Treatment of experimental murine colitis with CD40 antisense oligonucleotides delivered in amphoteric liposomes.

BACKGROUND AND AIMS: CD40-CD40L interactions appear to play an important role in the pathogenesis of experimental colitis. We tested the effect and investigated the underlying mechanism of action of systemically administered antisense oligonucleotide (ASO) targeting CD40 formulated in amphoteric liposomes (nov038/CD40). The charge characteristics of the amphoteric liposomes (anionic surface charge at physiological pH that becomes cationic at low pH), facilitate efficient sequestration of the ASO inside the liposomes at low pH and the direction of the carriers towards macrophages and dendritic cells under physiological conditions. METHODS: Colitis was induced in Balb/c mice using 2,4,6-Trinitrobenzene sulphonic acid (TNBS) and treated with nov038/CD40. Disease was monitored by body weight, histology, cytokine profiling and changes in immune cell populations. CD40 expression on different cell subsets was analyzed by flow cytometry. An antigen challenge model was used to determine neoimmunity under CD40 modulation. RESULTS: Administration of nov038/CD40 inhibited the development of TNBS colitis as assessed by weight loss, histology and cytokine profiles; unformulated CD40 ASO or nov038 encapsulating an unrelated ASO (nov038/SCR) were ineffective. The novel agent is potent as it completely suppressed even established colitis with a single treatment and significantly reduced T-cell activation as well as levels of pro-inflammatory mediators in serum. The inhibition of CD40 specifically occurred in macrophages, but not in B-cells. In contrast to prednisolone, standard treatment for inflammatory bowel diseases (IBD) that is effective in a single administration and involves extensive immunosuppression, nov038/CD40 did not affect the number of B- or Treg cells. Eventually, we observed a largely intact neoimmunity under conditions of a CD40 inhibition. CONCLUSIONS: Administration of nov038/CD40, but neither naked CD40 ASO nor nov038/SCR, prevents the development and treats established colitis in mice. Delivery of CD40 ASO in nov038 is highly cell-specific as it selectively suppresses CD40 on macrophages, but not on B-cells; the novel agent has strong anti-inflammatory characteristics without being immunosuppressive.

STAT-1 decoy oligonucleotide improves microcirculation and reduces acute rejection in allogeneic rat small bowel transplants.

During acute rejection leukocyte-endothelial cell interaction fuelled by costimulatory molecules such as the CD40/CD154 receptor/ligand dyad disrupts microcirculation of the small bowel. Downregulating endothelial CD40 expression by employing a decoy oligonucleotide (dODN) neutralizing the transcription factor signal transducer and activator of transcription-1 (STAT-1) may protect the graft. Therefore allogenic small bowel transplantation was performed in the Brown Norway to Lewis rat model. Graft vessels were pretreated with STAT-1 dODN, mutant control ODN (20 microM) or vehicle (n=8). CD40 antisense ODN and scrambled control ODN-treated transplants served as target control (n=3 each). Intravital microscopy, histology, immunohistochemistry and Western blot analyses were performed 7 days later. Functional capillary density, red blood cell velocity and perfusion index in STAT-1 dODN and CD40 antisense ODN-treated transplants were improved whereas stasis index was reduced. Leukocyte-endothelial cell interaction showed no difference. Histological parameters of rejection, infiltrating CD3-positive cells and apoptotic bodies were also reduced in STAT-1 dODN and CD40 antisense ODN-treated transplants 7 days post-transplantation. CD40 protein abundance was reduced to less than 10% of control in STAT-1 dODN-treated grafts. STAT-1 dODN blockade of CD40 expression improves mucosal perfusion, reduces graft rejection, T-cell infiltration and apoptosis in rat small bowel allografts during acute rejection.

CD40 antisense oligonucleotide inhibition of trinitrobenzene sulphonic acid induced rat colitis.

BACKGROUND: CD154/CD40 interactions play a pivotal role both in humoral and cellular immune responses. Their involvement in the pathogenesis of chronic inflammatory bowel disease (IBD) has been revealed by increased expression of CD40 and CD154 in the inflamed mucosa of patients and the therapeutic effects of anti-CD154 antibodies in experimental colitis. Because of adverse side effects however, the use of such antibodies in patients with IBD may be limited. AIMS: An alternative approach to blocking CD154/CD40 interactions by employing a CD40 antisense oligonucleotide (ODN) was explored. RESULTS: After sequencing of the rat CD40 gene, five antisense ODNs were designed, of which one (rAS3) effectively downregulated CD40 expression in rat vascular smooth muscle cells as well as the subsequent changes in gene expression in response to CD40 stimulation. The therapeutic potency of rAS3 was evaluated in the 2,4,6-trinitrobenzene sulphonic acid (TNBS) induced colitis model of the rat. Single intracolonic injection of a liposomal formulation of rAS3 either prior to or post colitis induction markedly suppressed the inflammatory reaction in these animals monitored both macroscopically and microscopically over one week, while application of a scrambled control ODN had no such effects. Moreover, reverse transcription-polymerase chain reaction analyses revealed reduced expression of vascular cell adhesion molecule 1, interleukin 12 p40, and monocyte chemoatractive protein 1 in the inflamed mucosa, which in turn may have contributed to the decrease in leucocyte infiltration judged by immunohistochemistry. CONCLUSIONS: These results suggest that CD40 antisense ODNs effectively interfere with CD154/CD40 interactions in vivo and, therefore, may provide a novel approach to the treatment of patients with chronic IBD.

