Accessing Antibody Reactivities in Serum or Plasma to (Auto-)antigens Using Multiplexed Bead-Based Protein Immunoassays.

Antibody (AB) testing or serotesting for reactive ABs against antigenic proteins is broadly used. Parallel examination of many antigens is of high interest to identify autoantibodies (AAB) or differential antigenic reactivities in many biological settings like allergy and infectious autoimmune, cancerous, or systemic disease. The resulting AAB profiles can be used for diagnosis, prognosis, and monitoring of such conditions. Protein microarrays have been used for AB profiling over the past decade but show some significant limitations which make them unsuitable for clinical applications. Alternative multiplexing platforms such as bead arrays were shown to provide a versatile tool for the confirmation and efficient analysis of high numbers of biological samples. Luminex' bead-based xMAP technology combines advantages such as multiplexing and lower demand for sample volume and at the same time overcomes the challenges of microarrays. It works faster, shows better antigen stability, is more reproducible, and allows the analysis of up to 500 analytes in one sample well. In this chapter we introduce our established workflow for the use of the xMAP technology for AB profiling including an overview of the method principle and protocols for the covalent immobilization of proteins to the MagPlex beads, confirmation of protein coupling, the execution of a multiplexed bead-based protein immunoassay, and subsequent data handling.

Multiplexed Bead-Based Peptide Immunoassays for the Detection of Antibody Reactivities.

Antigenic peptides are commonly used in serological test settings such as enzyme-linked immunosorbent assays (ELISA) to determine reactive antibodies (ABs) from serum or plasma samples. The use of synthetic peptides provides advantages like lower production effort and easier incorporation of specific chemical modifications compared to full-length antigenic proteins. Multiplexed antibody (AB) profiling methods such as microarray technologies enable the simultaneous identification of multiple novel biomarkers for the use in early disease diagnostics, vaccine development, or monitoring of immune responses. Despite various benefits they still show major limitations which can be overcome with bead-based assay technologies like the multi-analyte profiling (xMAP) technology developed by Luminex. In this chapter we introduce our established workflow for AB profiling with a multiplexed bead-based peptide immunoassay. The workflow is based on copper-catalyzed click chemistry to immobilize designed synthetic peptides onto uniquely color-coded paramagnetic beads in an orientation-specific manner. The individual peptide-coupled beads can be distinguished by their unique emission spectra during readout in the xMAP instrument and therefore allow testing of up to 500 different antigenic peptides in one multiplexed reaction. The multistep process described in this chapter is divided into separate sections for peptide design, coupling of functionalized peptides to MagPlex beads via click chemistry, confirmation of successful peptide immobilization, processing of serum or plasma samples, or preferably purified IgG thereof, with the multiplexed bead-based peptide immunoassay and subsequent data export and analysis.

Antibody Profiling and In Silico Functional Analysis of Differentially Reactive Antibody Signatures of Glioblastomas and Meningiomas.

Studies on tumor-associated antigens in brain tumors are sparse. There is scope for enhancing our understanding of molecular pathology, in order to improve on existing forms, and discover new forms, of treatment, which could be particularly relevant to immuno-oncological strategies. To elucidate immunological differences, and to provide another level of biological information, we performed antibody profiling, based on a high-density protein array (containing 8173 human transcripts), using IgG isolated from the sera of n = 12 preoperative and n = 16 postoperative glioblastomas, n = 26 preoperative and n = 29 postoperative meningiomas, and n = 27 healthy, cancer-free controls. Differentially reactive antigens were compared to gene expression data from an alternate public GBM data set from OncoDB, and were analyzed using the Reactome pathway browser. Protein array analysis identified approximately 350-800 differentially reactive antigens, and revealed different antigen profiles in the glioblastomas and meningiomas, with approximately 20-30%-similar and 10-15%-similar antigens in preoperative and postoperative sera, respectively. Seroreactivity did not correlate with OncoDB-derived gene expression. Antigens in the preoperative glioblastoma sera were enriched for signaling pathways, such as signaling by Rho-GTPases, COPI-mediated anterograde transport and vesicle-mediated transport, while the infectious disease, SRP-dependent membrane targeting cotranslational proteins were enriched in the meningiomas. The pre-vs. postoperative seroreactivity in the glioblastomas was enriched for antigens, e.g., platelet degranulation and metabolism of lipid pathways; in the meningiomas, the antigens were enriched in infectious diseases, metabolism of amino acids and derivatives, and cell cycle. Antibody profiling in both tumor entities elucidated several hundred antigens and characteristic signaling pathways that may provide new insights into molecular pathology and may be of interest for the development of new treatment strategies.

