TNFα inhibitors reduce bone loss in rheumatoid arthritis independent of clinical response by reducing osteoclast precursors and IL-20.

OBJECTIVES: About half of RA patients treated with TNFα inhibitors either do not respond or lose their initial therapeutic response over time. The clinical response is measured by reduction in DAS28, which primarily reflects inflammation. However, other effects of TNFα inhibitors, such as impact on bone erosion, are not assessed by DAS28. We aimed to examine the effect of TNFα inhibitors on bone density, bone biomarkers and cytokine production in responder and non-responder patients and assessed mechanisms of action. METHODS: BMD in the lumbar spine and femur neck of 117 RA patients was measured by DEXA scan. Bone turnover biomarkers CTX, osteoprotegerin (OPG), osteocalcin and RANKL were measured by ELISA. Levels of 16 cytokines in plasma and in tissue culture supernatants of ex vivo T cells were measured by multiplex assays and ELISA. The effect of treatment with TNFα inhibitors on blood mononuclear cell (MNC) differentiation to osteoclast precursors (OCP) was measured flow cytometry and microscopy. RESULTS: TNFα inhibitors improved lumbar spine BMD but had modest effects on blood bone biomarkers, irrespective of patients' clinical response. Blood OCP numbers and the ability of monocytes to differentiate to OCP in vitro declined after treatment. Treatment also reduced RANK expression and IL-20 production. BMD improvement correlated with reduced levels of IL-20 in responder patients. CONCLUSION: This study reveals that TNFα inhibitors reduce lumbar spine bone loss in RA patients irrespective of changes in DAS28. The reduction in bone loss is associated with reduction in IL-20 levels in responder patients.

Functional and phenotypic heterogeneity of Th17 cells in health and disease.

BACKGROUND: Th17 cells have nonredundant roles in maintaining immunity, particularly at mucosal surfaces. These roles are achieved principally through the production of cytokines and the recruitment of other immune cells to maintain the integrity of mucosal barriers and prevent the dissemination of microorganisms. Th17 cells are heterogeneous and exhibit a considerable degree of plasticity. This allows these cells to respond to changing environmental challenges. However, Th17 cells also play pro-inflammatory roles in chronic autoimmune diseases. The trigger(s) that initiate these Th17 responses in chronic autoimmune diseases remain unclear. DESIGN: In this report, we provide an overview of studies involving animal models, patient data, genome wide association studies and clinical trials targeting IL-17 for treatment of patients to gain a better understanding of the pathogenic roles of Th17 cells play in a range of autoimmune diseases. RESULTS: The report sheds light on likely triggers that initiate or perpetuate Th17 responses that promote chronic inflammation and autoimmunity. The divergent effects of tumour necrosis factor alpha blockade on Th17 cells in patients, is explored. Furthermore, we highlight the role of Th17 cells in inducing autoreactive B cells, leading to autoantibody production. Pathogenic bacterial species can change Th17 cell phenotype and responses. These findings provide insights into how Th17 cells could be induced to promoting autoimmune disease pathogenesis. CONCLUSION: This article provides an overview of the distinct roles Th17 cells play in maintaining immunity at mucosal surfaces and in skin mucosa and how their functional flexibility could be linked with chronic inflammation in autoimmune rheumatic diseases.

TNFα in the regulation of Treg and Th17 cells in rheumatoid arthritis and other autoimmune inflammatory diseases.

TNFα is a principal pro-inflammatory cytokine vital for immunity to infections. However, its excessive production is involved in chronic inflammation and disease pathology in autoimmune diseases. Evidence for its pathogenic role is validated by the fact that its neutralisation by therapeutic agents in vivo is beneficial in ameliorating disease and controlling symptoms. Paradoxically, however, treatment with TNFα inhibitors can either have no clinical effects, or even exacerbate disease in some patients. The explanation for such contradictory outcomes may lay in how and which downstream signalling pathways are activated and drive disease. TNFα causes its effects by binding to either or both of two membrane-bound receptors, TNFR1 and TNFR2. Engagement of the receptors can induce cell death or cell proliferation. T cells both produce and respond to TNFα and depending on whether the cytokine is membrane-bound or soluble and the level of expression of its two receptors, the biological outcome can be distinct. In addition, polymorphisms in genes encoding TNFα and T cell signalling proteins can significantly impact the outcome of TNFα receptor engagement. Early studies revealed that effector T cells in patients with rheumatoid arthritis (RA) are hyporesponsive due to chronic exposure to TNFα. However, recent evidence indicates that the relationship between TNFα and T cell responses is complex and, at times, can be paradoxical. In addition, there is controversy as to the specific effects of TNFα on different T cell subsets. This review will summarise knowledge on how TNFα modulates T cell responses and the effect of engaging either of its two receptors. Furthermore, we discuss how such interactions can dictate the outcome of treatment with TNFα inhibitors.

