Does the Use of the "Proseek® Multiplex Inflammation I Panel" Demonstrate a Difference in Local and Systemic Immune Responses in Endometriosis Patients with or without Deep-Infiltrating Lesions?

Endometriotic lesions are able to infiltrate surrounding tissue. This is made possible partly by an altered local and systemic immune response that helps achieve neoangiogenesis, cell proliferation and immune escape. Deep-infiltrating endometriosis (DIE) differs from other subtypes through the invasion of its lesions over 5 mm into affected tissue. Despite the invasive nature of these lesions and the wider range of symptoms they can trigger, DIE is described as a stable disease. This elicits the need for a better understanding of the underlying pathogenesis. We used the "Proseek® Multiplex Inflammation I Panel" in order to simultaneously detect 92 inflammatory proteins in plasma and peritoneal fluid (PF) of controls and patients with endometriosis, as well as in particular patients with DIE, in order to gain a better insight into the systemically and locally involved immune response. Extracellular newly identified receptor for advanced gycation end-products binding protein (EN-RAGE), C-C motif Chemokine ligand 23 (CCL23), Eukaryotic translation initiation factor 4-binding protein 1 (4E-BP1) and human glial cell-line derived neurotrophic factor (hGDNF) were significantly increased in plasma of endometriosis patients compared to controls, whereas Hepatocyte Growth factor (HGF) and TNF-related apoptosis inducing ligand (TRAIL) were decreased. In PF of endometriosis patients, we found Interleukin 18 (IL-18) to be decreased, yet Interleukin 8 (IL-8) and Interleukin 6 (IL-6) to be increased. TNF-related activation-induced cytokine (TRANCE) and C-C motif Chemokine ligand 11 (CCL11) were significantly decreased in plasma, whereas C-C motif Chemokine ligand 23 (CCL23), Stem Cell Factor (SCF) and C-X-C motif chemokine 5 (CXCL5) were significantly increased in PF of patients with DIE compared to endometriosis patients without DIE. Although DIE lesions are characterized by increased angiogenetic and pro-inflammatory properties, our current study seems to support the theory that the systemic immune system does not play a major role in the pathogenesis of these lesions.

Does the Use of the "Proseek® Multiplex Oncology I Panel" on Peritoneal Fluid Allow a Better Insight in the Pathophysiology of Endometriosis, and in Particular Deep-Infiltrating Endometriosis?

Endometriosis appears to share certain cancer-related processes, such as cell attachment, invasion, proliferation and neovascularization, some of which can also be found in other healthy tissues. In order to better understand the altered milieu of the peritoneal cavity, while acknowledging the reported similarities between endometriosis and neoplastic processes, we applied a multiplex oncology panel to search for specific biomarker signatures in the peritoneal fluid of women with endometriosis, women with deep-infiltrating endometriosis (DIE), as well as controls. In total, 84 patients were included in our study, 53 women with endometriosis and 31 controls. Ninety-two proteins were measured in prospectively collected peritoneal fluid (PF) samples, using the "Proseek® Multiplex Oncology I Panel". We first compared patients with endometriosis versus controls, and in a second step, DIE versus endometriosis patients without DIE. Out of the 92 analyzed proteins, few showed significant differences between the groups. In patients with endometriosis, ICOS ligand, Endothelial growth factor, E-selectin, Receptor tyrosine-protein kinase erbB-2, Interleukin-6 receptor alpha, Vascular endothelial growth factor receptor 2, Fms-related tyrosine kinase 3 ligand, C-X-C motif chemokine 10, Epididymal secretory protein E4 and Folate receptor-alpha were decreased, while Interleukin-6 and Interleukin-8 were increased compared to controls. Looking at patients with DIE, we found Chemokine ligand 19, Stem cell factor, Vascular endothelial growth factor D, Interleukin-6 receptor alpha and Melanoma inhibitory activity to be increased compared to endometriosis patients without DIE. We have shown a distinct regulation of the immune response, angiogenesis, cell proliferation, cell adhesion and inhibition of apoptosis in PF of patients with endometriosis compared to controls. The specific protein pattern in the PF of DIE patients provides new evidence that DIE represents a unique entity of extrauterine endometriosis with enhanced angiogenetic and pro-proliferative features.