Targeting epigenetic features in clear cell sarcomas based on patient-derived cell lines.

BACKGROUND: Clear cell sarcomas (CCSs) are translocated aggressive malignancies, most commonly affecting young adults with a high incidence of metastases and a poor prognosis. Research into the disease is more feasible when adequate models are available. By establishing CCS cell lines from a primary and metastatic lesion and isolating healthy fibroblasts from the same patient, the in vivo process is accurately reflected and aspects of clinical multistep carcinogenesis recapitulated. METHODS: Isolated tumor cells and normal healthy skin fibroblasts from the same patient were compared in terms of growth behavior and morphological characteristics using light and electron microscopy. Tumorigenicity potential was determined by soft agar colony formation assay and in vivo xenograft applications. While genetic differences between the two lineages were examined by copy number alternation profiles, nuclear magnetic resonance spectroscopy determined arginine methylation as epigenetic features. Potential anti-tumor effects of a protein arginine N-methyltransferase type I (PRMT1) inhibitor were elicited in 2D and 3D cell culture experiments using cell viability and apoptosis assays. Statistical significance was calculated by one-way ANOVA and unpaired t-test. RESULTS: The two established CCS cell lines named MUG Lucifer prim and MUG Lucifer met showed differences in morphology, genetic and epigenetic data, reflecting the respective original tissue. The detailed cell line characterization especially in regards to the epigenetic domain allows investigation of new innovative therapies. Based on the epigenetic data, a PRMT1 inhibitor was used to demonstrate the targeted antitumor effect; normal tissue cells isolated and immortalized from the same patient were not affected with the IC50 used. CONCLUSIONS: MUG Lucifer prim, MUG Lucifer met and isolated and immortalized fibroblasts from the same patient represent an ideal in vitro model to explore the biology of CCS. Based on this cell culture model, novel therapies could be tested in the form of PRMT1 inhibitors, which drive tumor cells into apoptosis, but show no effect on fibroblasts, further supporting their potential as promising treatment options in the combat against CCS. The data substantiate the importance of tailored therapies in the advanced metastatic stage of CCS.

Designed SARS-CoV-2 receptor binding domain variants form stable monomers.

The receptor binding domain (RBD) of the SARS-CoV-2 spike (S)-protein is a prime target of virus-neutralizing antibodies present in convalescent sera of COVID-19 patients and thus is considered a key antigen for immunosurveillance studies and vaccine development. Although recombinant expression of RBD has been achieved in several eukaryotic systems, mammalian cells have proven particularly useful. The authors aimed to optimize RBD produced in HEK293-6E cells towards a stable homogeneous preparation and addressed its O-glycosylation as well as the unpaired cysteine residue 538 in the widely used RBD (319-541) sequence. The authors found that an intact O-glycosylation site at T323 is highly relevant for the expression and maintenance of RBD as a monomer. Furthermore, it was shown that deletion or substitution of the unpaired cysteine residue C538 reduces the intrinsic propensity of RBD to form oligomeric aggregates, concomitant with an increased yield of the monomeric form of the protein. Bead-based and enzyme-linked immunosorbent assays utilizing these optimized RBD variants displayed excellent performance with respect to the specific detection of even low levels of SARS-CoV-2 antibodies in convalescent sera. Hence, these RBD variants could be instrumental for the further development of serological SARS-CoV-2 tests and inform the design of RBD-based vaccine candidates.

