Reduced cytotoxicity by mutation of lysine 590 of Pseudomonas exotoxin can be restored in an optimized, lysine-free immunotoxin

Immunotoxins, which are fusion proteins of an antibody fragment and a fragment of a bacterial or a plant toxin, induce apoptosis in target cells by inhibition of protein synthesis. ADP-ribosylating toxins often have few lysine residues in their catalytic domain. As they are the target for ubiquitination, the low number of lysines possibly prevents ubiquitin-dependent degradation of the toxin in the cytosol. To reduce this potential degradation, we aimed to generate a lysine-free (noK), Pseudomonas exotoxin (PE)-based immunotoxin. The new generation 24 kDa PE, which lacks all but the furin-cleavage site of domain II, was mutated at lysine 590 (K590) and at K606 in a CD22-targeting immunotoxin and activity was determined against various B cell malignancies in vitro and in vivo. On average, K590 mutated to arginine (R) reduced cytotoxicity by 1.3-fold and K606R enhanced cytotoxicity by 1.3-fold compared to wild type (wt). Mutating K590 to histidine or deleting K590 did not prevent this loss in cytotoxicity. Neither stability nor internalization rate of K590R could explain reduced cytotoxicity. These results highlight the relevance of lysine 590 for PE intoxication. In line with in vitro results, the K606R mutant was more than 1.8-fold more active than the other variants in vivo suggesting that this single mutation may be beneficial when targeting CD22-positive malignancies. Finally, reduced cytotoxicity by K590R was compensated for by K606R and the resulting lysine-free variant achieved wt-like activity in vitro and in vivo. Thus, PE24-noK may represent a promising candidate for down-stream applications that would interfere with lysines

PU.1 controls fibroblast polarization and tissue fibrosis

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs

Guanylate-binding protein 1 expression from embryonal endothelial progenitor cells reduces blood vessel density and cellular apoptosis in an axially vascularised tissue-engineered construct

BACKGROUND: Guanylate binding protein-1 (GBP-1) is a large GTPase which is actively secreted by endothelial cells. It is a marker and intracellular inhibitor of endothelial cell proliferation, migration, and invasion. We previously demonstrated that stable expression of GBP-1 in murine endothelial progenitor cells (EPC) induces their premature differentiation and decreases their migration capacity in vitro and in vivo. The goal of the present study was to assess the antiangiogenic capacity of EPC expressing GBP-1 (GBP-1-EPC) and their impact on blood vessel formation in an axially vascularized 3-D bioartificial construct in vivo. RESULTS: Functional in vitro testing demonstrated a significant increase in VEGF secretion by GBP-1-EPC after induction of cell differentiation. Undifferentiated GBP-1-EPC, however, did not secrete increased levels of VEGF compared to undifferentiated control EPC expressing an empty vector (EV-EPC). In our In vivo experiments, we generated axially vascularized tissue-engineered 3-D constructs. The new vascular network arises from an arterio-venous loop (AVL) embedded in a fibrin matrix inside a separation chamber. Total surface area of the construct as calculated from cross sections was larger after transplantation of GBP-1-EPC compared to control EV-EPC. This indicated reduced formation of fibrovascular tissue and less resorption of fibrin matrix compared to constructs containing EV-EPC. Most notably, the ratio of blood vessel surface area over total construct surface area in construct cross sections was significantly reduced in the presence of GBP-1-EPC. This indicates a significant reduction of blood vessel density and thereby inhibition of blood vessel formation from the AVL constructs caused by GBP-1. In addition, GBP-1 expressed from EPC significantly reduced cell apoptosis compared to GBP-1-negative controls. CONCLUSION: Transgenic EPC expressing the proinflammatory antiangiogenic GTPase GBP-1 can reduce blood vessel density and inhibit apoptosis in a developing bioartificial vascular network and may become a new powerful tool to manipulate angiogenetic processes in tissue engineering and other pathological conditions such as tumour angiogenesis

Intracellular Trafficking of Guanylate-Binding Proteins Is Regulated by Heterodimerization in a Hierarchical Manner