Fibrin encapsulated liposomes as protein delivery system. Studies on the in vitro release behavior.

The efficacy of biologically active proteins in medical therapy depends on the development of suitable drug delivery systems. These delivery systems need to overcome the severe problems connected with the use of proteins such as their usually short half lives in body fluids and their susceptibility to proteolysis and denaturation. Our delivery system combines two widespread devices by encapsulating liposomes containing the model protein horseradish peroxidase (HRP) inside the biopolymer fibrin. The liposomes enable the protein to remain in its preferred aqueous environment and protect it during the polymerization process. Further encapsulation of the liposomes inside fibrin was carried out in order to achieve a depot system with sustained protein release. In vitro experiments showed that the protein filled liposomes were absolutely stable within the fibrin network. In contrast to 'free' HRP, enzyme entrapped in liposomes was completely retained by the fibrin network and wasn't released from the device unless the fibrin was degraded by plasmin.

The homologue of mammalian SPC12 is important for efficient signal peptidase activity in Saccharomyces cerevisiae.

The multisubunit signal peptidase catalyzes the cleavage of signal peptides and the degradation of some membrane proteins within the endoplasmic reticulum (ER). The only subunit of this enzyme functionally examined to date, yeast Sec11p, is related to signal peptidase I from bacteria. Since bacterial signal peptidase is capable of processing both prokaryotic and eukaryotic signal sequences as a monomer, it is unclear why the analogous enzyme in the ER contains proteins unrelated to signal peptidase I. To address this issue, the gene encoding Spc1p, the yeast homologue to mammalian SPC12, is isolated from the yeast Saccharomyces cerevisiae. Spc1p co-purifies and genetically interacts with Sec11p, but unlike Sec11p, Spc1p is not required for cell growth or the proteolytic processing of tested proteins in yeast. This indicates that only a subset of the ER signal peptidase subunits is required for signal peptidase and protein degradation activities in vivo. Through both genetic and biochemical criteria, Spc1p appears, however, to be important for efficient signal peptidase activity.

A second trimeric complex containing homologs of the Sec61p complex functions in protein transport across the ER membrane of S. cerevisiae.

Yeast microsomes contain a heptameric Sec complex involved in post-translational protein transport that is composed of a heterotrimeric Sec61p complex and a tetrameric Sec62-Sec63 complex. The trimeric Sec61p complex also exists as a separate entity that probably functions in co-translational protein transport, like its homolog in mammals. We have now discovered in the yeast endoplasmic reticulum membrane a second, structurally related trimeric complex, named Ssh1p complex. It consists of Ssh1p1 (Sec sixty-one homolog 1), a rather distant relative of Sec61p, of Sbh2p, a homolog of the Sbh1p subunit of the Sec61p complex, and of Sss1p, a component common to both trimeric complexes. In contrast to Sec61p, Ssh1p is not essential for cell viability but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential, but cells lacking both proteins are impaired in their growth at elevated temperatures and accumulate precursors of secretory proteins; microsomes isolated from these cells also exhibit a reduced rate of post-translational protein transport. Like the Sec61p complex, the Ssh1p complex interacts with membrane-bound ribosomes, but it does not associate with the Sec62-Sec63p complex to form a heptameric Sec complex. We therefore propose that it functions exclusively in the co-translational pathway of protein transport.

Posttranslational protein transport in yeast reconstituted with a purified complex of Sec proteins and Kar2p.

We have reproduced the posttranslational mode of protein translocation across the endoplasmic reticulum membrane with reconstituted proteoliposomes containing a purified complex of seven yeast proteins. This Sec complex includes a heterotrimeric Sec61p complex, homologous to that in mammals, as well as all other membrane proteins found in genetic screens for translocation components. Efficient posttranslational translocation also requires the addition of lumenal Kar2p (BiP) and ATP. The trimeric Sec61p complex also exists as a separate entity that, in contrast with the large Sec complex, is associated with membrane-bound ribosomes. We therefore hypothesize that distinct membrane protein complexes function in co- and posttranslational translocation pathways.