Marjorie Boissinot

Cytokines as Biomarkers in Rheumatoid Arthritis

RA is a complex disease that develops as a series of events often referred to as disease continuum. RA would benefit from novel biomarker development for diagnosis where new biomarkers are still needed (even if progresses have been made with the inclusion of ACPA into the ACR/EULAR 2010 diagnostic criteria) and for prognostic notably in at risk of evolution patients with autoantibody-positive arthralgia. Risk biomarkers for rapid evolution or cardiovascular complications are also highly desirable. Monitoring biomarkers would be useful in predicting relapse. Finally, predictive biomarkers for therapy outcome would allow tailoring therapy to the individual. Increasing numbers of cytokines have been involved in RA pathology. Many have the potential as biomarkers in RA especially as their clinical utility is already established in other diseases and could be easily transferable to rheumatology. We will review the current knowledges relation to cytokine used as biomarker in RA. However, given the complexity and heterogeneous nature of RA, it is unlikely that a single cytokine may provide sufficient discrimination; therefore multiple biomarker signatures may represent more realistic approach for the future of personalised medicine in RA.

Autoantibodies to posttranslational modifications in rheumatoid arthritis.

Autoantibodies have been associated with human pathologies for a long time, particularly with autoimmune diseases (AIDs). Rheumatoid factor (RF) is known since the late 1930s to be associated with rheumatoid arthritis (RA). The discovery of anticitrullinated protein antibodies in the last century has changed this and other posttranslational modifications (PTM) relevant to RA have since been described. Such PTM introduce neoepitopes in proteins that can generate novel autoantibody specificities. The recent recognition of these novel specificities in RA provides a unique opportunity to understand human B-cell development in vivo. In this paper, we will review the three of the main classes of PTMs already associated with RA: citrullination, carbamylation, and oxidation. With the advancement of research methodologies it should be expected that other autoantibodies against PTM proteins could be discovered in patients with autoimmune diseases. Many of such autoantibodies may provide significant biomarker potential.

An immunological biomarker to predict MTX response in early RA

Objectives The therapeutic goal for patients with rheumatoid arthritis (RA) is clinical remission. This is best achieved by early diagnosis and appropriate therapeutic intervention. RA is associated with dysregulation of T-cell subsets (naïve, regulatory (Treg) and inflammation-related cells (IRC)) early in the disease. Our aim was to test the hypothesis that T-cell subset quantification can predict the achievement of clinical remission with early treatment in RA. Methods T-cell subsets were quantified in 108 drug-naïve, early RA patients commencing methotrexate (MTX) or MTX+antitumor necrosis factor (anti-TNF) and in 105 healthy controls (HC). The primary outcome assessed was remission (DAS28<2.6). A pilot study used frozen cells (38 patients and 35 HCs, see online supplementary material) and was validated with fresh blood (70 patients and 70 HCs). Results Immune dysregulation in early RA was confirmed with an association between age and reduced naïve cells compared with HCs (p=0.006), a lower age-adjusted Treg and higher IRC frequency (p=0.001). Anticitrullinated peptide antibody (ACPA) positivity was associated with lower naïve (p=0.031) and Treg frequencies (p=0.039). In 50 patients treated with MTX, ACPA/age-adjusted analysis demonstrated that higher naïve cell frequency (relative to HC) was associated with remission (OR 5.90 (1.66 to 20.98), p=0.006, sensitivity/specificity 62%/79%, Positive Predictive Value (PPV)/Negative Predictive Value (NPV) 66%/76%). Remission with MTX+anti-TNF (n=20) was not found to be associated with naïve cell frequency, and for patients with reduced naïve cells the remission rate increased from 24% (MTX) to 42% (MTX+anti-TNF). Conclusions Baseline T-cell subset analysis has a value in predicting early RA remission with first therapy with MTX. Immunological analysis could be used in conjunction with clinical/serological features to predict response to MTX and help select the most appropriate therapy at disease presentation.

