Bernhard Singer

<p>A) For quantification of CEACAM1 cell surface expression in confluent and log phase cultured A549 cells, samples were analyzed by flow cytometry utilizing the QuiFiKit approach as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a>. Briefly, cells were stained for CEACAM1 with mAb clone 283340. Fluorescence was quantitated using QuiFiKit calibration-beads as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a>. The data shown are means ± SD (*p≤0.007) of three different experiments. B) Immunoblot analyses of confluent and proliferating A549 cell lysates were done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a> using monospecific mAbs directed against CEACAM1 (clone 283340), CEACAM5 (Col-1), and CEACAM6 (9A6), respectively and visualized by HRP-coupled secondary antibody and ECL detection. Beta-actin served as a loading control. The blots shown are representative of three separate experiments. C) The fraction of CEACAM1 expressing A549 cells increased over time as cells were kept in the confluent state. To analyze the CEACAM1 expression in A549 cells grown to confluence (day 0), plus 1 day, plus 3 days, plus 5 days and plus 7 days, cells were stained with mAb clone 283340 (thick line) and isotype matched control antibody (thin line) followed by FITC-conjugated secondary antibody. Subsequently, samples were measured by flow cytometry. The data shown are representative of three different experiments.</p

The gastric pathogen Helicobacter pylori infects half of the world’s population and is a major risk factor for gastric cancer development. In order to attach to human gastric epithelial cells and inject the oncoprotein CagA into host cells, H. pylori utilizes the outer membrane protein HopQ that binds to the cell surface protein CEACAM, which can be expressed on the gastric mucosa. Once bound, H. pylori activates a number of signaling pathways, including canonical and non-canonical NF-κB. We investigated whether HopQ–CEACAM interaction is involved in activating the non-canonical NF-κB signaling pathway. Different gastric cancer cells were infected with the H. pylori wild type, or HopQ mutant strains, and the activation of non-canonical NF-κB was related to CEACAM expression levels. The correlation between CEACAM levels and the activation of non-canonical NF-κB was confirmed in human gastric tissue samples. Taken together, our findings show that the HopQ–CEACAM interaction is importa...

Growth factor receptors, extracellular matrix receptors, and cell-cell adhesion molecules co-operate in regulating the activities of intracellular signaling pathways. Here, we demonstrate that the cell adhesion molecule CEACAM1 co-regulates growth-factor-induced DNA synthesis in NBT-II epithelial cells in a cell-density-dependent manner. CEACAM1 exerted its effects by regulating the activity of the Erk 1/2 MAP kinase pathway and the expression levels of the cyclin-dependent kinase inhibitor p27(Kip1). Interestingly, both inhibitory and stimulatory effects were observed. Confluent cells continuously exposed to fetal calf serum showed little Erk activity and DNA synthesis compared with sparse cells. Under these conditions, anti-CEACAM1 antibodies strongly stimulated Erk activation, decreased p27 expression, and induced DNA synthesis. In serum-starved confluent cells, re-addition of 10% fetal calf serum activated the Erk pathway, decreased p27 expression, and stimulated DNA synthesis to the same levels as in sparse cells. Under these conditions anti-CEACAM1 antibodies de-activated Erk, restored the level of p27, and inhibited DNA synthesis. These data indicate that CEACAM1 mediates contact inhibition of proliferation in cells that are constantly exposed to growth factors, but co-activates growth-factor-induced proliferation in cells that have been starved for growth factors; exposure to extracellular CEACAM1 ligands reverts these responses.

