Publications by authors named "Diana Mechtcheriakova"

47 Publications

The Immune Phenotype of Isolated Lymphoid Structures in Non-Tumorous Colon Mucosa Encrypts the Information on Pathobiology of Metastatic Colorectal Cancer.

Cancers (Basel) 2020 Oct 25;12(11). Epub 2020 Oct 25.

Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna 1090, Austria.

The gut-associated lymphoid tissue represents an integral part of the immune system. Among the powerful players of the mucosa-associated lymphoid tissue are isolated lymphoid structures (ILSs), which as information centers, drive the local (and systemic) adaptive immune responses. Germinal center reactions, taking place within ILSs, involve the coordinated action of various immune cell types with a central role given to B cells. In the current study, we aimed at dissecting the impact of ILSs within non-tumorous colon tissue (NT) on the pathobiology of colorectal cancer (CRC) with metastasis in the liver (CRCLM). In particular, we focused on the immune phenotypes of ILSs and ectopic lymphoid structures (ELSs), built up at matching primary and metastatic tumor sites. We implemented an integrative analysis strategy on the basis of tissue image cytometry and clonality assessment to explore the immune phenotype of ILS/ELS at three tissue entities: NT, CRC, and CRCLM (69 specimens in total). Applying a panel of lineage markers used for immunostaining, we characterized and compared the anatomical features, the cellular composition, the activation, and proliferation status of ILSs and ELSs, and assessed the clinical relevance of staining-derived data sets. Our major discovery was that ILS characteristics at the NT site predefine the immune phenotype of ELSs at CRC and CRCLM. Thereby, B-cell-enriched (CD20) and highly proliferative (Ki67) ILSs and ELSs were found to be associated with improved clinical outcome in terms of survival and enabled patient stratification into risk groups. Moreover, the data revealed a linkage between B-cell clonality at the NT site and the metastatic characteristics of the tumor in the distant liver tissue. Consolidation of immunostaining-based findings with the results of compendium-wide transcriptomic analysis furthermore proposed CD27 as a novel marker of T follicular helper cells within lymphoid structures. Overall, the study nominates the ILS immune phenotype as a novel prognostic marker for patients with metastatic CRC.
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http://dx.doi.org/10.3390/cancers12113117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692185PMC
October 2020

Two common polymorphic variants of OATP4A1 as potential risk factors for colorectal cancer.

Oncol Lett 2020 Nov 17;20(5):252. Epub 2020 Sep 17.

Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, A-1090 Vienna, Austria.

Genetic variations in the organic-anion-transporting polypeptide (OATP)-encoding solute carrier of organic anions () genes can promote cancer development and progression. The overexpression of solute carrier organic anion transporter family member 4A1 (OATP4A1), a transporter for steroid hormones, prostaglandins, and bile acids, has been previously associated with tumor recurrence and progression in colorectal cancer (CRC). Therefore, the present study aimed to investigate the association between 2 frequent single nucleotide polymorphisms (SNPs) in (rs34419428, R70Q; rs1047099G, V78I) and CRC predisposition. Following restriction fragment length polymorphism-PCR analysis in 178 patients with CRC [Union for International Cancer Control (UICC) stage I/II] and 65 healthy controls, no significant difference was observed in allele frequency and the number of heterozygous/homozygous individuals between the groups. Notably, the R70Q minor allele was identified to be associated with the V78I minor allele in the genome. Comparing of the individual genotypes of CRC patients to clinical data, including sex, UICC-stage and relapse revealed no increased risk for CRC. In addition, the OATP4A1 immunoreactivity assay in paraffin-embedded CRC and adjacent non-tumorous mucosa sections, examined using quantitative microscopy image analysis, did not reveal any association with these polymorphisms. No significant differences were observed in the expression levels, localization, and sodium fluorescein transport capacity among the OATP4A1 variants, which was studied using functional assays in Sf9-insect and A431 tumor cells overexpressing the 2 single and a double mutant OATP4A1 SNP variants. These results suggested that the 2 most frequent polymorphisms located in the first intracellular loop of OATP4A1 do not associate with CRC predisposition and tumor recurrence. They are unlikely to affect the outcome of CRC in patients.
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http://dx.doi.org/10.3892/ol.2020.12115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509609PMC
November 2020

Metabolism of Estrogens: Turnover Differs Between Platinum-Sensitive and -Resistant High-Grade Serous Ovarian Cancer Cells.

Cancers (Basel) 2020 Jan 23;12(2). Epub 2020 Jan 23.

Division of Clinical Pharmacy and Diagnostics, Department of Pharmaceutical Chemistry, University of Vienna, 1090 Vienna, Austria.

High-grade serous ovarian cancer (HGSOC) is currently treated with cytoreductive surgery and platinum-based chemotherapy. The majority of patients show a primary response; however, many rapidly develop drug resistance. Antiestrogens have been studied as low toxic treatment options for HGSOC, with higher response rates in platinum-sensitive cases. Mechanisms for this difference in response remain unknown. Therefore, the present study investigated the impact of platinum resistance on steroid metabolism in six established HGSOC cell lines sensitive and resistant against carboplatin using a high-resolution mass spectrometry assay to simultaneously quantify the ten main steroids of the estrogenic metabolic pathway. An up to 60-fold higher formation of steroid hormones and their sulfated or glucuronidated metabolites was observed in carboplatin-sensitive cells, which was reversible by treatment with interleukin-6 (IL-6). Conversely, treatment of carboplatin-resistant cells expressing high levels of endogenous IL-6 with the monoclonal anti-IL-6R antibody tocilizumab changed their status to "platinum-sensitive", exhibiting a decreased IC value for carboplatin, decreased growth, and significantly higher estrogen metabolism. Analysis of these metabolic differences could help to detect platinum resistance in HGSOC patients earlier, thereby allowing more efficient interventions.
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http://dx.doi.org/10.3390/cancers12020279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072378PMC
January 2020

Interrelations of Sphingolipid and Lysophosphatidate Signaling with Immune System in Ovarian Cancer.

Comput Struct Biotechnol J 2019 10;17:537-560. Epub 2019 Apr 10.