Harnessing the Therapeutic Potential of Th17 Cells.

Th17 cells provide protective immunity to infections by fungi and extracellular bacteria as well as cancer but are also involved in chronic inflammation. The cells were first identified by their ability to produce interleukin 17A (IL-17A) and, subsequently, associated with chronic inflammation and autoimmunity. Th17 cells have some gene profile similarity with stem cells and can remain dormant in mucosal tissues for long periods. Indeed, recent studies suggest that functionally distinct subsets of pro- and anti-inflammatory Th17 cells can interchange phenotype and functions. For development, Th17 cells require activation of the transcription factors STAT3 and RORγt while RUNX1, c-Maf, and Aiolos are involved in changes of phenotype/functions. Attempts to harness Th17 cells against pathogens and cancer using vaccination strategies are being explored. The cells gain protective abilities when induced to produce interferon γ (IFNγ). In addition, treatment with antibodies to IL-17 is effective in treating patients with psoriasis, psoriatic arthritis, and refectory rheumatoid arthritis. Moreover, since RORγt is a nuclear receptor, it is likely to be a potential future drug target for modulating Th17 functions. This review explores pathways through which Th17 subsets are induced, the molecular basis of their plasticity, and potential therapeutic strategies for their modulation in diseases.

Targeted delivery of cytokine therapy to rheumatoid tissue by a synovial targeting peptide.

OBJECTIVES: The synovial endothelium targeting peptide (SyETP) CKSTHDRLC has been identified previously and was shown to preferentially localise to synovial xenografts in the human/severe combined immunodeficient (SCID) mouse chimera model of rheumatoid arthritis (RA). The objective of the current work was to generate SyETP-anti-inflammatory-cytokine fusion proteins that would deliver bioactive cytokines specifically to human synovial tissue. METHODS: Fusion proteins consisting of human interleukin (IL)-4 linked via a matrix metalloproteinase (MMP)-cleavable sequence to multiple copies of either SyETP or scrambled control peptide were expressed in insect cells, purified by Ni-chelate chromatography and bioactivity tested in vitro. The ability of SyETP to retain bioactive cytokine in synovial but not control skin xenografts in SCID mice was determined by in vivo imaging using nano-single-photon emission computed tomography-computed tomography (nano-SPECT-CT) and measuring signal transducer and activator of transcription 6 (STAT6) phosphorylation in synovial grafts following intravenous administration of the fusion protein. RESULTS: In vitro assays confirmed that IL-4 and the MMP-cleavable sequence were functional. IL-4-SyETP augmented production of IL-1 receptor antagonist (IL-1ra) by fibroblast-like synoviocytes (FLS) stimulated with IL-1ß  in a dose-dependent manner. In vivo imaging showed that IL-4-SyETP was retained in synovial but not in skin tissue grafts and the period of retention was significantly enhanced through increasing the number of SyETP copies from one to three. Finally, retention correlated with increased bioactivity of the cytokine as quantified by STAT6 phosphorylation in synovial grafts. CONCLUSIONS: The present work demonstrates that SyETP specifically delivers fused IL-4 to human rheumatoid synovium transplanted into SCID mice, thus providing a proof of concept for peptide-targeted tissue-specific immunotherapy in RA. This technology is potentially applicable to other biological treatments providing enhanced potency to inflammatory sites and reducing systemic toxicity.

Aberrant B-lymphocyte responses in lupus: inherent or induced and potential therapeutic targets.

