury during cold ischemic conditions. The aim of the present study is to introduce active and functional HO-1 protein conjugated to a cell penetrating peptide (CPP) directly in vitro to hepatocytes, and ex vivo to hepatocytes, endothelial cells, and Kupffer cells in hypothermic and anoxic conditions. This novel strategy may also be used for the ex vivo delivery of other protective proteins in hypothermic perfusion systems. Our preliminary experiments have determined that we are able to consistently produce a functional HO-1 protein fused to a CPP. To confirm the ability of our fusion protein to cross cell membranes, we have performed an in vitro experiment in which rodent (McA-RH7777) and human (Hep G2) hepatocytes were incubated with HO-1-CPP and later stained with anti-HO-1 and anti-His antibodies followed by fluorescent secondary antibodies. Localization of HO-1-CPP was revealed by (red) anti-His staining which shows a pattern consistent with HO-1 localization to the endoplasmic reticulum. Further evidence of the cell permeability of HO-1-CPP has been studied in ex vivo perfusion experiments. The HO-1-CPP perfused livers sections were paraffin embedded, sectioned and stained with an anti-HO-1 antibody which revealed intracellular localization of HO-1-CPP 4-5 cell layers deep surrounding the perfused vessel. Our ability to successfully deliver an active protein conjugated to a CPP to cells of a whole organ in an ex vivo hypothermic and hypoxic perfusion model holds great potential for future repair and protection of organs for transplantation. Future studies to determine the ability of HO-1-CPP to modulate the response to ischemia reperfusion injury (IRI) in our in vitro and ex vivo models are planned.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".