Molecular Systems Biology and Pathophysiology Research Group, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

The sphingolipid and lysophosphatidate regulatory networks impact diverse mechanisms attributed to cancer cells and the tumor immune microenvironment. Deciphering the complexity demands implementation of a holistic approach combined with higher-resolution techniques. We implemented a multi-modular integrative approach consolidating the latest accomplishments in gene expression profiling, prognostic/predictive modeling, next generation digital pathology, and systems biology for epithelial ovarian cancer. We assessed patient-specific transcriptional profiles using the sphingolipid/lysophosphatidate/immune-associated signature. This revealed novel sphingolipid/lysophosphatidate-immune gene-gene associations and distinguished tumor subtypes with immune high/low context. These were characterized by robust differences in sphingolipid-/lysophosphatidate-related checkpoints and the drug response. The analysis also nominates novel survival models for stratification of patients with , , , , and emerging as the most prognostically important genes. Alignment of proprietary data with curated transcriptomic data from public databases across a variety of malignancies (over 600 categories; over 21,000 arrays) showed specificity for ovarian carcinoma. Our systems approach identified novel sphingolipid-lysophosphatidate-immune checkpoints and networks underlying tumor immune heterogeneity and disease outcomes. This holds great promise for delivering novel stratifying and targeting strategies.
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http://dx.doi.org/10.1016/j.csbj.2019.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479272PMC
April 2019

Clinical Significance of Organic Anion Transporting Polypeptide Gene Expression in High-Grade Serous Ovarian Cancer.

Front Pharmacol 2018 7;9:842. Epub 2018 Aug 7.

Institute of Clinical Biometrics, Center for Medical Statistics, Informatics, and Intelligent Systems, Medical University of Vienna, Vienna, Austria.

High-grade serous ovarian cancer (HGSOC) is considered the most deadly and frequently occurring type of ovarian cancer and is associated with various molecular compositions and growth patterns. Evaluating the mRNA expression pattern of the organic anion transporters (OATPs) encoded by genes may allow for improved stratification of HGSOC patients for targeted invention. The expression of mRNA and genes coding for putative functionally related ABC-efflux pumps, enzymes, pregnane-X-receptor, and (coding for estrogen receptors ERα and ERß) and HER-2 were assessed using RT-qPCR. The expression levels were assessed in a cohort of 135 HGSOC patients to elucidate the independent impact of the expression pattern on the overall survival (OS). For identification of putative regulatory networks, Graphical Gaussian Models were constructed from the expression data with a tuning parameter K varying between meaningful borders (Pils et al., 2012; Auer et al., 2015, 2017; Kurman and Shih Ie, 2016; Karam et al., 2017; Labidi-Galy et al., 2017; Salomon-Perzynski et al., 2017; Sukhbaatar et al., 2017). The final value used ( = 4) was determined by maximizing the proportion of explained variation of the corresponding LASSO Cox regression model for OS. The following two networks of directly correlated genes were identified: (i) with implicated in estrogen homeostasis; and (ii) two ABC-efflux pumps in the immune regulation () with and . Combining LASSO Cox regression and univariate Cox regression analyses, coding for OATP5A1, an estrogen metabolite transporter located in the cytoplasm and plasma membranes of ovarian cancer cells, was identified as significant and independent prognostic factor for OS (HR = 0.68, CI 0.49-0.93; = 0.031). Furthermore, results indicated the benefits of patients with high expression by adding 5.1% to the 12.8% of the proportion of explained variation (PEV) for clinicopathological parameters known for prognostic significance (FIGO stage, age and residual tumor after debulking). Additionally, overlap with previously described signatures that indicated a more favorable prognosis for ovarian cancer patients was shown for , the network as well as . Furthermore, expression of and , which are important for PGE degradation, was associated with the non-miliary peritoneal tumor spreading. In conclusion, the present findings suggested that and the related molecules identified as potential biomarkers in HGSOC may be useful for the development of novel therapeutic strategies.
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http://dx.doi.org/10.3389/fphar.2018.00842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090214PMC
August 2018

Resveratrol Inhibits Key Steps of Steroid Metabolism in a Human Estrogen-Receptor Positive Breast Cancer Model: Impact on Cellular Proliferation.

Front Pharmacol 2018 10;9:742. Epub 2018 Jul 10.

Division of Clinical Pharmacy and Diagnostics, Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria.

The role of resveratrol (RES) in preventing breast cancer is controversial, as low concentrations may stimulate the proliferation of estrogen-receptor alpha positive (ERα+) breast cancer cells. As metabolism is the key factor in altering cellular estrogens, thereby influencing breast tumor growth, we investigated the effects of RES on the formation of estrogen metabolites, namely 4-androstene-3,17-dione (AD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone-3--sulfate (DHEA-S), estrone (E1), estrone-3-sulfate (E1-S), 17β-estradiol (E2), 17β-estradiol-3--(β-D-glucuronide) (E2-G), 17β-estradiol-3--sulfate (E2-S), 16α-hydroxy-17β-estradiol (estriol, E3), and testosterone (T) in ERα- MDA-MB-231 and ERα+ MCF-7 cells. Incubation of both of the cell lines with the hormone precursors DHEA and E1 revealed that sulfation and glucuronidation were preferred metabolic pathways for DHEA, E1 and E2 in MCF-7 cells, compared with in MDA-MB-231 cells, as the V values were significantly higher (DHEA-S: 2873.0 ± 327.4 fmol/10 cells/h, E1-S: 30.4 ± 2.5 fmol/10 cells/h, E2-S: 24.7 ± 4.9 fmol/10 cells/h, E2-G: 7.29 ± 1.36 fmol/10 cells/h). RES therefore significantly inhibited DHEA-S, E1-S, E2-S and E2-G formation in MCF-7, but not in MDA-MB-231 cells (Ks: E2-S, 0.73 ± 0.07 μM < E1-S, 0.94 ± 0.03 μM < E2-G, 7.92 ± 0.24 μM < DHEA-S, 13.2 ± 0.2 μM). Suppression of these metabolites subsequently revealed twofold higher levels of active E2, concomitant with an almost twofold increase in MCF-7 cell proliferation, which was the most pronounced upon the addition of 5 μM RES. As the content of RES in food is relatively low, an increased risk of breast cancer progression in women is likely to only be observed following the continuous consumption of high-dose RES supplements. Further long-term human studies simultaneously monitoring free estrogens and their conjugates are therefore highly warranted to evaluate the efficacy and safety of RES supplementation, particularly in patients diagnosed with ERα+ breast cancer.
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http://dx.doi.org/10.3389/fphar.2018.00742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048268PMC
July 2018

Reduced Levels of the Synaptic Functional Regulator FMRP in Dentate Gyrus of the Aging Sprague-Dawley Rat.

Front Aging Neurosci 2017 23;9:384. Epub 2017 Nov 23.

Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Vienna, Austria.

