Network pharmacology-based elucidation of the molecular mechanism underlying the anti-migraine effect of Asari Radix et Rhizoma

Purpose: To determine the molecular mechanism involved in the anti-migraine effect of Asari Radix et Rhizoma (ARR) using network pharmacology. Methods: The compounds present in ARR were identified through information retrieval from literature and public databases, and were screened based on absorption, distribution, metabolism, excretion and toxicity. Target genes related to the selected compounds and migraine were identified or predicted from public databases. Hub genes in ARR against migraine were identified through analysis of interactions in overlapping genes between compounds and migraine target genes, based on STRING database. Gene enrichment analysis of overlapping genes was performed using Database for Annotation, Visualization and Integrated Discovery. Results: A total of 138 compounds were selected as potential bioactive compounds in ARR. Target genes related to the selected compounds (611 genes) and migraine (278 genes) were obtained, including 71 overlapping genes. The hub genes in the anti-migraine effect of ARR were BDNF, IL6, COMT, APP and TNF. Gene enrichment analysis showed the top 10 biological processes or pathways involved in the mechanism of anti-migraine action of ARR. The tissue source of the overlapping genes was not limited to the brain. The results from gene enrichment analysis revealed that the effect of ARR on migraine was holistic, which is characteristic of traditional Chinese medicines. Conclusion: Network pharmacology has been used to decipher the molecular mechanism involved in the action of ARR against migraine. The results provide a scientific basis for the clinical effect of ARR on migraine.

Genome Wide Characterization & Phylogenetic Analysis of TNF Genes in Homo Sapiens

Tumor Necrosis Factor is very important inflammatory signaling unit that do an important role in immune system. It functions by attachment and stimulation of different receptor’s Cysteine Rich Domains (CDRs). A number of TNF receptors mediated factors have been identified having a major role in signal transduction pathways of TNF gene family. There are about 18 TNF homologues that are identified as a major cause of many disorders like cancer, Diabetes, AIDS and many other lethal inflammations. In this study the genome wide identification of TNF gene was done. Different tools and databases were used. Identification of conserved domains was done by using pfam and homology analysis showed that the TNF might be a member of TRAFs superfamily. Structural analysis of gene showed the number of introns and exons by a three-dimensional structure of TNF gene. The TNF gene family's exon-intron structure was found to be very similar in this study. The distribution of genes across chromosomes, on the other hand, was extremely varied. Collectively, the newly discovered genes can provide a wealth of information for manipulating the TNF genome to develop drugs and strategies to treat a variety of diseases.

P314 TNF gene activation used as criterion to discontinue biologic therapy – A patient series introducing the NovaPrime TNF kit

Background Severe inflammatory bowel disease can be successfully treated with biologic drugs such as anti-TNF. However, there is no consensus on if and how to stop treatment. Our group has earlier shown that normalization of TNF gene expression may be a beneficial prognostic factor when attempting to discontinue biologic therapy. We here present data from a series of patients in endoscopic remission and with normalized TNF gene expression. Methods Severe inflammatory bowel disease can be successfully treated with biologic drugs such as anti-TNF. However, there is no consensus on if and how to stop treatment. Our group has earlier shown that normalization of TNF gene expression may be a beneficial prognostic factor when attempting to discontinue biologic therapy. We here present data from a series of patients in endoscopic remission and with normalized TNF gene expression. A total of 91 patients were recruited at hospitals in Norway and Italy: 55 patients (32 UC, 23 CD) in remission (picked by normalization of TNF using our in-house TNF qPCR), and for comparison: 14 patients with active disease, and 22 normal controls. Mucosal samples taken prior to drug discontinuation from all patients were re-measured for TNF mRNA by the NovaPrime TNF kit. Results A clear difference could be seen between normal controls and patients with active disease, showing highly elevated TNF mRNA values in the active disease group. The patients in remission had values resembling the normal controls with values generally below a cut-off of 9060 copies/µg total RNA by the NovaPrime TNF kit. The patients in remission were followed for up to 5 years noting two endpoints: relapse triggering any adjustment of medication, and relapse triggering restart of biologics. Median survival time before adjusting medication was 12 months for ulcerative colitis and 17 months for Crohn’s disease. Median time to restart of biologics was 21 months (2 – 40) overall, and after 3 years 43% still were not in need of biologic therapy. Conclusion We conclude that the NovaPrime TNF kit yields consistent readings comparable to our in-house assay, and that this industrial standard kit enables any PCR lab to perform TNF gene expression in samples from intestinal mucosa. A considerable proportion of patients with normalized TNF gene expression can maintain long term remission without biologic therapy, relieving health care costs and potential side effects of long-term therapy.