Impact of Specific N-Glycan Modifications on the Use of Plant-Produced SARS-CoV-2 Antigens in Serological Assays.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in the virus-host cell interaction, and viral infection. The RBD is a major target for neutralizing antibodies, whilst recombinant RBD is commonly used as an antigen in serological assays. Such assays are essential tools to gain control over the pandemic and detect the extent and durability of an immune response in infected or vaccinated populations. Transient expression in plants can contribute to the fast production of viral antigens, which are required by industry in high amounts. Whilst plant-produced RBDs are glycosylated, N-glycan modifications in plants differ from humans. This can give rise to the formation of carbohydrate epitopes that can be recognized by anti-carbohydrate antibodies present in human sera. For the performance of serological tests using plant-produced recombinant viral antigens, such cross-reactive carbohydrate determinants (CCDs) could result in false positives. Here, we transiently expressed an RBD variant in wild-type and glycoengineered Nicotiana benthamiana leaves and characterized the impact of different plant-specific N-glycans on RBD reactivity in serological assays. While the overall performance of the different RBD glycoforms was comparable to each other and to a human cell line produced RBD, there was a higher tendency toward false positive results with sera containing allergy-related CCD-antibodies when an RBD carrying ß1,2-xylose and core α1,3-fucose was used. These rare events could be further minimized by pre-incubating sera from allergic individuals with a CCD-inhibitor. Thereby, false positive signals obtained from anti-CCD antibodies, could be reduced by 90%, on average.

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants.

Nicotiana benthamiana is used worldwide as production host for recombinant proteins. Many recombinant proteins such as monoclonal antibodies, growth factors or viral antigens require posttranslational modifications like glycosylation for their function. Here, we transiently expressed different variants of the glycosylated receptor binding domain (RBD) from the SARS-CoV-2 spike protein in N. benthamiana. We characterized the impact of variations in RBD-length and posttranslational modifications on protein expression, yield and functionality. We found that a truncated RBD variant (RBD-215) consisting of amino acids Arg319-Leu533 can be efficiently expressed as a secreted soluble protein. Purified RBD-215 was mainly present as a monomer and showed binding to the conformation-dependent antibody CR3022, the cellular receptor angiotensin converting enzyme 2 (ACE2) and to antibodies present in convalescent sera. Expression of RBD-215 in glycoengineered ΔXT/FT plants resulted in the generation of complex N-glycans on both N-glycosylation sites. While site-directed mutagenesis showed that the N-glycans are important for proper RBD folding, differences in N-glycan processing had no effect on protein expression and function.

In Planta Production of the Receptor-Binding Domain From SARS-CoV-2 With Human Blood Group A Glycan Structures.

Glycosylation of viral envelope proteins is important for infectivity and immune evasion. The SARS-CoV-2 spike protein is heavily glycosylated and host-derived glycan modifications contribute to the formation of specific immunogenic epitopes, enhance the virus-cell interaction or affect virus transmission. On recombinant viral antigens used as subunit vaccines or for serological assays, distinct glycan structures may enhance the immunogenicity and are recognized by naturally occurring antibodies in human sera. Here, we performed an in vivo glycoengineering approach to produce recombinant variants of the SARS-CoV-2 receptor-binding domain (RBD) with blood group antigens in Nicotiana benthamiana plants. SARS-CoV-2 RBD and human glycosyltransferases for the blood group ABH antigen formation were transiently co-expressed in N. benthamiana leaves. Recombinant RBD was purified and the formation of complex N-glycans carrying blood group A antigens was shown by immunoblotting and MS analysis. Binding to the cellular ACE2 receptor and the conformation-dependent CR3022 antibody showed that the RBD glycosylation variants carrying blood group antigens were functional. Analysis of sera from RBD-positive and RBD-negative individuals revealed further that non-infected RBD-negative blood group O individuals have antibodies that strongly bind to RBD modified with blood group A antigen structures. The binding of IgGs derived from sera of non-infected RBD-negative blood group O individuals to blood group A antigens on SARS-CoV-2 RBD suggests that these antibodies could provide some degree of protection from virus infection.

Ape1 guides DNA repair pathway choice that is associated with drug tolerance in glioblastoma.

Ape1 is the major apurinic/apyrimidinic (AP) endonuclease activity in mammalian cells, and a key factor in base-excision repair of DNA. High expression or aberrant subcellular distribution of Ape1 has been detected in many cancer types, correlated with drug response, tumor prognosis, or patient survival. Here we present evidence that Ape1 facilitates BRCA1-mediated homologous recombination repair (HR), while counteracting error-prone non-homologous end joining of DNA double-strand breaks. Furthermore, Ape1, coordinated with checkpoint kinase Chk2, regulates drug response of glioblastoma cells. Suppression of Ape1/Chk2 signaling in glioblastoma cells facilitates alternative means of damage site recruitment of HR proteins as part of a genomic defense system. Through targeting "HR-addicted" temozolomide-resistant glioblastoma cells via a chemical inhibitor of Rad51, we demonstrated that targeting HR is a promising strategy for glioblastoma therapy. Our study uncovers a critical role for Ape1 in DNA repair pathway choice, and provides a mechanistic understanding of DNA repair-supported chemoresistance in glioblastoma cells.