What's known on the subject? and What does the study add? Insulin-like growth factor mRNA-binding protein 3 (IMP3) is an oncofetal protein found to be re-expressed in a series of human cancers including bladder cancer. In vitro analyses showed an invasion and proliferation promoting effect for IMP3. Further in vitro studies suggested that IMP3 is able to bind to the mRNAs of CD44 and insulin-like growth factor 2 (IGF2), enhancing their stability and expression. However, this molecular interaction has not yet been analysed in tumour samples. In the present study, we identified for the first time high IMP3 tissue protein expression as an independent predictor of poor patients' survival in muscle-invasive bladder cancer. Furthermore, there was no correlation between IMP3 and its molecular targets in bladder carcinoma specimens and concluded that the tumour-promoting effect of IMP3 is not related to its regulatory action on IGF2 and CD44. To assess the prognostic value and molecular actions of the oncofetal protein insulin-like growth factor mRNA-binding protein 3 (IMP3) in muscle-invasive bladder cancer (BC). IMP3 expression was analysed by immunohistochemistry, real-time polymerase chain reaction and Western blot analysis in 224 patients with BC. The molecular targets of IMP3; CD44, insulin-like growth factor 2 (IGF2) and its receptor the IGF1 receptor (IGF1-R) were also investigated. Expression levels were correlated with clinical follow-up data by using both univariate and multivariate Cox regression analyses. IMP3 mRNA and protein levels were significantly elevated in high-stage and high-grade muscle-invasive BC. In muscle-invasive BC IMP3 protein but not gene expression proved to be an independent predictor of disease-specific (hazard ratio [HR] 2.58, 95% confidence interval [CI] 1.28-4.56, P = 0.004) and overall survival (HR 2.07, 95% CI 1.12-3.82, P = 0.020). The expression levels of IGF2 and CD44 showed no correlation with that of IMP3. High IMP3 protein levels may identify patients with BC at high risk of disease progression and may therefore select patients for a more intensive therapy or for a strict follow-up. Its high expression in high-grade bladder carcinoma cells makes IMP3 for an attractive target for therapy. The tumour promoting effect of IMP3 is independent from its regulatory action on IGF2 and CD44 expression.

Attachment to the host gastric mucosa is a key step in Helicobacter pylori infection. Recently, a novel adhesin, HopQ, was shown to bind distinct host CEACAM proteins—an interaction that was found to be essential for the translocation of CagA, a key virulence factor of H. pylori. The HopQ–CEACAM1 co-crystal structure revealed a binding mode dependent on loops in HopQ that are clasped by disulfide bonds. In this study, we investigated the importance of these cysteine residues for CEACAM1 engagement by H. pylori. We observed a loss of CEACAM1 binding and CagA translocation upon disruption of the disulfide bond in loop CL1 (connecting C103 to C132 in HopQ). Deletion of the Dsb-like oxidoreductase HP0231 did not affect cell surface expression of HopQ or alter the interaction of H. pylori with target cells. Although HP0231 deletion was previously described to impede CagA translocation, our results indicate that this occurs through a HopQ-independent mechanism. Together, our results open ...

Malignant melanoma is the most aggressive and treatment resistant type of skin cancer. It is characterized by continuously rising incidence and high mortality rate due to its high metastatic potential. Various types of cell adhesion molecules have been implicated in tumor progression in melanoma. One of these, the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), is a multi-functional receptor protein potentially expressed in epithelia, endothelia, and leukocytes. CEACAM1 often appears in four isoforms differing in the length of their extracellular and intracellular domains. Both the CEACAM1 expression in general, and the ratio of the expressed CEACAM1 splice variants appear very dynamic. They depend on both the cell activation stage and the cell growth phase. Interestingly, normal melanocytes are negative for CEACAM1, while melanomas often show high expression. As a cell–cell communication molecule, CEACAM1 mediates the direct interaction between tumor and immune...

A dysbalance between effector T cells (Tconv) and regulatory T cells (Tregs) and impaired Treg function can cause autoimmune liver disease. Therefore, it is important to identify molecular mechanisms that control Treg homeostasis. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a) is an immune coreceptor with dichotomous roles in T-cell regulation: its short isoform (CEACAM1S) can activate T cells and induce Tregs, whereas its long isoform (CEACAM1L), containing two intracellular immune receptor tyrosine-based inhibitory motifs, can inhibit activated T-cell function. In the liver, CEACAM1 has antifibrotic effects in models of nonalcoholic steatohepatitis. However, its role in immune-mediated hepatitis is unknown. In the mouse model of concanavalin A-induced CD4 T-cell-dependent liver injury, liver damage was aggravated and persisted in Ceacam1 mice. Concomitantly, we observed hyperexpansion of Tconv, but reduction of interleukin (IL)-2 production and hepatic ...