Fragile X mental retardation protein (FMRP) encoded by Fragile X mental retardation 1 () gene is a RNA-binding regulator of mRNA translation, transport and stability with multiple targets responsible for proper synaptic function. Epigenetic silencing of gene expression leads to the development of Fragile X syndrome (FXS) that is characterized by intellectual disability and other behavioral problems including autism. In the rat FXS model, the lack of FMRP caused a deficit in hippocampal-dependent memory. However, the hippocampal changes of FMRP in aging rats are not fully elucidated. The current study addresses the changes in FMRP levels in dentate gyrus (DG) from young (17 weeks) and aging (22 months) Sprague - Dawley rats. The aging animal group showed significant decline in spatial reference memory. Protein samples from five rats per each group were analyzed by quantitative proteomic analysis resulting in 153 significantly changed proteins. FMRP showed significant reduction in aging animals which was confirmed by immunoblotting and immunofluorescence microscopy. Furthermore, bioinformatic analysis of the differential protein dataset revealed several functionally related protein groups with individual interactions with FMRP. These include high representation of the RNA translation and processing machinery connected to FMRP and other RNA-binding regulators including CAPRIN1, the members of Pumilio (PUM) and CUG-BP, Elav-like (CELF) family, and YTH N(6)-methyladenosine RNA-binding proteins (YTHDF). The results of the current study point to the important role of FMRP and regulation of RNA processing in the rat DG and memory decline during the aging process.
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http://dx.doi.org/10.3389/fnagi.2017.00384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703695PMC
November 2017

Fasting metabolism modulates the interleukin-12/interleukin-10 cytokine axis.

PLoS One 2017 24;12(7):e0180900. Epub 2017 Jul 24.

Institute of Immunology, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

A crucial role of cell metabolism in immune cell differentiation and function has been recently established. Growing evidence indicates that metabolic processes impact both, innate and adaptive immunity. Since a down-stream integrator of metabolic alterations, mammalian target of rapamycin (mTOR), is responsible for controlling the balance between pro-inflammatory interleukin (IL)-12 and anti-inflammatory IL-10, we investigated the effect of upstream interference using metabolic modulators on the production of pro- and anti-inflammatory cytokines. Cytokine release and protein expression in human and murine myeloid cells was assessed after toll-like receptor (TLR)-activation and glucose-deprivation or co-treatment with 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators. Additionally, the impact of metabolic interference was analysed in an in-vivo mouse model. Glucose-deprivation by 2-deoxy-D-glucose (2-DG) increased the production of IL-12p40 and IL-23p19 in monocytes, but dose-dependently inhibited the release of anti-inflammatory IL-10. Similar effects have been observed using pharmacological AMPK activation. Consistently, an inhibition of the tuberous sclerosis complex-mTOR pathway was observed. In line with our in vitro observations, glycolysis inhibition with 2-DG showed significantly reduced bacterial burden in a Th2-prone Listeria monocytogenes mouse infection model. In conclusion, we showed that fasting metabolism modulates the IL-12/IL-10 cytokine balance, establishing novel targets for metabolism-based immune-modulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180900PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524343PMC
September 2017

Sphingosine 1-phosphate signaling in bone remodeling: multifaceted roles and therapeutic potential.

Expert Opin Ther Targets 2017 07 7;21(7):725-737. Epub 2017 Jun 7.

a Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology , Medical University of Vienna , Vienna , Austria.

Introduction: Sphingolipids belong to a complex class of lipid molecules that are crucially involved in the regulation of important biological processes including proliferation, migration and apoptosis. Given the significant progress made in understanding the sphingolipid pathobiology of several diseases, sphingolipid-related checkpoints emerge as attractive targets. Recent data indicate the multifaceted contribution of the sphingolipid machinery to osteoclast - osteoblast crosstalk, representing one of the pivotal interactions underlying bone homeostasis. Imbalances in the interplay of osteoblasts and osteoclasts might lead to bone-related diseases such as osteoporosis, rheumatoid arthritis, and bone metastases. Areas covered: We summarize and analyze the progress made in bone research in the context of the current knowledge of sphingolipid-related mechanisms regulating bone remodeling. Particular emphasis was given to bioactive sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs). Moreover, the mechanisms of how dysregulations of this machinery cause bone diseases, are covered. Expert opinion: In the context of bone diseases, pharmacological interference with sphingolipid machinery may lead to novel directions in therapeutic strategies. Implementation of knowledge derived from in vivo animal models and in vitro studies using pharmacological agents to manipulate the S1P/S1PRs axes suggests S1PR2 and S1PR3 as potential drug targets, particularly in conjunction with technology for local drug delivery.
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http://dx.doi.org/10.1080/14728222.2017.1332180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470107PMC
July 2017

Activation of the ileal neuroendocrine tumor cell line P-STS by acetylcholine is amplified by histamine: role of H3R and H4R.

Sci Rep 2017 05 2;7(1):1313. Epub 2017 May 2.

Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, 1090, Austria.

Neuroendocrine tumors may present with pseudoallergic reactions like diarrhea and idiopathic anaphylaxis. Here we present the P-STS human ileal neuroendocrine cell line as a model cell line for these tumors. Neuroendocrine markers and changes in cytoplasmic calcium concentration ([Ca]i) in response to several possible activators of 5-hydroxytryptamine (5-HT) release were analyzed. P-STS cells still expressed chromogranin A and synaptophysin after 2 years of culture. Tryptophan hydroxylase 1 mRNA and a low amount of 5-HT were also detected. Acetylcholine (ACh) caused a rise in [Ca]i. Somatostatin inhibited, whereas histamine (HA) but not the HA receptor ligand betahistine enhanced activation by ACh. The [Ca]i response to ACh/HA was inhibited by the HA receptor H3 (H3R) agonist methimepip and by the antidepressant imipramine. Further [Ca]i response studies indicated the presence of H4Rs and of a functional calcium sensing receptor. High or low affinity IgE receptor protein or mRNA were not detected. Taken together, neuroendocrine markers and response to intestinal neurotransmitters approve the P-STS cell line as a valuable model for enterochromaffin cells. Enhancement of their ACh-induced pro-secretory response by HA, with a role for H3R and H4R, suggests an amplifying role of neuroendocrine cells in allergen-induced diarrhea or anaphylaxis.
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http://dx.doi.org/10.1038/s41598-017-01453-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430954PMC
May 2017

ICAM-1 Binding Rhinoviruses Enter HeLa Cells via Multiple Pathways and Travel to Distinct Intracellular Compartments for Uncoating.

Viruses 2017 04 1;9(4). Epub 2017 Apr 1.

Department of Pathophysiology and Allergy Research, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Wien, Austria.

