Translational research in rheumatoid arthritis: Exploiting melanocortin receptors

The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the authorRheumatoid arthritis (RA) is a chronic inflammatory disease affecting 1% of the population. The aetiology of rheumatoid arthritis is unknown, although there are multiple postulated theories. In 1950, Philip Hench won the Nobel prize for treating patients with rheumatoid arthritis with cortisone. He also treated 6 patients with adrenocorticotropic hormone (ACTH) with good results. ACTH is a melanocortin. The melanocortin system describes the five melanocortin receptors, their ligands, agonists and antagonists and the accessory proteins. The aim of this study was to explore the melanocortin receptors in rheumatoid arthritis synovium. Methods HA-tagged stable cell lines were created for MC1R, MC3R and MC5R. Multiple antibodies were tested for their utility using Western Blot, immunohistochemistry and flow cytometry. Samples of synovium from 28 patients with RA were tested using RTPCR for the presence of MC1R and MC3R. Gene expression was correlated with clinical characteristics, cytokine (RTPCR) expression and immunohistochemical score. Results The stable cell lines expressed MC1R, MC3R and MC5R respectively. Of the antibodies tested none were found to be of utility in detecting MC1R or MC3R .The MC1R RQ values in rheumatoid synovium appear to split into two groups, high and low. The medians of the two groups are significantly different (p=0.0005). There is almost a 5 cycle, or 64 fold, difference in gene expression between the medians of the two groups (1.59 v 6.23). Of note no MC3R positive samples were CD138 high (i.e. no MC3R positive samples had a significant plasma cell infiltrate) (p=0.006). Categorical analysis using Fishers Exact test revealed an association between MC1R high samples and CD68 lining high scores, (i.e. MC1R high samples also had a high macrophage score in the lining of the sample) (p=0.02). MC1R low samples were associated with not being on combination therapy, 15 this did not quite reach significance (p=0.07). Linear regression analysis confirmed these associations for MC1R. PCA analysis did not show any grouping of samples according to any of the variables tested, likely due to sample size. Conclusion MC1R and MC3R are found in human synovium. Current commercial antibodies are not of utility in detecting MC1R or MC3R. Synovial samples can be split into high and low MC1R gene expression groups. MC3R was either present or absent. High expression of MC1R was associated with a high macrophage score and MC3R expression was associated with a low plasma cell score. MC1R and MC3R expression in RA synovium could be used as biomarkers of disease state or severity as well as a target for therapy

Investigating the effects of the disease-modifying anti-rheumatic drug methotrexate on the vascular endothelium

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease that associates with increased mortality from cardiovascular disease (CVD). Methotrexate (MTX), a folate analogue used as disease-modifying anti-rheumatic drug (DMARD), reduces CVD morbidity and mortality in RA patients, possibly by improving endothelial function. The aim of this study was to investigate the molecular effects of MTX in quiescent and activated vascular endothelial cells (EC). In EC pre-treated with tumour necrosis factor (TNF)-alpha, MTX independently increased the activity of mitogen-activated protein kinase (MAPK) p38 and Akt and caused cell cycle arrest due to folate depletion. In contrast, MTX did not affect cell signalling, proliferation or gene expression in EC exposed to different types of shear stress. To tease out the underlying mechanism, folate or one carbon metabolism (OCM) was investigated in EC cultivated under static or shear stress conditions. Endothelial OCM was fundamentally altered by shear stress, with potentially important implications for MTX uptake and function. Using endothelial colony forming cells (ECFC) isolated from RA patients before and after treatment initiation with MTX and hydroxychloroquine, it was shown that systemic inflammation primes ECFC towards a pro-inflammatory state that may be reversible with DMARD treatment. These data suggest that MTX acts on vascular EC in a folate-dependent manner. However, since endothelial OCM was largely downregulated by shear stress, this implies that MTX may have limited direct effects on EC in vivo and provides a potential explanation for the controversies around the vascular effects of folate and anti-folate therapy. Although findings in ECFC from patients with RA support anti-inflammatory effects of MTX containing therapy, the data in this thesis do not support a direct MTX-mediated anti-atherogenic effect on the endothelium but suggest that the drug may limit EC dysfunction in combination with other drugs indirectly by reducing systemic inflammation.Open Acces