New liver cancer biomarkers: PI3K/AKT/mTOR pathway members and eukaryotic translation initiation factors.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. The initiation of protein translation is an important rate-limiting step in eukaryotes and is crucial in many viral infections. Eukaryotic translation initiation factors (eIFs) are involved in the initiation step of protein translation and are linked to the phosphatidylinositol-3-kinases PI3K/AKT/mTOR pathway. Therefore we aimed to investigate a potential role of eIFs in HCC. We herein report on the immunohistochemical expression of the various eIF subunits in 235 cases of virus-related human HCC. Additionally, we used immunoblot analysis to investigate the expression of virus-related HCC and non-virus-related HCC in comparison to controls. Mammalian target of rapamycin (or mechanistic target of rapamycin as it is known now (mTOR) and activated mTOR were significantly increased in chronic hepatitis C (HCV)-associated HCC, in HCC without a viral background, in alcoholic liver disease and Wilson disease. pPTEN, phosphatase and tensin homologue (PTEN) and pAKT showed a significant increase in HBV- and HCV-associated HCC, chronic hepatitis B, HCC without a viral background, alcoholic steatohepatitis (ASH) and Wilson disease. Phosphorylated (p)-eIF2α, eIF2α, eiF3B, eIF3D, eIF3J, p-eIF4B, eIF4G and eIF6 were upregulated in HCV-associated HCC. eIF2α, p-eIF4B, eIF5 and various eIF3 subunits were significantly increased in chronic hepatitis B (HBV)-associated HCC. HCC without viral background displayed a significant increase for the eIF subunits p-2α, 3C, 3I, 4E and 4G. We noticed engraved differences in the expression pattern between chronic hepatitis B and C, HBV- and HCV-associated HCC and non-virus-related HCC.

Serum miRNA Signatures Are Indicative of Skeletal Fractures in Postmenopausal Women With and Without Type 2 Diabetes and Influence Osteogenic and Adipogenic Differentiation of Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro.

Standard DXA measurements, including Fracture Risk Assessment Tool (FRAX) scores, have shown limitations in assessing fracture risk in Type 2 Diabetes (T2D), underscoring the need for novel biomarkers and suggesting that other pathomechanisms may drive diabetic bone fragility. MicroRNAs (miRNAs) are secreted into the circulation from cells of various tissues proportional to local disease severity and were recently found to be crucial to bone homeostasis and T2D. Here, we studied, if and which circulating miRNAs or combinations of miRNAs can discriminate best fracture status in a well-characterized study of diabetic bone disease and postmenopausal osteoporosis (n = 80 postmenopausal women). We then tested the most discriminative and most frequent miRNAs in vitro. Using miRNA-qPCR-arrays, we showed that 48 miRNAs can differentiate fracture status in T2D women and that several combinations of four miRNAs can discriminate diabetes-related fractures with high specificity and sensitivity (area under the receiver-operating characteristic curve values [AUCs], 0.92 to 0.96; 95% CI, 0.88 to 0.98). For the osteoporotic study arm, 23 miRNAs were fracture-indicative and potential combinations of four miRNAs showed AUCs from 0.97 to 1.00 (95% CI, 0.93 to 1.00). Because a role in bone homeostasis for those miRNAs that were most discriminative and most present among all miRNA combinations had not been described, we performed in vitro functional studies in human adipose tissue-derived mesenchymal stem cells to investigate the effect of miR-550a-5p, miR-188-3p, and miR-382-3p on osteogenesis, adipogenesis, and cell proliferation. We found that miR-382-3p significantly enhanced osteogenic differentiation (p < 0.001), whereas miR-550a-5p inhibited this process (p < 0.001). Both miRNAs, miR-382-3p and miR-550a-5p, impaired adipogenic differentiation, whereas miR-188-3p did not exert an effect on adipogenesis. None of the miRNAs affected significantly cell proliferation. Our data suggest for the first time that miRNAs are linked to fragility fractures in T2D postmenopausal women and should be further investigated for their diagnostic potential and their detailed function in diabetic bone. © 2016 American Society for Bone and Mineral Research.