BACKGROUND: Lupus is a prototype autoimmune disease of unknown aetiology. The disease is complex; manifest diverse clinical symptoms and disease mechanisms. This complexity has provided many leads to explore: from disease mechanisms to approaches for therapy. B-lymphocytes play a central role in the pathogenesis of the disease. However, the cause of aberrant B-lymphocyte responses in patients and, indeed, its causal relationship with the disease remain unclear. DESIGN: This article provides a synopsis of current knowledge of immunological abnormalities in lupus with an emphasis on abnormalities in the B-lymphocyte compartment. RESULTS: There is evidence for abnormalities in most compartments of the immune system in animal models and patients with lupus including an ever expanding list of abnormalities within the B-lymphocyte compartment. In addition, recent genome-wide linkage analyses in large cohorts of patients have identified new sets of genetic association factors some with potential links with defective B-lymphocyte responses although their full pathophysiological effects remain to be determined. The accumulating knowledge may help in the identification and application of new targeted therapies for treating lupus disease. CONCLUSIONS: Cellular, molecular and genetic studies have provided significant insights into potential causes of immunological defects associated with lupus. Most of this insight relate to defects in B- and T-lymphocyte tolerance, signalling and responses. For B-lymphocytes, there is evidence for altered regulation of inter and intracellular signalling pathways at multiple levels. Some of these abnormalities will be discussed within the context of potential implications for disease pathogenesis and targeted therapies.

Incomplete response of inflammatory arthritis to TNFα blockade is associated with the Th17 pathway.

OBJECTIVES: To establish if changes in Th1/Th17 cell populations previously reported in experimental arthritis occur in patients with rheumatoid arthritis (RA) treated with anti-tumour necrosis factor α (TNFα) agents, and whether the therapeutic response to anti-TNFα is compromised in patients and mice because of elevated Th17/IL-17 levels. Finally, to assess the efficacy of combined blockade of anti-TNFα and anti-IL-17 in experimental arthritis. METHODS: A longitudinal study of two independent cohorts (cohort 1, n=24; cohort 2, n=19) of patients with RA treated with anti-TNFα biological agents was carried out to assess their Th17/IL-17 levels before and after the start of anti-TNFα therapy. IL-12/23p40 production was assessed in plasma Peripheral blood lymphocytes (PBLs) and monocytes. Mice with collagen-induced arthritis (CIA) were treated with anti-TNFα alone, anti-IL17 alone or a combination of the two. Efficacy of treatment and response was assessed from changes in Disease Activity Score 28-erythrocyte sedimentation rate scores in patients, and in clinical scores and histological analysis in CIA. RESULTS: Significant increases in circulating Th17 cells were observed in patients after anti-TNFα therapy and this was accompanied by increased production of IL-12/23p40. There was an inverse relationship between baseline Th17 levels and the subsequent response of patients with RA to anti-TNFα therapy. In addition, PBLs from non-responder patients showed evidence of increased IL-17 production. Similarly, in anti-TNFα-treated mice, there was a strong correlation between IL-17 production and clinical score. Finally combined blockade of TNFα and IL-17 in CIA was more effective than monotherapy, particularly with respect to the duration of the therapeutic effect. CONCLUSIONS: These findings, which need to be confirmed in a larger cohort, suggest that a Th17-targeted therapeutic approach may be useful for anti-TNFα non-responder patients or as an adjunct to anti-TNFα therapy, provided that safety concerns can be addressed.

Metabolic requirements of Th17 cells and of B cells: Regulation and defects in health and in inflammatory diseases.