Guanylate-binding proteins (GBPs) belong to the dynamin family of large GTPases and represent the major IFN-γ-induced proteins. Here we systematically investigated the mechanisms regulating the subcellular localization of GBPs. Three GBPs (GBP-1, GBP-2 and GBP-5) carry a C-terminal CaaX-prenylation signal, which is typical for small GTPases of the Ras family, and increases the membrane affinity of proteins. In this study, we demonstrated that GBP-1, GBP-2 and GBP-5 are prenylated in vivo and that prenylation is required for the membrane association of GBP-1, GBP-2 and GBP-5. Using co-immunoprecipitation, yeast-two-hybrid analysis and fluorescence complementation assays, we showed for the first time that GBPs are able to homodimerize in vivo and that the membrane association of GBPs is regulated by dimerization similarly to dynamin. Interestingly, GBPs could also heterodimerize. This resulted in hierarchical positioning effects on the intracellular localization of the proteins. Specifically, GBP-1 recruited GBP-5 and GBP-2 into its own cellular compartment and GBP-5 repositioned GBP-2. In addition, GBP-1, GBP-2 and GBP-5 were able to redirect non-prenylated GBPs to their compartment in a prenylation-dependent manner. Overall, these findings prove in vivo the ability of GBPs to dimerize, indicate that heterodimerization regulates sub-cellular localization of GBPs and underscore putative membrane-associated functions of this family of proteins

One Step Nucleic Acid Amplification (OSNA) - a new method for lymph node staging in colorectal carcinomas

<p>Abstract</p> <p>Background</p> <p>Accurate histopathological evaluation of resected lymph nodes (LN) is essential for the reliable staging of colorectal carcinomas (CRC). With conventional sectioning and staining techniques usually only parts of the LN are examined which might lead to incorrect tumor staging. A molecular method called OSNA (One Step Nucleic Acid Amplification) may be suitable to determine the metastatic status of the complete LN and therefore improve staging.</p> <p>Methods</p> <p>OSNA is based on a short homogenisation step and subsequent automated amplification of cytokeratin 19 (CK19) mRNA directly from the sample lysate, with result available in 30-40 minutes. In this study 184 frozen LN from 184 patients with CRC were investigated by both OSNA and histology (Haematoxylin & Eosin staining and CK19 immunohistochemistry), with half of the LN used for each method. Samples with discordant results were further analysed by RT-PCR for CK19 and carcinoembryonic antigen (CEA).</p> <p>Results</p> <p>The concordance rate between histology and OSNA was 95.7%. Three LN were histology+/OSNA- and 5 LN histology-/OSNA+. RT-PCR supported the OSNA result in 3 discordant cases, suggesting that metastases were exclusively located in either the tissue analysed by OSNA or the tissue used for histology. If these samples were excluded the concordance was 97.2%, the sensitivity 94.9%, and the specificity 97.9%. Three patients (3%) staged as UICC I or II by routine histopathology were upstaged as LN positive by OSNA. One of these patients developed distant metastases (DMS) during follow up.</p> <p>Conclusion</p> <p>OSNA is a new and reliable method for molecular staging of lymphatic metastases in CRC and enables the examination of whole LN. It can be applied as a rapid diagnostic tool to estimate tumour involvement in LN during the staging of CRC.</p

Repeated BCG treatment of mouse bladder selectively stimulates small GTPases and HLA antigens and inhibits single-spanning uroplakins

<p>Abstract</p> <p>Background</p> <p>Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following repeated intravesical BCG therapy.</p> <p>Methods</p> <p>Mice were transurethrally instilled with BCG or pyrogen-free on days 1, 7, 14, and 21. Seven days after the last instillation, urothelia along with the submucosa was removed and amplified ds-DNA was prepared from control- and BCG-treated bladder mucosa and used to generate suppression subtractive hybridization (SSH). Plasmids from control- and BCG-specific differentially expressed clones and confirmed by Virtual Northern were then purified and the inserts were sequenced and annotated. Finally, chromatin immune precipitation combined with real-time polymerase chain reaction assay (ChIP/Q-PCR) was used to validate SSH-selected transcripts.</p> <p>Results</p> <p>Repeated intravesical BCG treatment induced an up regulation of genes associated with antigen presentation (B2M, HLA-A, HLA-DQA1, HLA-DQB2, HLA-E, HLA-G, IGHG, and IGH) and representatives of two IFNγ-induced small GTPase families: the GBPs (GBP1, GBP2, and GBP5) and the p47GTPases (IIGTP1, IIGTP2, and TGTP). Genes expressed in saline-treated bladders but down-regulated by BCG included: the single-spanning uroplakins (UPK3a and UPK2), SPRR2G, GSTM5, and RSP 19.</p> <p>Conclusion</p> <p>Here we introduced a hypothesis-generator approach to determine key genes involved in the urothelium/sumbmucosa responses to BCG therapy. Urinary bladder responds to repeated BCG treatment by up-regulating not only antigen presentation-related genes, but also GBP and p47 small GTPases, both potentially serving to mount a resistance to the replication of the <it>Mycobacterium</it>. It will be of tremendous future interest to determine whether these immune response cascades play a role in the anti-cancer effects exerted by BCG.</p