Impact of Janus Kinase Inhibition on the Treatment of Axial Spondyloarthropathies.

Many immune cells and effector molecules (e.g. cytokines, Interferons, growth factors) utilize different combinations of Janus kinase (JAK) and signal transducer and activator of transcription (STAT) molecules to transduce signals from the cell surface to the nucleus, where they regulate transcription. This pathway is basically involved in almost all inflammatory diseases and also in the interleukin (IL)-23/IL-17 cascade, which is an essential part of the pathogenesis of spondyloarthropathies (SpA). Upon evidence from in vitro and in vivo experiments indicating disease-modifying effects of JAK inhibition in inflammatory joint disease, numerous inhibitors of the JAK/STAT pathway (= JAKinibs) with different selectivity against the four members of the JAK family [JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2)] were developed. Trials in rheumatoid arthritis were successful with respect to efficacy and safety, and currently, three JAKinibs are approved for the treatment of rheumatoid arthritis in the European Union. Although new treatment options (anti-IL-23, anti-IL-17, and phosphodiesterase 4 inhibitors) have become available for spondyloarthritis and especially psoriatic arthritis (PsA) within the last years, most of them are biologics and do not address all disease manifestations equally. Therefore, multiple trials were initiated to evaluate JAKinibs in PsA and axial spondyloarthritis (axSpA). A trial of Tofacitinib (OPAL) was successful in PsA and has led to the inclusion of JAKinibs into the treatment algorithm. Currently many trials with JAKinibs are ongoing for PsA and axSpA, with one phase III trial of upadacitinib (selective JAK1 inhibitor) showing good therapeutic response in active radiographic axSpA.

Inhibiting ex-vivo Th17 responses in Ankylosing Spondylitis by targeting Janus kinases.

Treatment options for Ankylosing Spondylitis (AS) are still limited. The T helper cell 17 (Th17) pathway has emerged as a major driver of disease pathogenesis and a good treatment target. Janus kinases (JAK) are key transducers of cytokine signals in Th17 cells and therefore promising targets for the treatment of AS. Here we investigate the therapeutic potential of four different JAK inhibitors on cells derived from AS patients and healthy controls, cultured in-vitro under Th17-promoting conditions. Levels of IL-17A, IL-17F, IL-22, GM-CSF and IFNγ were assessed by ELISA and inhibitory effects were investigated with Phosphoflow. JAK1/2/3 and TYK2 were silenced in CD4+ T cells with siRNA and effects analyzed by ELISA (IL-17A, IL-17F and IL-22), Western Blot, qPCR and Phosphoflow. In-vitro inhibition of CD4+ T lymphocyte production of multiple Th17 cytokines (IL-17A, IL-17F and IL-22) was achieved with JAK inhibitors of differing specificity, as well as by silencing of JAK1-3 and Tyk2, without impacting on cell viability or proliferation. Our preclinical data suggest JAK inhibitors as promising candidates for therapeutic trials in AS, since they can inhibit multiple Th17 cytokines simultaneously. Improved targeting of TYK2 or other JAK isoforms may confer tailored effects on Th17 responses in AS.

Silencing or inhibition of endoplasmic reticulum aminopeptidase 1 (ERAP1) suppresses free heavy chain expression and Th17 responses in ankylosing spondylitis.

OBJECTIVE: Human leucocyte antigen (HLA)-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly associated with ankylosing spondylitis (AS). ERAP1 is a key aminopeptidase in HLA class I presentation and can potentially alter surface expression of HLA-B27 free heavy chains (FHCs). We studied the effects of ERAP1 silencing/inhibition/variations on HLA-B27 FHC expression and Th17 responses in AS. METHODS: Flow cytometry was used to measure surface expression of HLA class I in peripheral blood mononuclear cells (PBMCs) from patients with AS carrying different ERAP1 genotypes (rs2287987, rs30187 and rs27044) and in ERAP1-silenced/inhibited/mutated HLA-B27-expressing antigen presenting cells (APCs). ERAP1-silenced/inhibited APCs were cocultured with KIR3DL2CD3ε-reporter cells or AS CD4+ T cells. Th17 responses of AS CD4+ T cells were measured by interleukin (IL)-17A ELISA and Th17 intracellular cytokine staining. FHC cell surface expression and Th17 responses were also measured in AS PBMCs following ERAP1 inhibition. RESULTS: The AS-protective ERAP1 variants, K528R and Q730E, were associated with reduced surface FHC expression by monocytes from patients with AS and HLA-B27-expressing APCs. ERAP1 silencing or inhibition in APCs downregulated HLA-B27 FHC surface expression, reduced IL-2 production by KIR3DL2CD3ε-reporter cells and suppressed the Th17 expansion and IL-17A secretion by AS CD4+ T cells. ERAP1 inhibition of AS PBMCs reduced HLA class I FHC surface expression by monocytes and B cells, and suppressed Th17 expansion. CONCLUSIONS: ERAP1 activity determines surface expression of HLA-B27 FHCs and potentially promotes Th17 responses in AS through binding of HLA-B27 FHCs to KIR3DL2. Our data suggest that ERAP1 inhibition has potential for AS treatment.

CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses.

Th17 responses are critical to a variety of human autoimmune diseases, and therapeutic targeting with monoclonal antibodies against IL-17 and IL-23 has shown considerable promise. Here, we report data to support selective bromodomain blockade of the transcriptional coactivators CBP (CREB binding protein) and p300 as an alternative approach to inhibit human Th17 responses. We show that CBP30 has marked molecular specificity for the bromodomains of CBP and p300, compared with 43 other bromodomains. In unbiased cellular testing on a diverse panel of cultured primary human cells, CBP30 reduced immune cell production of IL-17A and other proinflammatory cytokines. CBP30 also inhibited IL-17A secretion by Th17 cells from healthy donors and patients with ankylosing spondylitis and psoriatic arthritis. Transcriptional profiling of human T cells after CBP30 treatment showed a much more restricted effect on gene expression than that observed with the pan-BET (bromo and extraterminal domain protein family) bromodomain inhibitor JQ1. This selective targeting of the CBP/p300 bromodomain by CBP30 will potentially lead to fewer side effects than with the broadly acting epigenetic inhibitors currently in clinical trials.

Decreased expression of miR-146a and miR-155 contributes to an abnormal Treg phenotype in patients with rheumatoid arthritis.

OBJECTIVES: MicroRNAs (miRNAs) have been implicated in the pathogenesis of autoimmune diseases, not least for their critical role in the regulation of regulatory T cell (Treg) function. Deregulated expression of miR-146a and miR-155 has been associated with rheumatoid arthritis (RA). We therefore investigated miR-146a and miR-155 expression in Tregs of patients with RA and their possible impact on Treg function and disease activity. METHODS: Expression of miR-146a and miR-155 was assessed in RA patients and controls. MiRNA expression was correlated with disease activity and expression of target genes. Interference with biological activity of miRNAs was evaluated in functional Treg assays. RESULTS: Diminished upregulation of miR-146a and miR-155 in response to T cell stimulation was found in Tregs of RA patients. Diminution of miR-146a expression was observed in particular in patients with active disease, and correlated with joint inflammation. In patients with active RA, Tregs demonstrated a pro-inflammatory phenotype characterised by inflammatory cytokine expression. This was due to an augmented expression and activation of signal transducer and activator transcription 1 (STAT1), a direct target of miR-146a. CONCLUSIONS: Our results suggest that in RA miR-146a facilitates a pro-inflammatory phenotype of Tregs via increased STAT1 activation, and contributes thereby to RA pathogenesis.

Disease burden, disease manifestations and current treatment regimen of the SAPHO syndrome in Germany: results from a nationwide patient survey.

BACKGROUND: Due to diagnostic and therapeutic advances, quality of life of patients with spondyloarthritides (SpA) has improved substantially in recent years. However, little is known about how patients with the SAPHO syndrome, a heterogeneous disease counted among the SpAs, profit from these advances. OBJECTIVE: To investigate current aspects of patient care in a nationwide SAPHO cohort. METHODS: Patients were recruited in a university centre and via a nationwide SAPHO patient support group. Medical records were reviewed and patients were asked to complete a questionnaire on the course of diagnosis, disease burden and treatment regimen. RESULTS: A total of 64 patients were included in the analysis. The mean time from disease onset to diagnosis was 3.8 ± 5.3 years. The patients' overall satisfaction with the course of diagnosis was 23.0 ± 28.9 on a visual analogue scale (VAS) from 0 to 100. Musculoskeletal symptoms had the highest impact on the patients' wellbeing. The mean overall disease burden on a VAS for pain was 45.4 ± 25.9. Limitations in the quality of life were reported mainly in the general health, bodily pain and vitality dimensions of the SF-36 questionnaire. Current treatments consisted of NSAIDs (77%), DMARDs (27%), glucocorticoids (23%), TNF-inhibitors (16%) and bisphosphonates (11%). CONCLUSIONS: The SAPHO syndrome has a high impact on the patients' general health and quality of life. Establishing the diagnosis still takes years and expends multiple medical resources. Effective treatments such as TNF-inhibitors are rarely prescribed and current disease burden is not acceptable.