The immune system protects from infections and cancer through complex cellular networks. For this purpose, immune cells require well-developed mechanisms of energy generation. However, the immune system itself can also cause diseases when defective regulation results in the emergence of autoreactive lymphocytes. Recent studies provide insights into how differential patterns of immune cell responses are associated with selective metabolic pathways. This review will examine the changing metabolic requirements of Th17 cells and of B cells at different stages of their development and activation. Both cells provide protection but can also mediate diseases through the production of autoantibodies and the production of proinflammatory mediators. In health, B cells produce antibodies and cytokines and present antigens to T cells to mount specific immunity. Th17 cells, on the other hand, provide protection against extra cellular pathogens at mucosal surfaces but can also drive chronic inflammation. The latter cells can also promote the differentiation of B cells to plasma cells to produce more autoantibodies. Metabolism-regulated checkpoints at different stages of their development ensure the that self-reactive B cells clones and needless production of interleukin (IL-)17 are limited. The metabolic regulation of the two cell types has some similarities, e.g. the utility of hypoxia induced factor (HIF)1α during low oxygen tension, to prevent autoimmunity and regulate inflammation. There are also clear differences, as Th17 cells only are vulnerable to the lack of certain amino acids. B cells, unlike Th17 cells, are also dependent of mechanistic target of rapamycin 2 (mTORC2) to function. Significant knowledge has recently been gained, particularly on Th17 cells, on how metabolism regulates these cells through influencing their epigenome. Metabolic dysregulation of Th17 cells and B cells can lead to chronic inflammation. Disease associated alterations in the genome can, in addition, cause dysregulation to metabolism and, thereby, result in epigenetic alterations in these cells. Recent studies highlight how pathology can result from the cooperation between the two cell types but only few have so far addressed the key metabolic alterations in such settings. Knowledge of the impact of metabolic dysfunction on chronic inflammation and pathology can reveal novel therapeutic targets to treat such diseases.

Dysregulated B cell function and disease pathogenesis in systemic sclerosis.

Systemic sclerosis (SSc) is a complex, immune-mediated rheumatic disease characterised by excessive extracellular matrix deposition in the skin and internal organs. B cell infiltration into lesional sites such as the alveolar interstitium and small blood vessels, alongside the production of defined clinically relevant autoantibodies indicates that B cells play a fundamental role in the pathogenesis and development of SSc. This is supported by B cell and fibroblast coculture experiments revealing that B cells directly enhance collagen and extracellular matrix synthesis in fibroblasts. In addition, B cells from SSc patients produce large amounts of profibrotic cytokines such as IL-6 and TGF-ß, which interact with other immune and endothelial cells, promoting the profibrotic loop. Furthermore, total B cell counts are increased in SSc patients compared with healthy donors and specific differences can be found in the content of naïve, memory, transitional and regulatory B cell compartments. B cells from SSc patients also show differential expression of activation markers such as CD19 which may shape interactions with other immune mediators such as T follicular helper cells and dendritic cells. The key role of B cells in SSc is further supported by the therapeutic benefit of B cell depletion with rituximab in some patients. It is notable also that B cell signaling is impaired in SSc patients, and this could underpin the failure to induce tolerance in B cells as has been shown in murine models of scleroderma.

Transcriptional profiling of human Vδ1 T cells reveals a pathogen-driven adaptive differentiation program.

γδ T cells are generally considered innate-like lymphocytes, however, an "adaptive-like" γδ compartment has now emerged. To understand transcriptional regulation of adaptive γδ T cell immunobiology, we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult Vδ1+ T cells segregate into TCF7+LEF1+Granzyme Bneg (Tnaive) or T-bet+Eomes+BLIMP-1+Granzyme B+ (Teffector) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in Teffector cells. Transcriptional reprogramming mirrors changes within CD8+ αß T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive Vδ1+ and CD8+ T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive Vδ1 T cell differentiation from Tnaive to Teffector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant Vγ9+Vδ2+ T cells exhibit a distinct "innate-effector" transcriptional program established by early childhood. In summary, adaptive-like γδ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8+ T cells.

Protein phosphorylation and kinome profiling reveal altered regulation of multiple signaling pathways in B lymphocytes from patients with systemic lupus erythematosus.