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Inflammation in the 21st Century

The present book includes 17 chapters covering different fields of inflammation that can be classified into acute or chronic in response to trauma, infection, and exposure to other noninfectious agents, including allergens and xenobiotics. Inflammation is a self-healing process, upon the clearance of the foreign particle and helps to protect the host. However, when it is not resolved and becomes chronic, it may lead to cancer and autoimmune diseases. This book includes different topics of autoimmune diseases, cancer, and other sterile inflammatory conditions originating in the absence of allergens as well as autoimmune disease and generates inflammatory immune response. Hence, the book will prove beneficial to researchers and scientists involved in inflammation research

Peripheral-Blood Stem Cells versus Bone Marrow from Unrelated Donors

BACKGROUND Randomized trials have shown that the transplantation of filgrastim-mobilized peripheral-blood stem cells from HLA-identical siblings accelerates engraftment but increases the risks of acute and chronic graft-versus-host disease (GVHD), as compared with the transplantation of bone marrow. Some studies have also shown that peripheral-blood stem cells are associated with a decreased rate of relapse and improved survival among recipients with high-risk leukemia. METHODS We conducted a phase 3, multicenter, randomized trial of transplantation of peripheral-blood stem cells versus bone marrow from unrelated donors to compare 2-year survival probabilities with the use of an intention-to-treat analysis. Between March 2004 and September 2009, we enrolled 551 patients at 48 centers. Patients were randomly assigned in a 1:1 ratio to peripheral-blood stem-cell or bone marrow transplantation, stratified according to transplantation center and disease risk. The median follow-up of surviving patients was 36 months (interquartile range, 30 to 37). RESULTS The overall survival rate at 2 years in the peripheral-blood group was 51% (95% confidence interval [CI], 45 to 57), as compared with 46% (95% CI, 40 to 52) in the bone marrow group (P=0.29), with an absolute difference of 5 percentage points (95% CI, −3 to 14). The overall incidence of graft failure in the peripheral-blood group was 3% (95% CI, 1 to 5), versus 9% (95% CI, 6 to 13) in the bone marrow group (P=0.002). The incidence of chronic GVHD at 2 years in the peripheral-blood group was 53% (95% CI, 45 to 61), as compared with 41% (95% CI, 34 to 48) in the bone marrow group (P=0.01). There were no significant between-group differences in the incidence of acute GVHD or relapse. CONCLUSIONS We did not detect significant survival differences between peripheral-blood stem-cell and bone marrow transplantation from unrelated donors. Exploratory analyses of secondary end points indicated that peripheral-blood stem cells may reduce the risk of graft failure, whereas bone marrow may reduce the risk of chronic GVHD. (Funded by the National Heart, Lung, and Blood Institute–National Cancer Institute and others; ClinicalTrials.gov number, NCT00075816.

Advanced in vitro models for studying drug induced toxicity

Bringing safe medicines to the market has remained a major challenge to the pharmaceutical industry. Recent years have seen increased drug attrition rates due to toxicity - even after rigorous testing in both in vitro and in vivo test models. This is partly due to poor prediction of human-specific responses in these models. This thesis aims to address the issue by developing advanced in vitro models and methods that can complement and improve the predictive power of in vitro assays at preclinical level. Liver and kidneys are often susceptible to drug insult due to their respective roles in drug metabolism and reabsorption. We have developed a robust 3D in vitro model for liver toxicity studies, this model shows many hallmarks of in vivo hepatocytes, is applied in a 384-micro-well format and is compatible with standard medium- and high-throughput lab infrastructure for routine drug screening. This thesis also discusses the role of immune mediators in aggravating kidney toxicity and use of sophisticated high-content screening approach to measure apoptosis and necrosis in real time. These models are promising new tools for preclinical drug safety testingNetherlands Toxicogenomics centre (NTC) via Netherlands Genomics InitiativeUBL - phd migration 201

Psoriasis

We hope you enjoy and find the information provided in this book useful in your research or practice. We urge that you continue to keep abreast of the new developments in psoriasis and share your knowledge so that we may advance treatment and cures of psoriasis