The prostate cancer immunome: In silico functional analysis of antigenic proteins from microarray profiling with IgG.

The study of the immunome of prostate cancer (PCa) and characterization of autoantibody signature from differentially reactive antigens can uncover disease stage proteins, reveal enriched networks and even expose aberrant cellular mechanisms during the disease process. By conducting plasma IgG profiling on protein microarrays presenting 5449 unique human proteins expressed in 15 417 E. coli human cDNA expression clones, we elucidated 471 (21 higher reactive in PCa) differentially reactive antigens in 50 PCa versus 49 patients with benign prostate hyperplasia (BPH) at initial diagnosis. Functional analyzes show that the immune-profile of PCa compared to BPH control samples is significantly enriched in features targeting Cellular assembly, Cell death and pathways involved in Cell cycle, translation, and assembly of proteins as EIF2 signaling, PCa related genes as AXIN1 and TP53, and ribosomal proteins (e.g. RPS10). An overlap of 61 (out of 471) DIRAGs with the published 1545 antigens from the SEREX database has been found, however those were higher reactive in BPH. Clinical relevance is shown when antibody-reactivities against eight proteins were significantly (p < 0.001) correlated with Gleason-score. Herewith we provide a biological and pathophysiological characterization of the immunological layer of cancerous (PCa) versus benign (BPH) disease, derived from antibody profiling on protein microarrays.

Diagnostic Performance of Plasma DNA Methylation Profiles in Lung Cancer, Pulmonary Fibrosis and COPD.

Disease-specific alterations of the cell-free DNA methylation status are frequently found in serum samples and are currently considered to be suitable biomarkers. Candidate markers were identified by bisulfite conversion-based genome-wide methylation screening of lung tissue from lung cancer, fibrotic ILD, and COPD. cfDNA from 400 µl serum (n = 204) served to test the diagnostic performance of these markers. Following methylation-sensitive restriction enzyme digestion and enrichment of methylated DNA via targeted amplification (multiplexed MSRE enrichment), a total of 96 markers were addressed by highly parallel qPCR. Lung cancer was efficiently separated from non-cancer and controls with a sensitivity of 87.8%, (95%CI: 0.67-0.97) and specificity 90.2%, (95%CI: 0.65-0.98). Cancer was distinguished from ILD with a specificity of 88%, (95%CI: 0.57-1), and COPD from cancer with a specificity of 88% (95%CI: 0.64-0.97). Separation of ILD from COPD and controls was possible with a sensitivity of 63.1% (95%CI: 0.4-0.78) and a specificity of 70% (95%CI: 0.54-0.81). The results were confirmed using an independent sample set (n = 46) by use of the four top markers discovered in the study (HOXD10, PAX9, PTPRN2, and STAG3) yielding an AUC of 0.85 (95%CI: 0.72-0.95). This technique was capable of distinguishing interrelated complex pulmonary diseases suggesting that multiplexed MSRE enrichment might be useful for simple and reliable diagnosis of diverse multifactorial disease states.

Wound healing and longevity: lessons from long-lived αMUPA mice.

Does the longevity phenotype offer an advantage in wound healing (WH)? In an attempt to answer this question, we explored skin wound healing in the long-lived transgenic αMUPA mice, a unique model of genetically extended life span. These mice spontaneously eat less, preserve their body mass, are more resistant to spontaneous and induced tumorigenesis and live longer, thus greatly mimicking the effects of caloric restriction (CR). We found that αMUPA mice showed a much slower age-related decline in the rate of WH than their wild-type counterparts (FVB/N). After full closure of the wound, gene expression in the skin of old αMUPA mice returned close to basal levels. In contrast, old FVB/N mice still exhibited significant upregulation of genes associated with growth-promoting pathways, apoptosis and cell-cell/cell-extra cellular matrix interaction, indicating an ongoing tissue remodeling or an inability to properly shut down the repair process. It appears that the CR-like longevity phenotype is associated with more balanced and efficient WH mechanisms in old age, which could ensure a long-term survival advantage.

Human blood monocytes support persistence, but not replication of the intracellular pathogen C. pneumoniae.