OBJECTIVE: The cause of B lymphocyte hyperactivity and autoantibody production in systemic lupus erythematosus (SLE) remains unclear. Previously, we identified abnormalities in the level and translocation of signaling molecules in B cells in SLE patients. The present study was undertaken to examine the extent of signaling abnormalities that relate to altered B cell responses in SLE. METHODS: B lymphocytes from 88 SLE patients and 72 healthy controls were isolated from blood by negative selection. Protein tyrosine phosphorylation and cellular kinase levels were analyzed by Western blotting, flow cytometry, and a kinome array protocol. Changes in protein phosphorylation were determined in ex vivo B cells and following B cell receptor engagement. RESULTS: Differences in tyrosine phosphorylation in B cells from patients with SLE, compared with matched controls, were demonstrated. Further, the kinome array analysis identified changes in the activation of key kinases, i.e., the activity of phosphatidylinositol 3-kinase, which regulates survival and differentiation, was up-regulated and the activity of Rac and Rho kinases, which regulate the cytoskeleton and migration, was increased. In contrast, the activity of ATR, which regulates the cell cycle, was down-regulated in SLE patients compared with controls. Differences in signaling pathways were seen in all SLE B lymphocyte subsets that manifested phenotypic features of immature, mature, and memory cells. CONCLUSION: This study revealed dysregulation in multiple signaling pathways that control key responses in B cells of SLE patients. Data generated in this study provide a molecular basis for further analysis of the altered B lymphocyte responses in SLE.

Generation of Stable Isopentenyl Monophosphate Aryloxy Triester Phosphoramidates as Activators of Vγ9Vδ2 T Cells.

Aryloxy triester phosphoramidate prodrugs of the monophosphate derivatives of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) were synthesized as lipophilic derivatives that can improve cell uptake. Despite the structural similarity of IPP and DMAPP, it was noted that their phosphoramidate prodrugs exhibited distinct stability profiles in aqueous environments, which we show is due to the position of the allyl bond in the backbones of the IPP and DMAPP monophosphates. As the IPP monophosphate aryloxy triester phosphoramidates showed favorable stability, they were subsequently investigated for their ability to activate Vγ9/Vδ2 T cells and they showed promising activation of this subset of T cells. Together, these findings represent the first report of IPP and DMAPP monophosphate prodrugs and the ability of IPP aryloxy triester phosphoramidate prodrugs to activate Vγ9/Vδ2 T cells highlighting their potential as possible immunotherapeutics.

Aryloxy Diester Phosphonamidate Prodrugs of Phosphoantigens (ProPAgens) as Potent Activators of Vγ9/Vδ2 T-Cell Immune Responses.

Vγ9/Vδ2 T-cells are activated by pyrophosphate-containing small molecules known as phosphoantigens (PAgs). The presence of the pyrophosphate group in these PAgs has limited their drug-like properties because of its instability and polar nature. In this work, we report a novel and short Grubbs olefin metathesis-mediated synthesis of methylene and difluoromethylene monophosphonate derivatives of the PAg (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBP) as well as their aryloxy diester phosphonamidate prodrugs, termed ProPAgens. These prodrugs showed excellent stability in human serum (t1/2 > 12 h) and potent activation of Vγ9/Vδ2 T-cells (EC50 ranging from 5 fM to 73 nM), which translated into sub-nanomolar γδ T-cell-mediated eradication of bladder cancer cells in vitro. Additionally, a combination of in silico and in vitro enzymatic assays demonstrated the metabolism of these phosphonamidates to release the unmasked PAg monophosphonate species. Collectively, this work establishes HMBP monophosphonate ProPAgens as ideal candidates for further investigation as novel cancer immunotherapeutic agents.

Synthesis and Biological Evaluation of ( E)-4-Hydroxy-3-methylbut-2-enyl Phosphate (HMBP) Aryloxy Triester Phosphoramidate Prodrugs as Activators of Vγ9/Vδ2 T-Cell Immune Responses.

The aryloxy triester phosphoramidate prodrug approach has been used with success in drug discovery. Herein, we describe the first application of this prodrug technology to the monophosphate derivative of the phosphoantigen HMBPP and one of its analogues. Some of these prodrugs exhibited specific and potent activation of Vγ9/Vδ2 T-cells, which were then able to lyse bladder cancer cells in vitro. This work highlights the promise of this prodrug technology in the discovery of novel immunotherapeutics.

