Gene Expression Analysis in Peripheral Blood of Patients with Rheumatoid Arthritis
ABSTRACT: Peripheral blood cells in patients with rheumatoid arthritis before/after IL-6 inhibition The obtained conclusions on the change of each gene expression level before and after MRA medication were as follows. (1) In the case of IL-6, the specific time profile that the expression level dropped at day 21 and increased to some extent at day 42 was observed in 2 of 18 subjects. In other subjects, quite small change or small increase after day 7 was observed. (2) In the case of IL-6R as well as in IL-6, the specific time profile that the expression level dropped at day 21 and increased to some extent at day 42 was observed in 1 of 18 subjects. In other subjects, slow and small increase was observed in 6 of 18. (3) The ratio of mRNA expression level of membrane and soluble type of IL-6R showed marginal difference after tocilizumab treatment, (4) The distinct change of expression level of cytochrome P450 mRNA after tocilizumab treatment was hardly observed in most patients. Overall design: The measurement of expression level of several genes shown below in peripheral blood of human subjects (18 patients with rheumatic disease) before and after tocilizumab medication using DNA microarray (Hitachi human oligo chip) was conducted in this test, 1) Interleukin 6 (IL-6), membrane IL-6 receptor (IL-6R) and soluble type of the IL-6R, 2) several cytochrome P450 as follows: CYP3A4, CYP2D6, CYP2C9, CYP2C19. Number of human subjects was 18 and peripheral blood samples drawn at 4 time points: day 0 (the first Omeprazole administration was done), day 7 (Tocilizumab administration was done), day 21 and day 42 for each subject were analyzed. On day 0 and day 7, the blood sampling was done before drug administration.
INSTRUMENT(S): Hitachi human oligo chip - drug responsive genes
Project description:Peripheral blood cells in patients with rheumatoid arthritis before/after IL-6 inhibition The obtained conclusions on the change of each gene expression level before and after MRA medication were as follows. (1) In the case of IL-6, the specific time profile that the expression level dropped at day 21 and increased to some extent at day 42 was observed in 2 of 18 subjects. In other subjects, quite small change or small increase after day 7 was observed. (2) In the case of IL-6R as well as in IL-6, the specific time profile that the expression level dropped at day 21 and increased to some extent at day 42 was observed in 1 of 18 subjects. In other subjects, slow and small increase was observed in 6 of 18. (3) The ratio of mRNA expression level of membrane and soluble type of IL-6R showed marginal difference after tocilizumab treatment, (4) The distinct change of expression level of cytochrome P450 mRNA after tocilizumab treatment was hardly observed in most patients. The measurement of expression level of several genes shown below in peripheral blood of human subjects (18 patients with rheumatic disease) before and after tocilizumab medication using DNA microarray (Hitachi human oligo chip) was conducted in this test, 1) Interleukin 6 (IL-6), membrane IL-6 receptor (IL-6R) and soluble type of the IL-6R, 2) several cytochrome P450 as follows: CYP3A4, CYP2D6, CYP2C9, CYP2C19. Number of human subjects was 18 and peripheral blood samples drawn at 4 time points: day 0 (the first Omeprazole administration was done), day 7 (Tocilizumab administration was done), day 21 and day 42 for each subject were analyzed. On day 0 and day 7, the blood sampling was done before drug administration.
Project description:BACKGROUND - IL-6 mediates graft-versus-host disease (GVHD) in experimental allogeneic stem cell transplantation (alloSCT) and is an attractive therapeutic target. METHODS - A registered phase I/II study (ACTRN12612000726853) of IL-6 receptor (IL-6R) neutralizing antibody administration on day -1 to patients receiving full or reduced-intensity conditioning (RIC) and alloSCT from HLA-matched sibling or unrelated donors with standard cyclosporin and methotrexate GVHD prophylaxis. The primary endpoint was incidence of grade II-IV acute GVHD. Outcomes were compared to a non-randomized but contemporaneous group of study patients receiving the same alloSCT in the absence of IL-6R mAb. FINDINGS - Cytokine and pharmacokinetic analysis confirmed transient IL-6 dysregulation in the first month after alloSCT with complete inhibition following IL-6R mAb administration. With median follow up of 497 days, the incidence of grade II-IV GVHD was 12.5% in recipients of IL-6R inhibition (n = 48) versus 41.5% in the (n = 53) control cohort (P = 0.001). Low rates of acute GVHD were noted in patients receiving IL-6R inhibition relative to control patients following both myeloablative (12.5% vs. 46.4%, P = 0.03) and RIC (12.5% vs. 36.0%, P = 0.04). The incidence of severe (grade III/IV) acute GVHD was 4.2% in recipients of IL-6R inhibition versus 20.8% in the control cohort (P = 0.012). Relapse and chronic GVHD were unchanged. Immune reconstitution was preserved in recipients of IL-6R inhibition, but qualitatively modified with suppression of known pathogenic STAT3-dependent pathways. INTERPRETATION - IL-6 is the principal detectable and dysregulated cytokine secreted after alloSCT and its inhibition is a potential new and simple strategy to protect from acute GVHD despite robust immune reconstitution and a sustained graft-versus-leukemia effect. Single colour, Illumina Human HT12v4 Beadarrays.
