Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short-course costimulation blockade and rapamycin. This combination treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3-transduced Tregs, natural Tregs and TGF-beta induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.

Summary: The double‐stranded (ds)DNA virus φCh1 infects the haloalkaliphilic archaeon Natrialba magadii. The complete DNA sequence of 58 498 bp of the temperate virus was established, and the probable functions of 21 of 98 φCh1‐encoded open reading frames (ORFs) have been assigned. This knowledge has been used to propose functional modules each required for specific functions during virus development. The φCh1 DNA is terminally redundant and circularly permuted and therefore appears to be packaged by the so‐called headful mechanism. The presence of ORFs encoding homologues of proteins involved in plasmid replication as well as experimental evidence indicate a plasmid‐mediated replication strategy of the virus. Results from nanosequencing of virion components suggest covalent cross‐linking of monomers of at least one of the structural proteins during virus maturation. A comparison of the φCh1 genome with the partly sequenced genome of Halobacterium salinarum virus φH revealed a close relationship between the two viruses, although their host organisms live in distinct environments with respect to the different pH values required for growth.

A novel archaeal bacteriophage, ΦCh1, was isolated from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis. The phage-cured strain N. magadii (L13) was used to demonstrate in...

Fibroblasts are the prevalent cell type and main source for extracellular matrix (ECM) in connective tissue. Depending on their origin, fibroblasts play a central role in non-pathological tissue remodeling and disease like fibrosis. This study examined the effect of established culture conditions of primary human fibroblasts, from different origins on the myofibroblast-like phenotype formation. We isolated primary human fibroblasts from aortic adventitia, lung, juvenile- and adult skin and investigated the expression levels of CD90, alpha smooth muscle actin (αSMA) and procollagen I under different concentrations of fetal calf serum (FCS) and ascorbic acid (AA) in culture media by immunoblot and immunofluorescence assays. Furthermore, we determined the viability using XTT and migration/wound healing in scratch assays. Collagen 1 secretion was quantified by specific ELISA. Primary human fibroblasts show in part a myofibroblast-like phenotype even without addition of FCS. Supplemented AA reduces migration of cultured fibroblasts with no or low concentrations of FCS. Furthermore, AA and higher concentrations of FCS in culture media lead to higher levels of collagen 1 secretion instead of procollagen I accumulation. This study provides evidence for a partial switch of primary human fibroblasts of different origin to a myofibroblast-like phenotype under common culture conditions.

By differential screening we isolated genes upregulated in inflammatory cytokine-stimulated human skin microvascular endothelial cells. One of these cDNAs encoded RCC1 (regulator of chromosome condensation 1)-like repeats and a HECT (homologous to E6-AP C-terminus) domain, representing a member of the HERC (HECT and RCC1 domain protein) family of ubiquitin ligases. The mRNA level of this member, HERC5, is specifically upregulated in endothelial cells by the pro-inflammatory cytokines tumor necrosis factor α and interleukin 1β, and by lipopolysaccharide (LPS), but is hardly expressed in other cells of the vascular wall such as primary smooth muscle cells and fibroblasts. Regulation of HERC5 gene expression suggests a critical role for the transcription factor NF-κB. In contrast to mRNA expression HERC5 protein is subject of enhanced degradation upon LPS stimulation of endothelial cells. The time course of LPS-induced changes in HERC5 protein and mRNA levels suggests that the initial drop in HERC5 protein is balanced by increased protein synthesis due to upregulation of HERC5 mRNA. This leads to recovery of HERC5 protein levels within 12 hours of LPS stimulation and points at a tight control of HERC5 protein. To analyze functional activity of this putative member of the ubiquitin-conjugating pathway we performed in vitro assays with different ubiquitin-conjugating enzymes. We found that HERC5 possesses ubiquitin ligase activity and requires the presence of the ubiquitin-conjugating enzyme UbcH5a for its activity. These data show for the first time that a functionally active HECT ubiquitin ligase exhibits a tightly controlled cytosolic level under inflammatory conditions in endothelial cells.