Association of Defective Regulation of Autoreactive Interleukin-6-Producing Transitional B Lymphocytes With Disease in Patients With Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) has the highest case-specific mortality of any rheumatic disease, and no effective therapy is available. A clear manifestation of SSc is the presence of autoantibodies. However, the origin of autoantibody-producing B lymphocytes, their mechanisms of activation and autoantibody production, and their role remain unclear. This study was undertaken to identify mechanisms that contribute to pathogenic B cell generation and involvement in SSc and to assess the altered distribution and function of B cells in SSc patients. METHODS: Multicolor flow cytometry was performed to determine B cell subset distribution, cytokine production, and tolerance induction in SSc patients and healthy controls. Cytokine production following stimulation of the cells ex vivo was determined by multiplex assay. RESULTS: A range of defects in B lymphocyte tolerance and cytokine production in SSc were noted. There was evidence of altered distribution of transitional B cell subsets, increased production of interleukin-6 (IL-6) and IL-8, and defective tolerance induction in SSc B cells. In addition, B cells from SSc patients had a reduced ability to produce IL-10 when stimulated through innate immune pathways. In contrast to healthy individuals, tolerance checkpoints in SSc patients failed to suppress the emergence of B cells that produce autoantibodies with specificity to the Scl-70 antigen, which is strongly associated with SSc. These defects were paralleled by altered intracellular signaling and apoptosis following B cell receptor engagement. CONCLUSION: Our findings provide new insights into mechanisms underlying defective B lymphocyte responses in patients with SSc and their contribution to disease.

CD5 expression promotes IL-10 production through activation of the MAPK/Erk pathway and upregulation of TRPC1 channels in B lymphocytes.

CD5 is constitutively expressed on T cells and a subset of mature normal and leukemic B cells in patients with chronic lymphocytic leukemia (CLL). Important functional properties are associated with CD5 expression in B cells, including signal transducer and activator of transcription 3 activation, IL-10 production and the promotion of B-lymphocyte survival and transformation. However, the pathway(s) by which CD5 influences the biology of B cells and its dependence on B-cell receptor (BCR) co-signaling remain unknown. In this study, we show that CD5 expression activates a number of important signaling pathways, including Erk1/2, leading to IL-10 production through a novel pathway independent of BCR engagement. This pathway is dependent on extracellular calcium (Ca2+) entry facilitated by upregulation of the transient receptor potential channel 1 (TRPC1) protein. We also show that Erk1/2 activation in a subgroup of CLL patients is associated with TRPC1 overexpression. In this subgroup of CLL patients, small inhibitory RNA (siRNA) for CD5 reduces TRPC1 expression. Furthermore, siRNAs for CD5 or for TRPC1 inhibit IL-10 production. These findings provide new insights into the role of CD5 in B-cell biology in health and disease and could pave the way for new treatment strategies for patients with B-CLL.

Monitoring Promoter Activity by Flow Cytometry.

Genetic reporters have become invaluable tools for indirectly monitoring promoter activities. The quantitative measurement of promoter activities using reporter gene systems is fundamental for pharmaceutical, biomedical, and molecular biology research. Genetic reporters are used not only for measuring promoter activities but also for understanding the mechanisms controlling gene transcription and in the identification, and characterization of cis-acting regulatory elements. Fluorescent reporter proteins including enhanced green fluorescent protein (EGFP ) are reliable for monitoring quantitative underlying differences in promoter activities. The emitted fluorescence intensity of the expressed reporter is measured at the single-cell level by flow cytometry and represents a readout for the promoter activities. In this chapter, the protocol for measurement and analyzing of transfected cells expressing the reporter gene EGFP is thoroughly described and fully illustrated.

Autoantibody profiles in autoimmune hepatitis and chronic hepatitis C identifies similarities in patients with severe disease.

AIM: To determine how the auto-antibodies (Abs) profiles overlap in chronic hepatitis C infection (CHC) and autoimmune hepatitis (AIH) and correlate to liver disease. METHODS: Levels of antinuclear Ab, smooth muscle antibody (SMA) and liver/kidney microsomal-1 (LKM-1) Ab and markers of liver damage were determined in the sera of 50 patients with CHC infection, 20 AIH patients and 20 healthy controls using enzyme linked immunosorbent assay and other immune assays. RESULTS: We found that AIH patients had more severe liver disease as determined by elevation of total IgG, alkaline phosphatase, total serum bilirubin and serum transaminases and significantly higher prevalence of the three non-organ-specific autoantibodies (auto-Abs) than CHC patients. Antinuclear Ab, SMA and LKM-1 Ab were also present in 36% of CHC patients and related to disease severity. CHC cases positive for auto-Abs were directly comparable to AIH in respect of most markers of liver damage and total IgG. These cases had longer disease duration compared with auto-Ab negative cases, but there was no difference in gender, age or viral load. KLM-1+ Ab CHC cases showed best overlap with AIH. CONCLUSION: Auto-Ab levels in CHC may be important markers of disease severity and positive cases have a disease similar to AIH. Auto-Abs might have a pathogenic role as indicated by elevated markers of liver damage. Future studies will unravel any novel associations between these two diseases, whether genetic or other.