Of the more than 150 human rhinovirus (RV) serotypes, 89 utilize intercellular adhesion molecule-1 (ICAM-1) for cell entry. These belong either to species A or B. We recently demonstrated that RV-B14 and RV-A89, despite binding this same receptor, are routed into distinct endosomal compartments for release of their RNA into the cytosol. To gain insight into the underlying mechanism we now comparatively investigate the port of entry, temperature-dependence of uncoating, and intracellular routing of RV-B3, RV-B14, RV-A16, and RV-A89 in HeLa cells. The effect of various drugs blocking distinct stages on the individual pathways was determined via comparing the number of infected cells in a TissueFaxs instrument. We found that RV-B14 and RV-A89 enter via clathrin-, dynamin-, and cholesterol-dependent pathways, as well as by macropinocytosis. Drugs interfering with actin function similarly blocked entry of all four viruses, indicating their dependence on a dynamic actin network. However, uniquely, RV-A89 was able to produce progeny when internalized at 20 °C followed by neutralizing the endosomal pH and further incubation at 37 °C. Blocking dynein-dependent endosomal transport prevented uncoating of RV-A16 and RV-A89, but not of RV-B3 and RV-B14, indicative for routing of RV-A16 and RV-A89 into the endocytic recycling compartment for uncoating. Our results call for caution when developing drugs aimed at targeting entry or intracellular trafficking of all rhinovirus serotypes.
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http://dx.doi.org/10.3390/v9040068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408674PMC
April 2017

Human skin dendritic cell fate is differentially regulated by the monocyte identity factor Kruppel-like factor 4 during steady state and inflammation.

J Allergy Clin Immunol 2017 Jun 11;139(6):1873-1884.e10. Epub 2016 Oct 11.

Institute of Pathophysiology and Immunology, Medical University of Graz, Graz, Austria; Institute of Immunology, Medical University of Vienna, Vienna, Austria. Electronic address:

Background: Langerhans cell (LC) networks play key roles in immunity and tolerance at body surfaces. LCs are established prenatally and can be replenished from blood monocytes. Unlike skin-resident dermal DCs (dDCs)/interstitial-type DCs and inflammatory dendritic epidermal cells appearing in dermatitis/eczema lesions, LCs lack key monocyte-affiliated markers. Inversely, LCs express various epithelial genes critical for their long-term peripheral tissue residency.

Objective: Dendritic cells (DCs) are functionally involved in inflammatory diseases; however, the mechanisms remained poorly understood.

Methods: In vitro differentiation models of human DCs, gene profiling, gene transduction, and immunohistology were used to identify molecules involved in DC subset specification.

Results: Here we identified the monocyte/macrophage lineage identity transcription factor Kruppel-like factor 4 (KLF4) to be inhibited during LC differentiation from human blood monocytes. Conversely, KLF4 is maintained or induced during dermal DC and monocyte-derived dendritic cell/inflammatory dendritic epidermal cell differentiation. We showed that in monocytic cells KLF4 has to be repressed to allow their differentiation into LCs. Moreover, respective KLF4 levels in DC subsets positively correlate with proinflammatory characteristics. We identified epithelial Notch signaling to repress KLF4 in monocytes undergoing LC commitment. Loss of KLF4 in monocytes transcriptionally derepresses Runt-related transcription factor 3 in response to TGF-β1, thereby allowing LC differentiation marked by a low cytokine expression profile.

Conclusion: Monocyte differentiation into LCs depends on activation of Notch signaling and the concomitant loss of KLF4.
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http://dx.doi.org/10.1016/j.jaci.2016.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538449PMC
June 2017

AID/APOBEC-network reconstruction identifies pathways associated with survival in ovarian cancer.

BMC Genomics 2016 08 16;17(1):643. Epub 2016 Aug 16.

Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Background: Building up of pathway-/disease-relevant signatures provides a persuasive tool for understanding the functional relevance of gene alterations and gene network associations in multifactorial human diseases. Ovarian cancer is a highly complex heterogeneous malignancy in respect of tumor anatomy, tumor microenvironment including pro-/antitumor immunity and inflammation; still, it is generally treated as single disease. Thus, further approaches to investigate novel aspects of ovarian cancer pathogenesis aiming to provide a personalized strategy to clinical decision making are of high priority. Herein we assessed the contribution of the AID/APOBEC family and their associated genes given the remarkable ability of AID and APOBECs to edit DNA/RNA, and as such, providing tools for genetic and epigenetic alterations potentially leading to reprogramming of tumor cells, stroma and immune cells.

Results: We structured the study by three consecutive analytical modules, which include the multigene-based expression profiling in a cohort of patients with primary serous ovarian cancer using a self-created AID/APOBEC-associated gene signature, building up of multivariable survival models with high predictive accuracy and nomination of top-ranked candidate/target genes according to their prognostic impact, and systems biology-based reconstruction of the AID/APOBEC-driven disease-relevant mechanisms using transcriptomics data from ovarian cancer samples. We demonstrated that inclusion of the AID/APOBEC signature-based variables significantly improves the clinicopathological variables-based survival prognostication allowing significant patient stratification. Furthermore, several of the profiling-derived variables such as ID3, PTPRC/CD45, AID, APOBEC3G, and ID2 exceed the prognostic impact of some clinicopathological variables. We next extended the signature-/modeling-based knowledge by extracting top genes co-regulated with target molecules in ovarian cancer tissues and dissected potential networks/pathways/regulators contributing to pathomechanisms. We thereby revealed that the AID/APOBEC-related network in ovarian cancer is particularly associated with remodeling/fibrotic pathways, altered immune response, and autoimmune disorders with inflammatory background.

Conclusions: The herein study is, to our knowledge, the first one linking expression of entire AID/APOBECs and interacting genes with clinical outcome with respect to survival of cancer patients. Overall, data propose a novel AID/APOBEC-derived survival model for patient risk assessment and reconstitute mapping to molecular pathways. The established study algorithm can be applied further for any biologically relevant signature and any type of diseased tissue.
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http://dx.doi.org/10.1186/s12864-016-3001-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986275PMC
August 2016

A detailed proteomic profiling of plasma membrane from zebrafish brain.

Proteomics Clin Appl 2016 12 12;10(12):1264-1268. Epub 2016 Aug 12.

Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Vienna, Austria.

Zebrafish (Danio rerio) is a well-established model organism in developmental biology and disease modeling. In recent years, an increasing amount of studies used zebrafish to analyze the genetic changes underlying various neurological disorders. The brain plasma membrane proteome represents the major subsets of signaling proteins and promising drug targets, but is often understudied due to traditional experimental difficulties including problems with solubility, detergent removal, or low abundance. Here, we report a comprehensive dataset of the proteins identified in the enriched plasma membrane of the zebrafish brain by applying sequential trypsin/chymotrypsin digestion with multidimensional LC-MS/MS. A total number of 97 017 peptide groups corresponding to 9201 proteins were identified. These were annotated in various molecular functions or neurological disorders. The dataset of the current study provides a useful data source for further utilizing zebrafish in basic and clinical neuroscience.
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http://dx.doi.org/10.1002/prca.201600081DOI Listing
December 2016

Exploring the role of sphingolipid machinery during the epithelial to mesenchymal transition program using an integrative approach.

Oncotarget 2016 Apr;7(16):22295-323

Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.