Project description:IL-6 is a proinflammatory cytokine implicated in multiple autoimmune diseases. Here we show that IL-6 induced STAT3 and STAT1 phosphorylation is enhanced in CD4 and CD8 T cells from patients with T1D compared to healthy controls. Enhanced IL-6/pSTAT3 is associated with increased surface IL-6R and early clinical disease. The transcriptome of IL-6 treated CD4 T cells from T1D patients reveals upregulation of genes involved in T cell migration. The data suggest that individuals with type 1 diabetes may benefit from therapeutic targeting of the IL-6 pathway. Overall design: CD4+CD25- T cells were purified from thawed PBMC of seven subjects with type 1 diabetes. Cells were left unstimulated or were treated with 10ng/ml IL-6 for 24 hours. RNA was extracted and RNA sequencing was performed.
Project description:We found a unique subset of effector memory (EM) CD8+ T cells that expressed high levels of IL-6 receptor in human peripheral blood. These cells which also expressed high levels of IL-7Ra (referred to as IL-6R high IL-7Rahigh cells) had the a distinct gene expression profile and cellular characteristics compared to other EM CD8+ T cells. IL-6R high IL-7Ra high cells were early differentiated EM CD8+ T cells with decreased expression of T-bet, KLRG1, perforin and granzyme B. These cells had increased cell proliferation likely secondary to enhanced IL-2 production and high affinity IL-2R expression. IL-6R high IL-7Ra high EM CD8+ T cells exclusively produced high levels of IL-2, IL-5, IL-9 and IL-13 although IFN-r was produced by this cell subset and other EM CD8+ T cells. Of interest, IL-6R high IL-7Ra high EM CD8+ T cells expanded in the peripheral blood of patients with chronic obstructive pulmonary disease (COPD) and asthma where CD8+ T cells, IL-13 and IFN-r are suggested to be involved in the pathogenesis. Being the early-differentiated EM CD8+ T cells with a potent capacity to proliferate, survive and generate multiple cytokines, IL-6R high IL-7Ra high EM CD8+ T cells may serve as a primary reservoir for effector CD8+ T cells which potently expand and produce cytokines upon immune stimulation. Duplicate experiments were performed for each condition. In each condition, we independently prepared total RNA using the RNeasy mini kit (Qiagen) and assessed RNA integrity using Bioanalyzer 2100 (Agilent)- RINs were close to 10 for all samples. RNA was then amplified and hybridized to the Illumina HumanHT-12 v4.0 BeadChip, according to Illumina standard protocols.
Project description:To understand the molecular mechanism underlying the IL-6-mediated attenuation of Th1 differentiation in tumor-bearing animals, we have employed whole genome microarray expression profiling of CD4+ T cells isolated from tumor-bearing mice. In order to distingish the effect of IL-6/sIL-6R signaling augmented in tumor-bearing mice from the effecct of other tumor-derived signals, we isolated CD4+ T cells that were primed in tumor free-mice and in the mice inoculated with ovalbumin (OVA)-expressing MCA205 fibrosamcoma, in conjunction of anti-IL-6R Ab injection. We found that Th2-like gene signature such as Il4, IL10, Ccr4, and c-maf was up-regulated in CD4+ T cells primed in tumor-bearing mice. However,the expression of other Th2 master regulator Gata3 gene, or Tfh-related genes (Bcl6, Cxcr5, and Batf) was not substantially augmented in tumor-bearing mice as compared with those in tumor-free mice. These expressions were abrogated by IL-6R blockade, suggesting that the expression of these Th2-like gene set and the other IL-6 signal-dependent genes like Klf1, Bcl11a, and Klf4 are candidate regulators responsible for IL-6 signaling-mediated Th1 inhibition in tumor-bearing animals. Overall design: The differential gene expressions in CD4+ T cells primed in tumor-free vs MCA205-OVA-bearing mice, and in control Ab-treated vs anti-IL-6R Ab-treated MCA-OVA tumor-bearing mice were measured. Two independent experiments were performed in each condition.