Response to Treatment with TNFα Inhibitors in Rheumatoid Arthritis Is Associated with High Levels of GM-CSF and GM-CSF+ T Lymphocytes.

Biologic TNFα inhibitors are a mainstay treatment option for patients with rheumatoid arthritis (RA) refractory to other treatment options. However, many patients either do not respond or relapse after initially responding to these agents. This study was carried out to identify biomarkers that can distinguish responder from non-responder patients before the initiation of treatment. The level of cytokines in plasma and those produced by ex vivo T cells, B cells and monocytes in 97 RA patients treated with biologic TNFα inhibitors was measured before treatment and after 1 and 3 months of treatment by multiplex analyses. The frequency of T cell subsets and intracellular cytokines were determined by flow cytometry. The results reveal that pre-treatment, T cells from patients who went on to respond to treatment with biologic anti-TNFα agents produced significantly more GM-CSF than non-responder patients. Furthermore, immune cells from responder patients produced higher levels of IL-1ß, TNFα and IL-6. Cytokine profiling in the blood of patients confirmed the association between high levels of GM-CSF and responsiveness to biologic anti-TNFα agents. Thus, high blood levels of GM-CSF pre-treatment had a positive predictive value of 87.5% (61.6 to 98.5% at 95% CI) in treated RA patients. The study also shows that cells from most anti-TNFα responder patients in the current cohort produced higher levels of GM-CSF and TNFα pre-treatment than non-responder patients. Findings from the current study and our previous observations that non-responsiveness to anti-TNFα is associated with high IL-17 levels suggest that the disease in responder and non-responder RA patients is likely to be driven/sustained by different inflammatory pathways. The use of biomarker signatures of distinct pro-inflammatory pathways could lead to evidence-based prescription of the most appropriate biological therapies for different RA patients.

Intracellular B Lymphocyte Signalling and the Regulation of Humoral Immunity and Autoimmunity.

B lymphocytes are critical for effective immunity; they produce antibodies and cytokines, present antigens to T lymphocytes and regulate immune responses. However, because of the inherent randomness in the process of generating their vast repertoire of antigen-specific receptors, B cells can also cause diseases through recognizing and reacting to self. Therefore, B lymphocyte selection and responses require tight regulation at multiple levels and at all stages of their development and activation to avoid diseases. Indeed, newly generated B lymphocytes undergo rigorous tolerance mechanisms in the bone marrow and, subsequently, in the periphery after their migration. Furthermore, activation of mature B cells is regulated through controlled expression of co-stimulatory receptors and intracellular signalling thresholds. All these regulatory events determine whether and how B lymphocytes respond to antigens, by undergoing apoptosis or proliferation. However, defects that alter regulated co-stimulatory receptor expression or intracellular signalling thresholds can lead to diseases. For example, autoimmune diseases can result from altered regulation of B cell responses leading to the emergence of high-affinity autoreactive B cells, autoantibody production and tissue damage. The exact cause(s) of defective B cell responses in autoimmune diseases remains unknown. However, there is evidence that defects or mutations in genes that encode individual intracellular signalling proteins lead to autoimmune diseases, thus confirming that defects in intracellular pathways mediate autoimmune diseases. This review provides a synopsis of current knowledge of signalling proteins and pathways that regulate B lymphocyte responses and how defects in these could promote autoimmune diseases. Most of the evidence comes from studies of mouse models of disease and from genetically engineered mice. Some, however, also come from studying B lymphocytes from patients and from genome-wide association studies. Defining proteins and signalling pathways that underpin atypical B cell response in diseases will help in understanding disease mechanisms and provide new therapeutic avenues for precision therapy.