The epithelial to mesenchymal transition (EMT) program is activated in epithelial cancer cells and facilitates their ability to metastasize based on enhanced migratory, proliferative, anti-apoptotic, and pluripotent capacities. Given the fundamental impact of sphingolipid machinery to each individual process, the sphingolipid-related mechanisms might be considered among the most prominent drivers/players of EMT; yet, there is still limited knowledge. Given the complexity of the interconnected sphingolipid system, which includes distinct sphingolipid mediators, their synthesizing enzymes, receptors and transporters, we herein apply an integrative approach for assessment of the sphingolipid-associated mechanisms underlying EMT program. We created the sphingolipid-/EMT-relevant 41-gene/23-gene signatures which were applied to denote transcriptional events in a lung cancer cell-based EMT model. Based on defined 35-gene sphingolipid/EMT-attributed signature of regulated genes, we show close associations between EMT markers, genes comprising the sphingolipid network at multiple levels and encoding sphingosine 1-phosphate (S1P)-/ceramide-metabolizing enzymes, S1P and lysophosphatidic acid (LPA) receptors and S1P transporters, pluripotency genes and inflammation-related molecules, and demonstrate the underlying biological pathways and regulators. Mass spectrometry-based sphingolipid analysis revealed an EMT-attributed shift towards increased S1P and LPA accompanied by reduced ceramide levels. Notably, using transcriptomics data across various cell-based perturbations and neoplastic tissues (24193 arrays), we identified the sphingolipid/EMT signature primarily in lung adenocarcinoma tissues; besides, bladder, colorectal and prostate cancers were among the top-ranked. The findings also highlight novel regulatory associations between influenza virus and the sphingolipid/EMT-associated mechanisms. In sum, data propose the multidimensional contribution of sphingolipid machinery to pathological EMT and may yield new biomarkers and therapeutic targets.
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http://dx.doi.org/10.18632/oncotarget.7947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5008362PMC
April 2016

Quantitative proteomics reveals protein kinases and phosphatases in the individual phases of contextual fear conditioning in the C57BL/6J mouse.

Behav Brain Res 2016 Apr 31;303:208-17. Epub 2015 Dec 31.

Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria.

A series of protein kinases and phosphatases (PKPs) have been linked to contextual fear conditioning (cFC) but information is mainly derived from immunochemical studies. It was therefore decided to use an explorative label-free quantitative proteomics approach to concomitantly determine PKPs in hippocampi of mice in the individual phases of cFC. C57BL/6J mice were divided into four groups: three training groups representing the acquisition, consolidation and retrieval phases of cFC and a foot shock control group. Using this approach we identified 32 protein kinases or phosphatases/phosphatase subunits with significantly changed protein levels in one or more training groups as compared to foot shock control. These include members of PKP signalling modules of mitogen-activated protein kinase (MAP3K10, RAF1, KSR2), Ca2+/calmodulin-dependent protein kinase (CaMKIIα, DAPK1), protein kinase C (PRKCD) and protein phosphatases 1, 2A, 2B(3) previously implicated in various learning paradigms. In addition, our analysis showed protein kinases WNK1, LYN, VRK1, ABL1, CDK4, CDKL3, SgK223 and ADCK1, and protein phosphatases PTPRF, ACP1, DNAJC6, SSH2 and UBASH3B that have not been directly linked to fear memory processes so far. Determination of PKPs in the individual cFC phases represents a valuable resource for interpretation of previous and design of future studies on PKPs in memory mechanisms.
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http://dx.doi.org/10.1016/j.bbr.2015.12.033DOI Listing
April 2016

Immunology of Osteoporosis: A Mini-Review.

Gerontology 2016 17;62(2):128-37. Epub 2015 Jun 17.

Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University Vienna, Vienna, Austria.

Osteoporosis is a major cause of fractures and associated morbidity in the aged population. The pathogenesis of osteoporosis is multifactorial; whereas traditional pathophysiological concepts emphasize endocrine mechanisms, it has been recognized that also components of the immune system have a significant impact on bone. Since 2000, when the term 'osteoimmunology' was coined, novel insights into the role of inflammatory cytokines by influencing the fine-tuned balance between bone resorption and bone formation have helped to explain the occurrence of osteoporosis in conjunction with chronic inflammatory reactions. Moreover, the phenomenon of a low-grade, chronic, systemic inflammatory state associated with aging has been defined as 'inflamm-aging' by Claudio Franceschi and has been linked to age-related diseases such as osteoporosis. Given the tight anatomical and physiological coexistence of B cells and the bone-forming units in the bone marrow, a role of B cells in osteoimmunological interactions has long been suspected. Recent findings of B cells as active regulators of the RANK/RANKL/OPG axis, of altered RANKL/OPG production by B cells in HIV-associated bone loss or of a modulated expression of genes linked to B-cell biology in response to estrogen deficiency support this assumption. Furthermore, oxidative stress and the generation of advanced glycation end products have emerged as links between inflammation and bone destruction.
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http://dx.doi.org/10.1159/000431091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821368PMC
December 2016

The calcium-sensing receptor suppresses epithelial-to-mesenchymal transition and stem cell- like phenotype in the colon.

Mol Cancer 2015 Mar 18;14:61. Epub 2015 Mar 18.

Department of Pathophysiology and Allergy Research, Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria.

Background: The calcium sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is expressed also in tissues not directly involved in calcium homeostasis like the colon. We have previously reported that CaSR expression is down-regulated in colorectal cancer (CRC) and that loss of CaSR provides growth advantage to transformed cells. However, detailed mechanisms underlying these processes are largely unknown.

Methods And Results: In a cohort of 111 CRC patients, we found significant inverse correlation between CaSR expression and markers of epithelial-to-mesenchymal transition (EMT), a process involved in tumor development in CRC. The colon of CaSR/PTH double-knockout, as well as the intestine-specific CaSR knockout mice showed significantly increased expression of markers involved in the EMT process. In vitro, stable expression of the CaSR (HT29(CaSR)) gave a more epithelial-like morphology to HT29 colon cancer cells with increased levels of E-Cadherin compared with control cells (HT29(EMP)). The HT29(CaSR) cells had reduced invasive potential, which was attributed to the inhibition of the Wnt/β-catenin pathway as measured by a decrease in nuclear translocation of β-catenin and transcriptional regulation of genes like GSK-3β and Cyclin D1. Expression of a spectrum of different mesenchymal markers was significantly down-regulated in HT29(CaSR) cells. The CaSR was able to block upregulation of mesenchymal markers even in an EMT-inducing environment. Moreover, overexpression of the CaSR led to down-regulation of stem cell-like phenotype.