BACKGROUND: Intracellular pathogens have devised various mechanisms to subvert the host immune response in order to survive and replicate in host cells. Here, we studied the infection of human blood monocytes with the intracellular pathogen C. pneumoniae and the effect on cytokine and chemokine profiles in comparison to stimulation with LPS. RESULTS: Monocytes purified from peripheral blood mononuclear cells by negative depletion were infected with C. pneumoniae. While immunofluorescence confirmed the presence of chlamydial lipopolysaccharide (LPS) in the cytoplasm of infected monocytes, real-time PCR did not provide evidence for replication of the intracellular pathogen. Complementary to PCR, C. pneumoniae infection was confirmed by an oligonucleotide DNA microarray for the detection of intracellular pathogens. Raman microspectroscopy revealed different molecular fingerprints for infected and non-infected monocytes, which were mainly due to changes in lipid and fatty acid content. Stimulation of monocytes with C. pneumoniae or with LPS induced similar profiles of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6, but higher levels of IL-1ß, IL-12p40 and IL-12p70 for C. pneumoniae which were statistically significant. C. pneumoniae also induced release of the chemokines MCP-1, MIP-1α and MIP-1ß, and CXCL-8, which correlated with TNF-α secretion. CONCLUSION: Infection of human blood monocytes with intracellular pathogens triggers altered cytokine and chemokine pattern as compared to stimulation with extracellular ligands such as LPS. Complementing conventional methods, an oligonucleotide DNA microarray for the detection of intracellular pathogens as well as Raman microspectroscopy provide useful tools to trace monocyte infection.

Strategies for validation and testing of DNA methylation biomarkers.

DNA methylation is a stable covalent epigenetic modification of primarily CpG dinucleotides that has recently gained considerable attention for its use as a biomarker in different clinical settings, including disease diagnosis, prognosis and therapeutic response prediction. Although the advent of genome-wide DNA methylation profiling in primary disease tissue has provided a manifold resource for biomarker development, only a tiny fraction of DNA methylation-based assays have reached clinical testing. Here, we provide a critical overview of different analytical methods that are suitable for biomarker validation, including general study design considerations, which might help to streamline epigenetic marker development. Furthermore, we highlight some of the recent marker validation studies and established markers that are currently commercially available for assisting in clinical management of different cancers.

Increased antioxidant defense mechanism in human adventitia-derived progenitor cells is associated with therapeutic benefit in ischemia.

AIMS: Vascular wall-resident progenitor cells hold great promise for cardiovascular regenerative therapy. This study evaluates the impact of oxidative stress on the viability and functionality of adventitia-derived progenitor cells (APCs) from vein remnants of coronary artery bypass graft (CABG) surgery. We also investigated the antioxidant enzymes implicated in the resistance of APCs to oxidative stress-induced damage and the effect of interfering with one of them, the extracellular superoxide dismutase (EC-SOD/SOD3), on APC therapeutic action in a model of peripheral ischemia. RESULTS: After exposure to hydrogen peroxide, APCs undergo apoptosis to a smaller extent than endothelial cells (ECs). This was attributed to up-regulation of antioxidant enzymes, especially SODs and catalase. Pharmacological inhibition of SODs increases reactive oxygen species (ROS) levels in APCs and impairs their survival. Likewise, APC differentiation results in SOD down-regulation and ROS-induced apoptosis. Oxidative stress increases APC migratory activity, while being inhibitory for ECs. In addition, oxidative stress does not impair APC capacity to promote angiogenesis in vitro. In a mouse limb ischemia model, an injection of naïve APCs, but not SOD3-silenced APCs, helps perfusion recovery and neovascularization, thus underlining the importance of this soluble isoform in protection from ischemia. INNOVATION: This study newly demonstrates that APCs are endowed with enhanced detoxifier and antioxidant systems and that SOD3 plays an important role in their therapeutic activity in ischemia. CONCLUSIONS: APCs from vein remnants of CABG patients express antioxidant defense mechanisms, which enable them to resist stress. These properties highlight the potential of APCs in cardiovascular regenerative medicine.

Age influence on hypersensitivity pneumonitis induced in mice by exposure to Pantoea agglomerans.