Autoantibodies as diagnostic markers and potential drivers of inflammation in ulcerative colitis.

To date, no comprehensive analysis of autoantibodies in sera of patients with ulcerative colitis has been conducted. To analyze the spectrum of autoantibodies and to elucidate their role serum-IgG from UC patients (n = 49) and non-UC donors (n = 23) were screened by using a human protein microarray. Screening yielded a remarkable number of 697 differentially-reactive at the nominal 0·01 significance level (FDR<0·1) of the univariate test between the UC and the non-UC group. CD99 emerged as a biomarker to discriminate between both groups (p = 1e-04, AUC = 0·8). In addition, cytokines, chemokines and growth factors were analyzed by Olink's Proseek® Multiplex Inflammation-I 96×96 immuno-qPCR assay and 31 genes were significant at the nominal 0.05 level of the univariate test to discriminate between UC and non-UC donors. MCP-3, HGF and CXCL-9 were identified as the most significant markers to discriminate between UC patients with clinically active and inactive disease. Levels of CXCL10 (cor = 0.3; p = 0.02), CCL25 (cor = 0.25; p = 0.04) and CCL28 (cor = 0.3; p = 0.02) correlated positively with levels of anti CD99. To assess whether autoantibodies are detectable prior to diagnosis with UC, sera from nine donors at two different time points (T-early, median 21 months and T-late, median 6 months) were analyzed. 1201 features were identified with higher reactivity in samples at time points closer to clinical UC presentation. In vitro, additional challenge of peripheral mononuclear cells with CD99 did not activate CD4+ T cells but induced the secretion of IL-10 (-CD99: 20.21±20.25; +CD99: 130.20±89.55; mean ±sd; p = 0.015). To examine the effect of CD99 in vivo, inflammation and autoantibody levels were examined in NOD/ScidIL2Rγnull mice reconstituted with PBMC from UC donors (NSG-UC). Additional challenge with CD99 aggravated disease symptoms and pathological phenotype as indicated by the elevated clinical score (-CD99: 1·85 ± 1·94; +CD99: 4·25 ± 1·48) and histological score (-CD99: 2·16 ± 0·83; +CD99: 3·15 ± 1·16, p = 0·01). Furthermore, levels of anti-CD99 antibodies increased (Control: 398 ± 323; mean MFI ± sd; Ethanol + PBS: 358 ±316; Ethanol + CD99: 1363 ± 1336; Control versus Ethanol + CD99: p = 0.03). In a highly inflammatory environment, frequencies of pro-inflammatory M1 monocytes (CD14+ CD64+: unchallenged 8.09±4.72; challenged 14.2±8.62; p = 0.07; CD14+ CD1a+: unchallenged 16.29 ±6.97; challenged 43.81±14.4, p = 0.0003) increased and levels of autoantibodies in serum decreased in the NSG-UC mouse model. These results suggest that autoantibodies are potent biomarkers to discriminate between UC and non-UC and indicate risk to develop UC. In an inflammatory environment, auto-antibodies may promote the pathological phenotype by activating M1 monocytes in the NSG-UC animal model and also in patients with UC.

Multiplexed and Sensitive DNA Methylation Testing Using Methylation-Sensitive Restriction Enzymes "MSRE-qPCR".