Project description:Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a major role in responding to injury or infection as well as immune response, inflammation, and hematopoiesis. High levels of circulating IL-6 are observed in many tumor types and are associated with poor outcomes. We show that knockdown of IL-6 or IL-6 receptor (IL-6R) inhibits IL-6 signaling and cell viability. In contrast, over-expression of IL-6 enhances tumor growth in vitro and in vivo, thereby supporting the role of IL-6 in tumorigenesis. We developed a human monoclonal antibody against human IL-6 (MEDI5117) that bears Fc mutations (YTE) to extend its half-life. We tested this antibody in several cancer cell lines that secrete high levels of IL-6, soluble IL-6R, and express gp130. High constitutive pSTAT3 (phosphorylated signal transducer and activator of transcription 3) activation is seen in several of these cell lines, suggesting autocrine growth stimulation by IL-6. Treating these cell lines with MEDI5117 effectively blocked phosphorylation of STAT3 and inhibited IL-6-induced cell proliferation. In vivo, MEDI5117 suppressed the growth of multiple cancer xenograft models and specifically modulated IL-6 signaling and downstream gene expression. Combining MEDI5117 with chemotherapy or gefitinib demonstrated significantly enhanced anti-tumor activities in vivo. Taken together, our data suggest that IL-6 signaling contributes to tumor growth, thereby supporting the development of MEDI5117 as a therapy to treat solid tumors. Xenograft tumors from DU145, KPL4, and NCI-H1650 were treated with 30 mg/kg of MEDI5117 or IgG isotype control IgG1 for a total of two doses. Differentially expressed genes were identified and canonical pathway enrichment analysis was performed
Project description:Here, we performed a comprehensive proteome profiling of platelets collected from 18 healthy subjects before and after administration of sarpogrelate. For each subject, platelets were isolated before and then again after drug administration to investigate the alteration in the platelet proteome by sarpogrelate. A sample preparation method involving filter-aided sample preparation (FASP) was used to improve extraction of proteins including both soluble and membraneous proteins. Moreover, an ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) coupled with extensive fractionation was used to generate a comprehensive proteome of platelets.
Project description:Epigenetic changes, including histone methylation, control T cell differentiation and memory formation, though the enzymes that mediate these processes are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, promotes the generation of T follicular helper (Tfh) cells, a CD4+ T cell subset essential for B cell antibody generation and clearance of chronic viral infections. Mice with T cell specific UTX deletion (UTX TKO mice) had fewer Tfh cells, showed defective germinal center formation, lacked virus-specific IgG production, and were unable to resolve chronic lymphocytic choriomeningitis virus infection. In UTX TKO T cells, decreased expression of IL-6R±, and other Tfh-related loci, was associated with increased H3K27 methylation. Additionally, Turner Syndrome subjects, who are predisposed to chronic ear infections, have reduced UTX expression in immune cells and decreased Tfh frequency. Thus, we identify a critical link between UTX in T cells and immunity to infection. Mice were infected with chronic LCMV and sorted for Tfh on day 21 pi (sorted for: CD4+CXCR5+CD62Llo T cell). ChIP-Seq and RNA-Seq on WT and Utx KO cells.
Project description:Dwivedi2014 - Crohns IL6 Disease model -
This model is comprised of four models:
Healthy Volunteer model
Crohn's Disease - IL-6 Antibody
Crohn's Disease - sgp130FC
Crohn's Disease - IL-6Ra Antibody
Possible avenues for Interleukin-6 (IL-6) inhibition in
treating Crohn's disease are compared here. Each model refers to
separate ligands. The system simulates differential activity of the
ligands on the signalling of IL-6.
This affects Signal Transducer and Activator of
Transcription 3 (STAT3) activity on the production of
biomarker C-Reactive Protein (CRP) expression.
The figure referring to this Crohn's Disease model is 6b.
This model is described in the article:
A multiscale model of
interleukin-6-mediated immune regulation in Crohn's disease and
its application in drug discovery and development.
Dwivedi G, Fitz L, Hegen M, Martin
SW, Harrold J, Heatherington A, Li C.
CPT Pharmacometrics Syst Pharmacol
2014; 3: e89
In this study, we have developed a multiscale systems model
of interleukin (IL)-6-mediated immune regulation in Crohn's
disease, by integrating intracellular signaling with
organ-level dynamics of pharmacological markers underlying the
disease. This model was linked to a general pharmacokinetic
model for therapeutic monoclonal antibodies and used to
comparatively study various biotherapeutic strategies targeting
IL-6-mediated signaling in Crohn's disease. Our work
illustrates techniques to develop mechanistic models of disease
biology to study drug-system interaction. Despite a sparse
training data set, predictions of the model were qualitatively
validated by clinical biomarker data from a pilot trial with
tocilizumab. Model-based analysis suggests that strategies
targeting IL-6, IL-6R?, or the IL-6/sIL-6R? complex are less
effective at suppressing pharmacological markers of Crohn's
than dual targeting the IL-6/sIL-6R? complex in addition to
IL-6 or IL-6R?. The potential value of multiscale system
pharmacology modeling in drug discovery and development is also
discussed.CPT: Pharmacometrics & Systems Pharmacology
(2014) 3, e89; doi:10.1038/psp.2013.64; advance online
publication 8 January 2014.
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