Relevance of grade 1 gray-scale ultrasound findings in wrists and small joints to the assessment of subclinical synovitis in rheumatoid arthritis.

OBJECTIVE: To investigate the clinical relevance of grade 1 findings on gray-scale ultrasound (GSUS) of the joints in patients with rheumatoid arthritis (RA). METHODS: We examined the wrists and small joints of 100 patients with early or established RA and 30 healthy controls, using GSUS and power Doppler ultrasound (PDUS). Independent clinical assessment of all joints for tenderness and swelling according to the European League Against Rheumatism examination technique was performed. Joints with grade 1 findings on GSUS were identified, and associations with swelling, pain, and findings on PDUS were assessed. Grade 1 findings on GSUS in patients with early RA were reassessed after 6 months of antirheumatic treatment. RESULTS: Grade 1 results represented the majority of all GSUS findings in patients with RA and were also frequently recorded in healthy controls. Grade 1 GSUS findings were not associated with tenderness, swelling, or positive results on PDUS. In comparison to joints with grade 2 and grade 3 findings on GSUS, joints with grade 1 findings were less likely to respond to treatment. CONCLUSION: The present results indicate that grade 1 findings on GSUS have limited clinical relevance.

Evaluation of effectiveness of instruction and study habits in two consecutive clinical semesters of the medical curriculum Munich (MeCuM) reveals the need for more time for self study and higher frequency of assessment.

BACKGROUND: Seven years after implementing a new curriculum an evaluation was performed to explore possibilities for improvements. PURPOSES: To analyze students' study habits in relation to exam frequency and to evaluate effectiveness of instruction. METHODS: Time spent on self study (TSS) and the quantity of instruction (QI) was assessed during the internal medicine and the surgical semester. Students and faculty members were asked about study habits and their evaluation of the current curriculum. RESULTS: The TSS/QI ratio as a measure of effectiveness of instruction ranges mainly below 1.0 and rises only prior to exams. Students and teachers prefer to have multiple smaller exams over the course of the semester. Furthermore, students wish to have more time for self-guided study. CONCLUSIONS: The TSS/QI ratio is predominantly below the aspired value of 1.0. Furthermore, the TSS/QI ratio is positively related to test frequency. We therefore propose a reduction of compulsory lessons and an increase in test frequency.

Suppression of DNA-PKcs enhances FGF-2 dependent human endothelial cell proliferation via negative regulation of Akt.

Angiogenesis initiation is crucially dependent on endothelial proliferation and can be stimulated by the fibroblast growth factor 2 (FGF-2). The DNA dependent protein kinase (DNA-PK), long known for its importance in repairing DNA double strand breaks, belongs to the phosphatidylinositol-3 kinase (PI3-K) super family and has recently been identified as one of the enzymes phosphorylating and activating Akt. Due to its similarity with PI3-K, we hypothesized that DNA-PK may have similar effects on endothelial angiogenic processes and signalling. We used primary endothelial cells (HUVEC and PAEC) and human microvascular endothelial cells (HMEC) to study the role of DNA-PK in endothelial proliferation and signalling. DNA-PKcs suppression with the compound NU7026 or with siRNA induced basal endothelial cell proliferation as well as enhanced FGF-2 dependent proliferation. This was associated with an increase in phosphorylated Akt. Tube formation was not affected by DNA-PKcs clearly showing that the role of DNA-PK in endothelial processes differs from that of PI3-K. Our findings indicate DNA-PK as an important enzyme maintaining the quiescent endothelial phenotype by actively inhibiting Akt thus restraining endothelial cell proliferation preventing excessive growth.