Conclusions: The results from this study demonstrate that the CaSR inhibits epithelial-to-mesenchymal transition and the acquisition of a stem cell-like phenotype in the colon of mice lacking the CaSR as well as colorectal cancer cells, identifying the CaSR as a key molecule in preventing tumor progression. Our results support the rationale to develop new strategies either preventing CaSR loss or reversing its silencing.
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http://dx.doi.org/10.1186/s12943-015-0330-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405849PMC
March 2015

The major birch pollen allergen Bet v 1 induces different responses in dendritic cells of birch pollen allergic and healthy individuals.

PLoS One 2015 30;10(1):e0117904. Epub 2015 Jan 30.

Department of Pathophysiology and Allergy Research; Center for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Vienna, Austria.

Dendritic cells play a fundamental role in shaping the immune response to allergens. The events that lead to allergic sensitization or tolerance induction during the interaction of the major birch pollen allergen Bet v 1 and dendritic cells are not very well studied. Here, we analyzed the uptake of Bet v 1 and the cross-reactive celery allergen Api g 1 by immature monocyte-derived dendritic cells (iMoDCs) of allergic and normal donors. In addition, we characterized the allergen-triggered intracellular signaling and transcriptional events. Uptake kinetics, competitive binding, and internalization pathways of labeled allergens by iMoDCs were visualized by live-cell imaging. Surface-bound IgE was detected by immunofluorescence microscopy and flow cytometry. Allergen- and IgE-induced gene expression of early growth response genes and Th1 and Th2 related cytokines and chemokines were analyzed by real-time PCR. Phosporylation of signaling kinases was analyzed by Western blot. Internalization of Bet v 1 by iMoDCs of both donor groups, likely by receptor-mediated caveolar endocytosis, followed similar kinetics. Bet v 1 outcompeted Api g 1 in cell surface binding and uptake. MoDCs of allergic and healthy donors displayed surface-bound IgE and showed a pronounced upregulation of Th2 cytokine- and NFκB-dependent genes upon non-specific Fcε receptor cross-linking. In contrast to these IgE-mediated responses, Bet v 1-stimulation increased transcript levels of the Th2 cytokines IL-4 and IL-13 but not of NFκB-related genes in MoDCs of BP allergic donors. Cells of healthy donors were either unresponsive or showed elevated mRNA levels of Th1-promoting chemokines. Moreover, Bet v 1 was able to induce Erk1/2 and p38 MAPK activation in BP allergics but only a slight p38 activation in normal donors. In conclusion, our data indicate that Bet v 1 favors the activation of a Th2 program only in DCs of BP allergic individuals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117904PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311984PMC
January 2016

B cells and ectopic follicular structures: novel players in anti-tumor programming with prognostic power for patients with metastatic colorectal cancer.

PLoS One 2014 6;9(6):e99008. Epub 2014 Jun 6.

Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Remarkably limited information is available about biological mechanisms that determine the disease entity of metastatic colorectal cancer in the liver (CRCLM) with no good clinical parameters to estimate prognosis. For the last few years, understanding the relationship between tumor characteristics and local immune response has gained increasing attention. Given the multifaceted roles of B-cell-driven responses, we aimed to elucidate the immunological imprint of B lymphocytes at the metastatic site, the interrelation with macrophages, and their prognostic relevance. Here we present novel algorithm allowing to assess a link between the local patient-specific immunological capacity and clinical outcome. The microscopy-based imaging platform was used for automated scanning of large-scale tissue sections and subsequent qualitative and quantitative analyses of immune cell subtypes using lineage markers and single-cell recognition strategy. Results indicate massive infiltration of CD45-positive leukocytes confined to the metastatic border. We report for the first time the accumulation of CD20-positive B lymphocytes at the tumor-liver interface comprising the major population within the large CD45-positive aggregates. Strikingly, functionally active, activation-induced cytidine deaminase (AID)-positive ectopic lymphoid structures were found to be assembled within the metastatic margin. Furthermore, the CD20-based data set revealed a strong prognostic power: patients with high CD20 content and/or ectopic follicles had significantly lower risk for disease recurrence as revealed by univariate analysis (p<0.001 for both) and in models adjusted for clinicopathological variables (p<0.001 and p = 0.01, respectively), and showed prolonged overall survival. In contrast, CD68 staining-derived data set did not show an association with clinical outcome. Taken together, we nominate the magnitude of B lymphocytes, including those organized in ectopic follicles, as novel prognostic marker which is superior to clinicopathological parameters. Findings emphasize anti-tumoral role of B cell-driven mechanism(s) and thus indicate a new way of thinking about potential treatment strategies for CRCLM patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099008PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048213PMC
January 2015

Generation of a canine anti-EGFR (ErbB-1) antibody for passive immunotherapy in dog cancer patients.

Mol Cancer Ther 2014 Jul 22;13(7):1777-1790. Epub 2014 Apr 22.

Comparative Immunology and Oncology, Institute of Pathophysiology and Allergy Research, Medical University of Vienna.

Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a "caninized" version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer.
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http://dx.doi.org/10.1158/1535-7163.MCT-13-0288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174294PMC
July 2014

Phenotyping of human melanoma cells reveals a unique composition of receptor targets and a subpopulation co-expressing ErbB4, EPO-R and NGF-R.

PLoS One 2014 29;9(1):e84417. Epub 2014 Jan 29.

Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria ; Division of Hematology & Hemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Austria.

Malignant melanoma is a life-threatening skin cancer increasingly diagnosed in the western world. In advanced disease the prognosis is grave. Growth and metastasis formation in melanomas are regulated by a network of cytokines, cytokine-receptors, and adhesion molecules. However, little is known about surface antigens and target expression profiles in human melanomas. We examined the cell surface antigen profile of human skin melanoma cells by multicolor flow cytometry, and compared their phenotype with 4 melanoma cell lines (A375, 607B, Mel-Juso, SK-Mel28). Melanoma cells were defined as CD45-/CD31- cells co-expressing one or more melanoma-related antigens (CD63, CD146, CD166). In most patients, melanoma cells exhibited ErbB3/Her3, CD44/Pgp-1, ICAM-1/CD54 and IGF-1-R/CD221, but did not express CD20, ErbB2/Her2, KIT/CD117, AC133/CD133 or MDR-1/CD243. Melanoma cell lines were found to display a similar phenotype. In most patients, a distinct subpopulation of melanoma cells (4-40%) expressed the erythropoietin receptor (EPO-R) and ErbB4 together with PD-1 and NGF-R/CD271. Both the EPO-R+ and EPO-R- subpopulations produced melanoma lesions in NOD/SCID IL-2Rgamma(null) (NSG) mice in first and secondary recipients. Normal skin melanocytes did not express ErbB4 or EPO-R, but expressed a functional KIT receptor (CD117) as well as NGF-R, ErbB3/Her3, IGF-1-R and CD44. In conclusion, melanoma cells display a unique composition of surface target antigens and cytokine receptors. Malignant transformation of melanomas is accompanied by loss of KIT and acquisition of EPO-R and ErbB4, both of which are co-expressed with NGF-R and PD-1 in distinct subfractions of melanoma cells. However, expression of EPO-R/ErbB4/PD-1 is not indicative of a selective melanoma-initiating potential.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084417PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906015PMC
September 2014

Eicosanoid modulation by the short-chain fatty acid n-butyrate in human monocytes.