The human gastric pathogen is a major causative agent of gastritis, peptic ulcer disease, and gastric cancer. As part of its adhesive lifestyle, the bacterium targets members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family by the conserved outer membrane adhesin HopQ. The HopQ-CEACAM1 interaction is associated with inflammatory responses and enables the intracellular delivery and phosphorylation of the CagA oncoprotein via a yet unknown mechanism. Here, we generated crystal structures of HopQ isotypes I and II bound to the N-terminal domain of human CEACAM1 (C1ND) and elucidated the structural basis of specificity toward human CEACAM receptors. Both HopQ alleles target the β-strands G, F, and C of C1ND, which form the dimerization interface in homo- and heterophilic CEACAM interactions. Using SAXS, we show that the HopQ ectodomain is sufficient to induce C1ND monomerization and thus providing a route to influence CEACAM-mediated cell adherence and sign...

Dysfunction of CD8 T cells can lead to the development of chronic viral infection. Identifying mechanisms responsible for such T cell dysfunction is therefore of great importance to understand how to prevent persistent viral infection. Here we show using lymphocytic choriomeningitis virus (LCMV) infection that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is fundamental for recruiting lymphocyte-specific protein kinase (Lck) into the T cell receptor complex to form an efficient immunological synapse. CEACAM1 is essential for activation of CD8 T cells, and the absence of CEACAM1 on virus-specific CD8 T cells limits the antiviral CD8 T cell response. Treatment with anti-CEACAM1 antibody stabilizes Lck in the immunological synapse, prevents CD8 T cell exhaustion, and improves control of virus infection in vivo. Treatment of human virus-specific CD8 T cells with anti-CEACAM1 antibody similarly enhances their proliferation. We conclude that CEACAM1 is an important r...

Candida albicans colonizes human mucosa, including the gastrointestinal tract, as a commensal. In immunocompromised patients, C. albicans can breach the intestinal epithelial barrier and cause fatal invasive infections. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a), CEACAM5 (CEA), and CEACAM6 (CD66c) are immunomodulatory receptors expressed on human mucosa and are recruited by bacterial and viral pathogens. Here we show for the first time that a fungal pathogen (i.e., C. albicans ) also binds directly to the extracellular domain of human CEACAM1, CEACAM3, CEACAM5, and CEACAM6. Binding was specific for human CEACAMs and mediated by the N-terminal IgV-like domain. In enterocytic C2BBe1 cells, C. albicans caused a transient tyrosine phosphorylation of CEACAM1 and induced higher expression of membrane-bound CEACAM1 and soluble CEACAM6. Lack of the CEACAM1 receptor after short hairpin RNA (shRNA) knockdown abolished CXCL8 (interleukin-8) secretion by C2BBe1 c...

CEACAM1 and CEACAM6 belong to the carcinoembryonic antigen (CEA) family and may play an immune-modulatory role during pregnancy. The aim of the study was to determine the blood serum levels of soluble CEACAM1 and CEACAM6 over the course of pregnancy and postpartum. CEACAM1 and CEACAM6 levels were determined with customized in-house Sandwich-enzyme-linked immunosorbent assay (ELISA) systems. The study population (n=125) was divided into four groups according to the pregnancy trimester and postpartum. Additionally, samples of non-pregnant women (n=14) were analyzed. Serum levels of CEACAM1 in healthy pregnant women were much lower than in non-pregnant women, a difference not seen for CEACAM6. Comparison between the trimesters and postpartum revealed a significant difference in CEACAM1 serum levels. The highest CEACAM1 levels were detected in third trimester. These levels were statistically significantly different from the CEACAM1 levels in first trimester and second trimester. The low...

A major challenge in corneal tissue engineering and lamellar corneal transplantation is to develop synthetic scaffolds able to simulate the optical and mechanical properties of the native cornea. As a carrier, the graft scaffolds should provide the basis for anchorage, repair and regeneration. Although quite a number of scaffolds have been engineered to date, they have not been able to simultaneously recapitulate chemical, mechanical, and structural properties of the corneal extracellular matrix (ECM). Here, we examined different compositions of elastomeric biodegradable poly (glycerol sebacate) (PGS)-poly (ε-caprolactone) (PCL) nanofibrous scaffolds with respect to their cyto- and immunocompatibility. These scaffolds were semi-transparent with well-defined mechanical properties and direct positive effects on viability of human corneal endothelial cells (HCEC) and human conjunctival epithelial cells (HCjEC). Moreover, within 3days HCEC established monolayers with the hexagonal morph...

