Cdc42 (cell division cycle 42) is a member of the Rho GTPase family which regulates a variety of cellular activities by controlling actin cytoskeleton and gene expression. Cdc42 is expressed in the form of two splice variants. The canonical Cdc42 isoform is prenylated (Cdc42-prenyl), whereas the brainspecific isoform can be palmitoylated (Cdc42-palm). In the present study we have demonstrated palmitoylation of endogenous Cdc42 in rodent and human brains and identified Cys(188) and Cys(189) as acylation sites of Cdc42-palm. Moreover, we have shown that Cys(188) can also be prenylated. Analysis of acylation-deficient mutants revealed that lipidation of Cys(188) is essential for proper membrane binding of Cdc42-palm as well as for Cdc42-mediated regulation of gene transcription and induction of densely packed filopodia in neuroblastoma cells. We also found that Cdc42-prenyl is a dominant splice variant in a wide range of commonly used cell lines as well as in the cerebellum, whereas Cdc42-palm is the main Cdc42 isoform in hippocampus, where it is critically involved in the formation of dendritic filopodia and spines. Replacement of endogenous Cdc42 by its acylation-deficient mutants revealed the importance of Cdc42-palm lipidation for its morphogenic and synaptogenic effects in neurons. These findings demonstrate that dual lipidation of Cdc42-palm represents an important regulator of morphogenic signalling in hippocampal neurons.
Hepatocyte transplantation is a promising therapeutic approach for various liver diseases. Despite the liver's tolerogenic potential, early immune‐mediated loss of transplanted cells is observed, and longterm acceptance has not been achieved yet. Patients deemed tolerant after liver transplantation presented an increased frequency of regulatory T cells (Tregs), which therefore also might enable reduction of posttransplant cell loss and enhance longterm allograft acceptance. We hence characterized hepatocyte‐induced immune reactions and evaluated the immunomodulatory potential of Tregs applying mixed lymphocyte cultures and mixed lymphocyte hepatocyte cultures. These were set up using peripheral blood mononuclear cells and primary human hepatocytes, respectively. Polyclonally expanded CD4+CD25highCD127low Tregs were added to cocultures in single‐/trans‐well setups with/without supplementation of anti‐interferon γ (IFNγ) antibodies. Hepatocyte‐induced alloresponses were then analyzed by multicolor flow cytometry. Measurements indicated that T cell response upon stimulation was associated with IFNγ‐induced major histocompatibility complex (MHC) class II up‐regulation on hepatocytes and mediated by CD4+ T cells. An indirect route of antigen presentation could be ruled out by use of fragmented hepatocytes and culture supernatants of hepatocytes. Allospecific proliferation was accompanied by inflammatory cytokine secretion. CD8+ T cells showed early up‐regulation of CD69 despite lack of cell proliferation in the course of coculture. Supplementation of Tregs effectively abrogated hepatocyte‐induced alloresponses and was primarily cell contact dependent. In conclusion, human hepatocytes induce a CD4+ T cell alloresponse in vitro, which is associated with MHC class II up‐regulation on hepatocytes and is susceptible to suppression by Tregs. Liver Transplantation 24 407–419 2018 AASLD.
In contrast to the whole liver, primary hepatocytes are highly immunogenic. Thus, alternative strategies of immunomodulation after hepatocyte transplantation are of special interest. Silencing of HLA class I expression is expected to reduce the strength of allogeneic immune responses and to improve graft survival. In this study, primary human hepatocytes (PHH) were isolated using a two‐step‐collagenase perfusion‐technique and co‐cultured with allogeneic lymphocytes in terms of a mixed lymphocyte hepatocyte culture. Expression of HLA class I on PHH was silenced using lentiviral vectors encoding for β2‐microglobulin‐specific short hairpin RNA (shβ2m) or non‐specific shRNA (shNS) as control. The delivery of shβ2m into PHH caused a decrease by up to 96% in β2m transcript levels and a down‐regulation of HLA class I cell surface expression on PHH by up to 57%. Proliferative T cell alloresponses against HLA‐silenced PHH were significantly lower than those observed form fully HLA‐expressing PHH. In addition, significantly lower secretion of pro‐inflammatory cytokines was observed. Levels of albumin, urea and aspartate‐aminotransferase did not differ in supernatants of cultured PHH. In conclusion, silencing HLA class I expression on PHH might represent a promising approach for immunomodulation in the transplant setting without compromising metabolic function of silenced hepatocytes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.