DNA methylation is a chemically stable key-player in epigenetics. In the vertebrate genome the 5-methyl cytosine (5mC) has been found almost exclusively in the CpG dinucleotide context. CpG dinucleotides are enriched in CpG islands very frequently located within or close to gene promoters. Analyses of DNA methylation changes in human diagnostics have been conducted classically using methylation-sensitive restriction enzymes (MSRE). Since the discovery of bisulfite conversion-based sequencing and PCR assays, MSRE-based PCR assays have been less frequently used, although especially in the field of cancer epigenetics MSRE-based genome-wide discovery and targeted screening applications have been and are still performed successfully. Even though epigenome-wide discovery of altered DNA methylation patterns has found its way into various fields of human disease and molecular genetics research, the validation of findings upon discovery is still a bottleneck. Usually several multiples of 10 up to 100 candidate biomarkers from discovery have to be confirmed or are of interest for further work. In particular, bisulfite PCR assays are often limited in the number of candidates which can be analyzed, due to their low multiplexing capability, especially, if only small amounts of DNA are available from for example clinical specimens. In clinical research and diagnostics a similar situation arises for the analyses of cell-free DNA (cfDNA) in body fluids or circulating tumor cells (CTCs). Although tissue- or disease- (e.g., cancer) specific DNA methylation patterns can be deduced very efficiently in a genome-wide manner if around 100 ng of DNA are available, confirming these candidates and selecting target-sequences for studying methylation changes in liquid biopsies using cfDNA or CTCs remains a big challenge. Along these lines we have developed MSRE-qPCR and introduce here method details, which have been found very suitable for the efficient confirmation and testing of DNA methylation in a quantitative multiplexed manner (e.g., 48-96 plex) from ng amounts of DNA. The method is applicable in a standard qPCR setting as well for nanoliter scaled high-throughput qPCR, enabling detection of <10 copies of targets, thus suitable to pick up 0.1-1% of specific methylated DNA in an unmethylated background.

Inhibitors of DNA methylation support TGF-ß1-induced IL11 expression in gingival fibroblasts.

PURPOSE: Oral wound healing requires gingival fibroblasts to respond to local growth factors. Epigenetic silencing through DNA methylation can potentially decrease the responsiveness of gingival fibroblasts to local growth factors. In this study, our aim was to determine whether the inhibition of DNA methylation sensitized gingival fibroblasts to transforming growth factor-ß1 (TGF-ß1). METHODS: Gingival fibroblasts were exposed to 5-aza-2'-deoxycytidine (5-aza), a clinically approved demethylating agent, before stimulation with TGF-ß1. Gene expression changes were evaluated using quantitative polymerase chain reaction (PCR) analysis. DNA methylation was detected by methylation-sensitive restriction enzymes and PCR amplification. RESULTS: We found that 5-aza enhanced TGF-ß1-induced interleukin-11 (IL11) expression in gingival fibroblasts 2.37-fold (P=0.008). 5-aza had no significant effects on the expression of proteoglycan 4 (PRG4) and NADPH oxidase 4 (NOX4). Consistent with this, 5-aza caused demethylation of the IL11 gene commonly next to a guanosine (CpG) island in gingival fibroblasts. The TGF-ß type I receptor kinase inhibitor SB431542 impeded the changes in IL11 expression, indicating that the effects of 5-aza require TGF-ß signaling. 5-aza moderately increased the expression of TGF-ß type II receptor (1.40-fold; P=0.009), possibly enhancing the responsiveness of fibroblasts to TGF-ß1. As part of the feedback response, 5-aza increased the expression of the DNA methyltransferases 1 (DNMT1) (P=0.005) and DNMT3B (P=0.002), which are enzymes responsible for gene methylation. CONCLUSIONS: These in vitro data suggest that the inhibition of DNA methylation by 5-aza supports TGF-ß-induced IL11 expression in gingival fibroblasts.

MSP-HTPrimer: a high-throughput primer design tool to improve assay design for DNA methylation analysis in epigenetics.