Immunology 2013 Jul;139(3):395-405

Institute of Immunology, Centre of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

n-Butyrate deriving from bacterial fermentation in the mammalian intestine is a key determinant in gastrointestinal homeostasis. We examined the effects of this short-chain fatty acid and Toll-like receptor 2 (TLR) and TLR4 engagement on inflammatory/immunity-associated genes, cyclo-oxygenases (COXs), prostaglandins (PGs) and leukotrienes (LTs) in human monocytes. Before RNA isolation, freshly isolated human monocytes were co-incubated for different time-points with 1 mm n-butyrate alone or in combination with bacterial stimuli. Based on a knowledge-driven approach, a signature of 180 immunity/inflammation-associated genes was picked and real-time PCR analysis was performed. Pathway analysis was carried out using a web-based database analysing program. Based on these gene expression studies the findings were evaluated at the protein/mediator level by Western blot analysis, FACS and ELISA. Following co-incubation with n-butyrate and lipopolysaccharide, key enzymes of the eicosanoid pathway, like PTGS2 (COX-2), TXS, ALOX5, LTA4H and LTC4S, were significantly up-regulated compared with stimulation with lipopolysaccharide alone. Furthermore, release of the lipid mediators PGE(2), 15d-PGJ(2), LTB(4) and thromboxane B(2) was increased by n-butyrate. Regarding signalling, n-butyrate had no additional effect on mitogen-activated protein kinase and interfered differently with early and late phases of nuclear factor-κB signalling. Our results suggest that among many other mediators of eicosanoid signalling n-butyrate massively induces PGE(2) production by increasing the expression of PTGS2 (COX-2) in monocytes following TLR4 and TLR2 activation and induces secretion of LTB(4) and thromboxane B(2). This underscores the role of n-butyrate as a crucial mediator of gut-specific immunity.
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http://dx.doi.org/10.1111/imm.12089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701186PMC
July 2013

Activation-induced cytidine deaminase (AID) linking immunity, chronic inflammation, and cancer.

Cancer Immunol Immunother 2012 Sep 19;61(9):1591-8. Epub 2012 Apr 19.

Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria.

Activation-induced cytidine deaminase (AID) is critically involved in class switch recombination and somatic hypermutation of Ig loci resulting in diversification of antibodies repertoire and production of high-affinity antibodies and as such represents a physiological tool to introduce DNA alterations. These processes take place within germinal centers of secondary lymphoid organs. Under physiological conditions, AID is expressed predominantly in activated B lymphocytes. Because of the mutagenic and recombinogenic potential of AID, its expression and activity is tightly regulated on different levels to minimize the risk of unwanted DNA damage. However, chronic inflammation and, probably, combination of other not-yet-identified factors are able to create a microenvironment sufficient for triggering an aberrant AID expression in B cells and, importantly, in non-B-cell background. Under these circumstances, AID may target also non-Ig genes, including cancer-related genes as oncogenes, tumor suppressor genes, and genomic stability genes, and modulate both genetic and epigenetic information. Despite ongoing progress, the complete understanding of fundamental aspects is still lacking as (1) what are the crucial factors triggering an aberrant AID expression/activity including the impact of Th2-driven inflammation and (2) to what extent may aberrant AID in human non-B cells lead to abnormal cell state associated with an increased rate of genomic alterations as point mutations, small insertions or deletions, and/or recurrent chromosomal translocations during solid tumor development and progression.
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http://dx.doi.org/10.1007/s00262-012-1255-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427704PMC
September 2012

Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting.

Mol Immunol 2012 Apr;50(4):200-9

Institute of Pathophysiology and Allergy Research, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.

To facilitate comparative oncology trials we compared the biological and molecular homologies of canine (dog; Canis lupus familiaris) and human tumor-associated antigens ErbB-1 and -2. Further, we investigated whether they could serve as targets for anti-ErbB-1 (cetuximab) and anti-ErbB-2 antibodies (trastuzumab), which are highly relevant in human clinical oncology. Immunohistochemistry of canine mammary cancer showed ErbB-1 overexpression in 3/10 patients and ErbB-2 in 4/10. We report 91% amino acid homology for ErbB-1 and 92% for ErbB-2 between canine and human molecules. Modeling of canine on human ErbB-1 revealed that the cetuximab epitope only differs by 4 amino acids: Lys443 is replaced by Arg, Ser468 by Asn, Gly471 by Asp, and Asn473 by Lys in canines. The trastuzumab binding site is identical in human and canine ErbB-2 apart from a single amino acid change (Pro557 to Ser). Binding of cetuximab and trastuzumab to canine mammary carcinoma cells CF33, CF41, Sh1b and P114 was confirmed by flow cytometry. Both antibodies significantly inhibited canine tumor cell proliferation partly due to growth arrest in G(0)/G(1) phase. We explain the lower efficiency on the tested canine than on human SKBR3 and A431 cells, by a 2-log lower expression level of the canine ErbB-1 and -2 molecules. Our results indicate significant homology of human and canine Erb-1 and -2 tumor associated antigens. The fact that the canine homologues express the cetuximab and trastuzumab epitopes may facilitate antibody-based immunotherapy in dogs. Importantly, the striking similarities of ErbB-1 and -2 molecules open up avenues towards comparative strategies for targeted drug development.
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http://dx.doi.org/10.1016/j.molimm.2012.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318186PMC
April 2012

Activation-induced cytidine deaminase (AID)-associated multigene signature to assess impact of AID in etiology of diseases with inflammatory component.

PLoS One 2011 3;6(10):e25611. Epub 2011 Oct 3.

Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Activation-induced cytidine deaminase (AID) is expressed in B cells within germinal centers and is critically involved in class switch recombination and somatic hypermutation of immunoglobulin loci. Functionally active AID can additionally be detected within ectopic follicular structures developed at sites of chronic inflammation. Furthermore, AID may target non-Ig genes in B- and non-B-cell background. Therefore, AID-associated effects are of increasing interest in disease areas such as allergy, inflammation, autoimmunity, and cancer.Pathway- or disease-relevant multigene signatures have attracted substantial attention for therapeutic target proposal, diagnostic tools, and monitoring of therapy response. To delineate the impact of AID in etiology of multifactorial diseases, we designed the AID-associated 25-gene signature. Chronic rhinosinusitis with nasal polyps was used as an inflammation-driven airway disease model; high levels of IgE have been previously shown to be present within polyp tissue. Expression levels of 16 genes were found to be modulated in polyps including AID, IgG and IgE mature transcripts which reflect AID activity; clustering algorithm revealed an AID-specific gene signature for the disease state with nasal polyp. Complementary, AID-positive ectopic lymphoid structures were detected within polyp tissues by in situ immunostaining. Our data demonstrate the class switch recombination and somatic hypermutation events likely taking place locally in the airways and in addition to the previously highlighted markers and/or targets as IL5 and IgE suggest novel candidate genes to be considered for treatment of nasal polyposis including among others IL13 and CD23. Thus, the algorithm presented herein including the multigene signature approach, analysis of co-regularities and creation of AID-associated functional network gives an integrated view of biological processes and might be further applied to assess role of altered AID expression in etiology of other diseases, in particular, aberrant immunity and cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025611PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184987PMC
January 2012

Subcellular localization of ceramide kinase and ceramide kinase-like protein requires interplay of their Pleckstrin Homology domain-containing N-terminal regions together with C-terminal domains.

Biochim Biophys Acta 2009 Oct 6;1791(10):1023-30. Epub 2009 Jun 6.

Novartis Institutes for BioMedical Research, Brunnerstrasse 59, A-1235 Vienna, Austria.

Ceramide kinase (CERK) and the ceramide kinase-like protein (CERKL), two related members of the diacylglycerol kinase family, are ill-defined at the molecular level. In particular, what determines their distinctive subcellular localization is not well understood. Here we show that the Pleckstrin Homology (PH) domain of CERK, which is required for Golgi complex localization, can substitute for the N-terminal region of CERKL and allow for wild-type CERKL localization, which is typified by nucleolar accumulation. This demonstrates that determinants for localization of these two enzymes do not lie solely in their PH domain-containing N-terminal regions. Moreover, we present evidence for a previously unrecognized participation of CERK distal sequences in structural stability, localization and activity of the full-length protein. Progressive deletion of CERK and CERKL from the C-terminus revealed similar sequential organization in both proteins, with nuclear import signals in their N-terminal part, and nuclear export signals in their C-terminal part. Furthermore, mutagenesis of individual cysteine residues of a CERK-specific CXXXCXXC motif severely compromised both exportation of CERK from the nucleus and its association with the Golgi complex. Altogether, this work identifies conserved domains in CERK and CERKL as well as new determinants for their subcellular localization. It further suggests a nucleocytoplasmic shuttling mechanism for both proteins that may be defective in CERKL mutant proteins responsible for retinal degenerative diseases.
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http://dx.doi.org/10.1016/j.bbalip.2009.05.009DOI Listing
October 2009

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells.

Cancer Immunol Immunother 2009 Jun 22;58(6):915-30. Epub 2008 Oct 22.

Cutaneous Medicine and Immunotherapy Unit, St. John's Institute of Dermatology, Division of Genetics and Molecular Medicine, King's College London School of Medicine, Guy's Tower, Guy's Hospital, London, UK.

Trastuzumab (Herceptin), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.
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http://dx.doi.org/10.1007/s00262-008-0607-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017872PMC
June 2009

FTY720 rescue therapy in the dark agouti rat model of experimental autoimmune encephalomyelitis: expression of central nervous system genes and reversal of blood-brain-barrier damage.

Brain Pathol 2009 Apr 4;19(2):254-66. Epub 2008 Jun 4.

Novartis Institutes for BioMedical Research, Brunner Strasse 59, Vienna, Austria.

FTY720 (fingolimod) is an oral sphingosine-1 phosphate (S1P) receptor modulator in phase III development for the treatment of multiple sclerosis. To further investigate its mode of action, we analyzed gene expression in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE). FTY720 downregulated inflammatory genes in addition to vascular adhesion molecules. It decreased the matrix metalloproteinase gene MMP-9 and increased its counterregulator--tissue inhibitor of metalloproteinase, TIMP-1--resulting in a proteolytic balance that favors preservation of blood-brain-barrier (BBB) integrity. Furthermore, FTY720 reduced S1P lyase that increases the S1P concentration in the brain, in line with a marked reversal of neurological deficits and raising the possibility for enhanced triggering of S1P receptors on resident brain cells. This is accompanied by an increase in S1P(1) and S1P(5) in contrast with the attenuation of S1P(3) and S1P(4). Late-stage rescue therapy with FTY720, even up to 1 month after EAE onset, reversed BBB leakiness and reduced demyelination, along with normalization of neurologic function. Our results indicate rapid blockade of ongoing disease processes by FTY720, and structural restoration of the CNS parenchyma, which is likely caused by the inhibition of autoimmune T cell infiltration and direct modulation of microvascular and/or glial cells.
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http://dx.doi.org/10.1111/j.1750-3639.2008.00182.xDOI Listing
April 2009

Neutropenia with impaired immune response to Streptococcus pneumoniae in ceramide kinase-deficient mice.

J Immunol 2008 Mar;180(5):3457-66

Novartis Institutes for BioMedical Research, Vienna, Austria.

In mammals, ceramide kinase (CerK)-mediated phosphorylation of ceramide is the only known pathway to ceramide-1-phosphate (C1P), a recently identified signaling sphingolipid metabolite. To help delineate the roles of CerK and C1P, we knocked out the gene of CerK in BALB/c mice by homologous recombination. All in vitro as well as cell-based assays indicated that CerK activity is completely abolished in Cerk-/- mice. Labeling with radioactive orthophosphate showed a profound reduction in the levels of de novo C1P formed in Cerk-/- macrophages. Consistently, mass spectrometry analysis revealed a major contribution of CerK to the formation of C16-C1P. However, the significant residual C1P levels in Cerk-/- animals indicate that alternative routes to C1P exist. Furthermore, serum levels of proapoptotic ceramide in these animals were significantly increased while levels of dihydroceramide as the biosynthetic precursor were reduced. Previous literature pointed to a role of CerK or C1P in innate immune cell function. Using a variety of mechanistic and disease models, as well as primary cells, we found that macrophage- and mast cell-dependent readouts are barely affected in the absence of CerK. However, the number of neutrophils was strikingly reduced in blood and spleen of Cerk-/- animals. When tested in a model of fulminant pneumonia, Cerk-/- animals developed a more severe disease, lending support to a defect in neutrophil homeostasis following CerK ablation. These results identify ceramide kinase as a key regulator of C1P, dihydroceramide and ceramide levels, with important implications for neutrophil homeostasis and innate immunity regulation.
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http://dx.doi.org/10.4049/jimmunol.180.5.3457DOI Listing
March 2008