Helicobacter pylori specifically colonizes the human gastric epithelium and is the major causative agent for ulcer disease and gastric cancer development. Here, we identify members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family as receptors of H. pylori and show that HopQ is the surface-exposed adhesin that specifically binds human CEACAM1, CEACAM3, CEACAM5 and CEACAM6. HopQ-CEACAM binding is glycan-independent and targeted to the N-domain. H. pylori binding induces CEACAM1-mediated signalling, and the HopQ-CEACAM1 interaction enables translocation of the virulence factor CagA into host cells and enhances the release of pro-inflammatory mediators such as interleukin-8. Based on the crystal structure of HopQ, we found that a β-hairpin insertion (HopQ-ID) in HopQ's extracellular 3+4 helix bundle domain is important for CEACAM binding. A peptide derived from this domain competitively inhibits HopQ-mediated activation of the Cag virulence pathway, as ...

EpCAM is overexpressed in many neoplasms including ovarian cancer. We screened the EpCAM-coding gene TACSTD1 for single nucleotide polymorphisms (SNPs), which could alter ovarian cancer risk, impact upon disease progression, or alter binding of the therapeutic EpCAM-binding antibody, catumaxomab. DNA fragments of 10 healthy volunteers were analyzed to identify SNPs. Subsequently, DNA of ovarian cancer patients (n = 117) and age-matched healthy controls (n = 115) was genotyped by restriction fragment length polymorphism and pyrosequencing. TACSTD1 genotypes 4461T>C were cloned into a gene expression vector; Hek293 cells were subsequently used for stable transfection. FACS analysis of the transfected Hek293 cells was conducted with HO-3-the EpCAM binding site of catumaxomab-to determine antibody binding. One SNP was detected in exon 3 (4461T>C; rs1126497), resulting in an amino acid exchange at position 115 (Met115Thr). Another polymorphism was found in the 3'UTR (17225A>...

Family members, friends and colleagues of Prof. Dr. Dr. h.c. Björn Öbrink were deeply saddened to learn of his sudden death on August 21, 2015. Björn was one of the pioneers in cell adhesion research. Reading an article written by Björn, one quickly recognizes his outstanding qualities as a scientist: knowledgeable, innovative, precise and detailed in order to enable reproduction of his reported results. When meeting Björn, you were captured by his personality: warm-hearted, modest, and, above all, devoted to truth.

In the past three decades many efforts have been undertaken to understand the mechanisms of tumor angiogenesis. The introduction of the anti-angiogenic drugs in tumor therapy during the last few years necessitates the establishment of new techniques enabling molecular imaging of vascular remodeling. Tumor imaging by X-ray, CT, MRI and ultrasound has to be improved by coupling with molecular markers targeting the tumor vessels. The determination of tumor size as commonly used is not appropriate since the extended necrosis under anti-angiogenic therapy does not result in a reduction of tumor diameter. But remodeling of the tumor vessels under anti-angiogenic therapy obviously occurs at an early stage and seems to be a convincing parameter for tumor imaging. Despite the enormous progress in this field during the last few years the resolution is still not high enough to evaluate the remodeling of the microtumor vessels. Thus, new imaging approaches are needed to overcome this issue.

In the past three decades many efforts have been undertaken to understand the mechanisms of tumor angiogenesis. The introduction of anti-angiogenic drugs in tumor therapy during the last few years necessitates the establishment of new techniques enabling molecular imaging of tumor vascular remodelling. The determination of tumor size as commonly used is not appropriate since the extended necrosis under anti-angiogenic therapy does not necessarily result in the reduction of tumor diameter. The basis for the molecular imaging of tumor blood vessels is the remodelling of the tumor vessels under anti-angiogenic therapy which obviously occurs at an early stage and seems to be a convincing parameter. Beside the enormous progress in this field during the last few years the resolution is still not high enough to evaluate the remodelling of the micro tumor vessels. New imaging approaches combining specific molecular markers for tumor vessels with the different imaging techniques are needed to overcome this issue as exemplarily discussed for prostate cancer in this review. Molecular contrast agents targeting the vasculature will allow clinicians the visualization of vascular remodelling processes taking place under anti-angiogenic therapy and improve tumor diagnosis and follow-up.