BACKGROUND: Bisulfite (BS) conversion-based and methylation-sensitive restriction enzyme (MSRE)-based PCR methods have been the most commonly used techniques for locus-specific DNA methylation analysis. However, both methods have advantages and limitations. Thus, an integrated approach would be extremely useful to quantify the DNA methylation status successfully with great sensitivity and specificity. Designing specific and optimized primers for target regions is the most critical and challenging step in obtaining the adequate DNA methylation results using PCR-based methods. Currently, no integrated, optimized, and high-throughput methylation-specific primer design software methods are available for both BS- and MSRE-based methods. Therefore an integrated, powerful, and easy-to-use methylation-specific primer design pipeline with great accuracy and success rate will be very useful. RESULTS: We have developed a new web-based pipeline, called MSP-HTPrimer, to design primers pairs for MSP, BSP, pyrosequencing, COBRA, and MSRE assays on both genomic strands. First, our pipeline converts all target sequences into bisulfite-treated templates for both forward and reverse strand and designs all possible primer pairs, followed by filtering for single nucleotide polymorphisms (SNPs) and known repeat regions. Next, each primer pairs are annotated with the upstream and downstream RefSeq genes, CpG island, and cut sites (for COBRA and MSRE). Finally, MSP-HTPrimer selects specific primers from both strands based on custom and user-defined hierarchical selection criteria. MSP-HTPrimer produces a primer pair summary output table in TXT and HTML format for display and UCSC custom tracks for resulting primer pairs in GTF format. CONCLUSIONS: MSP-HTPrimer is an integrated, web-based, and high-throughput pipeline and has no limitation on the number and size of target sequences and designs MSP, BSP, pyrosequencing, COBRA, and MSRE assays. It is the only pipeline, which automatically designs primers on both genomic strands to increase the success rate. It is a standalone web-based pipeline, which is fully configured within a virtual machine and thus can be readily used without any configuration. We have experimentally validated primer pairs designed by our pipeline and shown a very high success rate of primer pairs: out of 66 BSP primer pairs, 63 were successfully validated without any further optimization step and using the same qPCR conditions. The MSP-HTPrimer pipeline is freely available from http://sourceforge.net/p/msp-htprimer.

Erratum to: MSRE-HTPrimer: a high-throughput and genome-wide primer design pipeline optimized for epigenetic research.

[This corrects the article DOI: 10.1186/s13148-016-0190-9.].

MSRE-HTPrimer: a high-throughput and genome-wide primer design pipeline optimized for epigenetic research.

BACKGROUND: Methylation-sensitive restriction enzymes-polymerase chain reaction (MSRE-PCR) has been used in epigenetic research to identify genome-wide and gene-specific DNA methylation. Currently, epigenome-wide discovery studies provide many candidate regions for which the MSREqPCR approach can be very effective to confirm the findings. MSREqPCR provides high multiplexing capabilities also when starting with limited amount of DNA-like cfDNA to validate many targets in a time- and cost-effective manner. Multiplex design is challenging and cumbersome to define specific primers in an effective manner, and no suitable software tools are freely available for high-throughput primer design in a time-effective manner and to automatically annotate the resulting primers with known SNPs, CpG, repeats, and RefSeq genes. Therefore a robust, powerful, high-throughput, optimized, and methylation-specific primer design tool with great accuracy will be very useful. RESULTS: We have developed a novel pipeline, called MSRE-HTPrimer, to design MSRE-PCR and genomic PCR primers pairs in a very efficient manner and with high success rate. First, our pipeline designs all possible PCR primer pairs and oligos, followed by filtering for SNPs loci and repeat regions. Next, each primer pair is annotated with the number of cut sites in primers and amplicons, upstream and downstream genes, and CpG islands loci. Finally, MSRE-HTPrimer selects resulting primer pairs for all target sequences based on a custom quality matrix defined by the user. MSRE-HTPrimer produces a table for all resulting primer pairs as well as a custom track in GTF file format for each target sequence to visualize it in UCSC genome browser. CONCLUSIONS: MSRE-HTPrimer, based on Primer3, is a high-throughput pipeline and has no limitation on the number and size of target sequences for primer design and provides full flexibility to customize it for specific requirements. It is a standalone web-based pipeline, which is fully configured within a virtual machine and thus can be readily used without any configuration. We have experimentally validated primer pairs designed by our pipeline and shown a very high success rate of primer pairs: out of 190 primer pairs, 71 % could be successfully validated. The MSRE-HTPrimer software is freely available from http://sourceforge.net/p/msrehtprimer/wiki/Virtual_Machine/ as a virtual machine.