P148 Differential methylation of the TNF gene promoter, potential for the development of a diagnostic biomarker for RA

Background/Aims Diagnosing rheumatoid arthritis (RA) early is difficult despite the use of anti-citrullinated protein antibodies in the new EULAR-2010 classification criteria, particularly for antibody-negative patients. Alterations in epigenetic patterns have been associated with RA CD4+T-cells at the TNF gene locus (1). The second aim of my PhD project was to select a CpG site to develop a quantitative methylation sensitive PCR (qMSP) assay as a diagnostic biomarker test for RA classification. Methods Patients attending an early arthritis clinic were used (with full ethical approval). An 450K Illumina methylation genome-wide dataset analysing DNA from CD4+T-cells identified CpG on TNF promoter region as a differentially methylated CpG between controls and RA patients (1). A qMSP assay was designed using a TaqMan approach (primers and probe), using a reference gene (GAPHD) for normalisation. The assay was validated in DNA extracted from PBMC. The performance of the assay for RA classification was accessed using binary logistic regression. Results We noted differences in levels of methylation in CD4+T-cells in RA, notably affecting the TNF gene promoter in about 15% of cells. We validated this using bisulphite sequencing showing about 30% of cells with differential methylation in early RA compared to controls. This allowed us to select cg11484872, cg21370522, and cg01569083 as candidates. We optimised a qMSP assay using fully methylated/un-methylated DNA. PBMC samples were retrieved from our tissue bank for 65 early RA, and 64 non-RA patients (including 11 reactive arthritis, 37 undifferentiated arthritis, and 16 psoriatic arthritis). The methylation levels (%) detected by the TNF-qMSP assay were significantly lower in RA (Δ-DM =-10.28%, p = 7x10-7). The loss of TNF promoter gene methylation was associated with RA classification (p < 0.0001, unadjusted OR = 0.900 (95% CI: 0.85-0.94)) with a good classification performance (AUROC = 0.76 (95% CI: 0.67, 0.84)). An adjusted regression model using the demographic and clinical parameters associated with RA classification in this group of patients (age, smoking, counts for tender and swollen joints) showed that adding TNF to the clinical model improved the model’s performance (increasing AUROC from 0.86 , to 0.91). Comparing classification accuracy, adding the TNF-qMSP resulted in an increased correct classification of 5.7 % compared to the clinical model. A similar result was obtained in the antibody negative patients, with TNF-qMSP assay improving the model’s classification from AUROC 0.91 to 0.96 and 3.3% more patients correctly classified. Conclusion The loss of DNA methylation on the TNF gene promoter identified in early RA patients provided a valuable target for a biomarker assay development. The results will need to be confirmed in a larger cohort but the TNF-qMSP assay offers good opportunities for RA versus non-RA classification particular in antibody negative disease. Disclosure R. Pitaksalee: Grants/research support; ROYAL THAI GOVERNMENT SCHOLARSHIP. P. Emery: None. R. Hodgett: None. F. Ponchel: None.

The role of the TNF (rs1800629), TNFB (rs2239704) and TNFRSF1B (rs652625) genes polymorphic variants in the allergic and infectious disease's development

В настоящем исследовании установлены ассоциации полиморфных вариантов генов TNF (rs1800629), TNFB (rs2239704) и TNFRSF1B (rs652625) с развитием бронхиальной астмы и туберкулеза. Различия в характере транскрипции гена TNF в зависимости от генотипа и воздействия стимуляторов микробного/немикробного происхождения (LPS, IFN-γ) предполагают функциональную значимость однонуклеотидной замены G>A (rs1800629) в гене TNF. In the present study, we established associations of the genes TNF (rs1800629); TNFB (rs2239704) and TNFRSF1B (rs652625) with the development of bronchial asthma and tuberculosis. Differences in the TNF gene transcription pattern depending on the genotype and the effect of stimulators of microbial/non-microbial origin (LPS, IFN-γ) suggest the functional significance of the single nucleotide substitution G>A (rs1800629) of the TNF gene.