Hypersensitivity pneumonitis (HP) represents the immunologically mediated lung disease induced by repeated inhalations of a wide variety of certain finely dispersed organic antigens. In susceptible subjects, these inhalations provoke a hypersensitivity reaction characterized by intense inflammation of the terminal bronchioles, the interstitium and the alveolar tree. The inflammation often organizes into granulomas and may progress to pulmonary fibrosis. Our previous work indicated that cell extract of gram-negative bacteria Pantoea agglomerans (SE-PA) causes, in young C57BL/6J mice, pulmonary changes that are very similar to the clinical manifestations of HP in men. The purpose of presented studies was to describe the response of mice immune system while exposed to SE-PA. Particular attention was paid to examine the age influence on SE-PA induced inflammation and fibrosis in lung tissue. We used 3- and 18-month-old C57BL/6J mice. Lung samples were collected from untreated mice and animals exposed to harmful agent for 7 and 28 days. HP development was monitored by histological and biochemical evaluation. Using ELISA tests, we examined concentration of pro- and anti-inflammatory cytokines in lung homogenates. Our study demonstrated again that SE-PA provokes in mice changes typical for the clinical picture of HP, and that successive stages of disease (acute, subacute and chronic) might be obtained by modulation of time exposure. Furthermore, we found that animals' age at the time of sensitization influences the nature of observed changes (cytokine expression pattern) and the final outcome (reaction intensity and scale of fibrosis).

Meta-analysis of gene expression signatures defining the epithelial to mesenchymal transition during cancer progression.

The epithelial to mesenchymal transition (EMT) represents a crucial event during cancer progression and dissemination. EMT is the conversion of carcinoma cells from an epithelial to a mesenchymal phenotype that associates with a higher cell motility as well as enhanced chemoresistance and cancer stemness. Notably, EMT has been increasingly recognized as an early event of metastasis. Numerous gene expression studies (GES) have been conducted to obtain transcriptome signatures and marker genes to understand the regulatory mechanisms underlying EMT. Yet, no meta-analysis considering the multitude of GES of EMT has been performed to comprehensively elaborate the core genes in this process. Here we report the meta-analysis of 18 independent and published GES of EMT which focused on different cell types and treatment modalities. Computational analysis revealed clustering of GES according to the type of treatment rather than to cell type. GES of EMT induced via transforming growth factor-ß and tumor necrosis factor-α treatment yielded uniformly defined clusters while GES of models with alternative EMT induction clustered in a more complex fashion. In addition, we identified those up- and downregulated genes which were shared between the multitude of GES. This core gene list includes well known EMT markers as well as novel genes so far not described in this process. Furthermore, several genes of the EMT-core gene list significantly correlated with impaired pathological complete response in breast cancer patients. In conclusion, this meta-analysis provides a comprehensive survey of available EMT expression signatures and shows fundamental insights into the mechanisms that are governing carcinoma progression.

Soluble ST2 is regulated by p75 neurotrophin receptor and predicts mortality in diabetic patients with critical limb ischemia.

OBJECTIVE: The p75 neurotrophin receptor (p75(NTR)) contributes to diabetes mellitus-induced defective postischemic neovascularization. The interleukin-33 receptor ST2 is expressed as transmembrane (ST2L) and soluble (sST2) isoforms. Here, we studied the following: (1) the impact of p75(NTR) in the healing of ischemic and diabetic calf wounds; (2) the link between p75(NTR) and ST2; and (3) circulating sST2 levels in critical limb ischemia (CLI) patients. METHODS AND RESULTS: Diabetes mellitus was induced in p75(NTR) knockout (p75KO) mice and wild-type (WT) littermates by streptozotocin. Diabetic and nondiabetic p75KO and WT mice received left limb ischemia induction and a full-thickness wound on the ipsilateral calf. Diabetes mellitus impaired wound closure and angiogenesis and increased ST2 expression in WT, but not in p75KO wounds. In cultured endothelial cells, p75(NTR) promoted ST2 (both isoforms) expression through p38(MAPK)/activating transcription factor 2 pathway activation. Next, sST2 was measured in the serum of patients with CLI undergoing either revascularization or limb amputation and in the 2 nondiabetic groups (with CLI or nonischemic individuals). Serum sST2 increased in diabetic patients with CLI and was directly associated with higher mortality at 1 year from revascularization. CONCLUSIONS: p75(NTR) inhibits the healing of ischemic lower limb wounds in diabetes mellitus and promotes ST2 expression. Circulating sST2 predicts mortality in diabetic CLI patients.