Induction of differentiation and apoptosis in leukaemic cell lines by the novel benzamide family histone deacetylase 2 and 3 inhibitor MI-192

Histone deacetylase inhibitors (HDACIs) are in advanced clinical development as cancer therapeutic agents. However, first generation HDACIs such as butyrate and valproate are simple short chain aliphatic compounds with moieties resembling acetyl groups, and have a broad spectrum of activity against HDACs. More complex second generation HDACIs undergoing clinical trials, such as the benzamide group compounds MS-275 and MGCD0103, are specific primarily for HDAC1 and HDAC2. To expand the repertoire of available HDACIs and HDAC specificities we created a novel benzamide-based compound named MI-192. When tested against purified recombinant HDACs, MI-192 had marked selectivity for the class I enzymes, HDAC2 and HDAC3. Screening in the NCI60 screen demonstrated that MI-192 had greatly enhanced efficacy against cells of leukaemic origin. When tested in culture against the acute myeloid leukaemic cell lines U937, HL60 and Kasumi-1, MI-192 induced differentiation and was cytotoxic through promotion of apoptosis. MI-192 therefore justifies further investigation and development as a potential therapeutic agent for use in leukaemia.

The Hepatocyte Growth Factor (HGF)/Met Axis: A Neglected Target in the Treatment of Chronic Myeloproliferative Neoplasms?

Met is the receptor of hepatocyte growth factor (HGF), a cytoprotective cytokine. Disturbing the equilibrium between Met and its ligand may lead to inappropriate cell survival, accumulation of genetic abnormalities and eventually, malignancy. Abnormal activation of the HGF/Met axis is established in solid tumours and in chronic haematological malignancies, including myeloma, acute myeloid leukaemia, chronic myelogenous leukaemia (CML), and myeloproliferative neoplasms (MPNs). The molecular mechanisms potentially responsible for the abnormal activation of HGF/Met pathways are described and discussed. Importantly, inCML and in MPNs, the production of HGF is independent of Bcr-Abl and JAK2V617F, the main molecular markers of these diseases. In vitro studies showed that blocking HGF/Met function with neutralizing antibodies or Met inhibitors significantly impairs the growth of JAK2V617F-mutated cells. With personalised medicine and curative treatment in view, blocking activation of HGF/Met could be a useful addition in the treatment of CML and MPNs for those patients with high HGF/MET expression not controlled by current treatments (Bcr-Abl inhibitors in CML; phlebotomy, hydroxurea, JAK inhibitors in MPNs).

Prior epigenetic priming of cytokine genes in naive T cells is required for their subsequent activation by inducible enhancers

The inducible lL-3 and GM-CSF genes can only be efficiently expressed in T cells once they have been through a previous cycle of activation and undergone a process termed blast cell transformation [1]. An initial stimulus is required to bring naive T cells out of the resting state and into the cell cycle. This process is triggered by T cell receptor (TCR) stimulation, takes about 24 hours, and is associated with extensive nuclear remodeling. Once primed by a cycle of activation, T blast cells can maintain their ability to express lL-3 and GM-CSF for many cell divisions without the continual need for additional stimuli. In contrast, the lL-3 and GM-CSF genes cannot be induced in naive T cells that have never received a TCR stimulus. We show that this pattern of regulation of the IL-3/GM-CSF locus is controlled at two distinct levels: (1) During blast cell transformation, the IL-3/GM-CSF locus acquires an extensive array of DNase I Hypersensitive Sites (DHSs) which are then maintained indefinitely for many cell cycles [1,2]. These primed DHSs are marked by me2K4 histone H3, and they also persist in non-dividing memory T cells in the peripheral blood [1]. These DHSs are absent in the thymus, spleen T cells, and naive T cells in the blood. These DHSs do not function as classical enhancers, and we propose that they serve to maintain an active chromatin structure in previously activated T cells. (2) The expression of the lL-3 and GM-CSF genes is in each case dependent on the activation of inducible upstream enhancers. The lL-3 and GM-CSF enhancers appear as inducible DHSs T blast cells within 20 min of stimulation, and only acquire the me2K4H3 modification after stimulation. These enhancers are dependant on the TCR inducible factors NFAT and AP-1. However, although AP-1 and NFAT family mRNAs are efficiently expressed in both naive T cells and in T blast cells, the enhancers only respond to induction of these factors in T blast cells. The inducible DHSs remain completely undetectable in naive T cells even after 4 hours of stimulation with direct activators of TCR signaling pathways (PMA and Calcium ionophore). We propose that T blast cells and memory T cells have developed a strategy of maintaining a discrete class of DHS as epigenetic memory modules that support an accessible chromatin environment and thereby prime inducible genes for efficient re-activation when memory cells re-encounter Ag stimuli. In the absence of the priming elements, the lL-3/GM-CSF locus appears to remain inaccessible to transcription factors that are otherwise very efficient at recruiting the chromatin remodelers that create DHSs within the inducible enhancers.