Epigenetic profiling demarcates molecular subtypes of muscle-invasive bladder cancer.

Muscle-invasive bladder cancer (MIBC) is a heterogeneous disease that often recurs despite aggressive treatment with neoadjuvant chemotherapy and (radical) cystectomy. Basal and luminal molecular subtypes have been identified that are linked to clinical characteristics and have differential sensitivities to chemotherapy. While it has been suggested that epigenetic mechanisms play a role in defining these subtypes, a thorough understanding of the biological mechanisms is lacking. This report details the first genome-wide analysis of histone methylation patterns of human primary bladder tumours by chromatin immunoprecipitations and next-generation sequencing (ChIP-seq). We profiled multiple histone marks: H3K27me3, a marker for repressed genes, and H3K4me1 and H3K4me3, which are indicators of active enhancers and active promoters. Integrated analysis of ChIP-seq data and RNA sequencing revealed that H3K4 mono-methylation demarcates MIBC subtypes, while no association was found for the other two histone modifications in relation to basal and luminal subtypes. Additionally, we identified differentially methylated H3K4me1 peaks in basal and luminal tumour samples, suggesting that active enhancers play a role in defining subtypes. Our study is the first analysis of histone modifications in primary bladder cancer tissue and provides an important resource for the bladder cancer community.

Exploring B → π π , π K decays at the high-precision frontier.

The B → π π , π K system offers a powerful laboratory to probe strong and weak interactions. Using the isospin symmetry, we determine hadronic B → π π parameters from data where new measurements of direct CP violation in B d 0 → π 0 π 0 resolve a discrete ambiguity. With the help of the SU(3) flavour symmetry, the B → π π parameters can be converted into their B → π K counterparts, thereby allowing us to make predictions of observables. A particularly interesting decay is B d 0 → π 0 K S as it exhibits mixing-induced CP violation. Using an isospin relation, complemented with a robust SU(3) input, we calculate correlations between the direct and mixing-induced CP asymmetries of B d 0 → π 0 K S , which are the theoretically cleanest B → π K probes. Interestingly, they show tensions with respect to the Standard Model. Should this B → π K puzzle originate from New Physics, electroweak penguins offer an attractive scenario for new particles to enter. We present a strategy to determine the parameters characterising these topologies and obtain the state-of-the-art picture from current data. In the future, this method will allow us to reveal the B → π K dynamics and to obtain insights into the electroweak penguin sector with unprecedented precision.

Association Of Plasma And Urinary Mutant DNA With Clinical Outcomes In Muscle Invasive Bladder Cancer.

Muscle Invasive Bladder Cancer (MIBC) has a poor prognosis. Whilst patients can achieve a 6% improvement in overall survival with Neo-Adjuvant Chemotherapy (NAC), many do not respond. Body fluid mutant DNA (mutDNA) may allow non-invasive identification of treatment failure. We collected 248 liquid biopsy samples including plasma, cell pellet (UCP) and supernatant (USN) from spun urine, from 17 patients undergoing NAC. We assessed single nucleotide variants and copy number alterations in mutDNA using Tagged-Amplicon- and shallow Whole Genome- Sequencing. MutDNA was detected in 35.3%, 47.1% and 52.9% of pre-NAC plasma, UCP and USN samples respectively, and urine samples contained higher levels of mutDNA (p = <0.001). Longitudinal mutDNA demonstrated tumour evolution under the selective pressure of NAC e.g. in one case, urine analysis tracked two distinct clones with contrasting treatment sensitivity. Of note, persistence of mutDNA detection during NAC predicted disease recurrence (p = 0.003), emphasising its potential as an early biomarker for chemotherapy response.

FOXP1 acts through a negative feedback loop to suppress FOXO-induced apoptosis.

Transcriptional activity of Forkhead box transcription factor class O (FOXO) proteins can result in a variety of cellular outcomes depending on cell type and activating stimulus. These transcription factors are negatively regulated by the phosphoinositol 3-kinase (PI3K)-protein kinase B (PKB) signaling pathway, which is thought to have a pivotal role in regulating survival of tumor cells in a variety of cancers. Recently, it has become clear that FOXO proteins can promote resistance to anti-cancer therapeutics, designed to inhibit PI3K-PKB activity, by inducing the expression of proteins that provide feedback at different levels of this pathway. We questioned whether such a feedback mechanism may also exist directly at the level of FOXO-induced transcription. To identify critical modulators of FOXO transcriptional output, we performed gene expression analyses after conditional activation of key components of the PI3K-PKB-FOXO signaling pathway and identified FOXP1 as a direct FOXO transcriptional target. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that FOXP1 binds enhancers that are pre-occupied by FOXO3. By sequencing the transcriptomes of cells in which FOXO is specifically activated in the absence of FOXP1, we demonstrate that FOXP1 can modulate the expression of a specific subset of FOXO target genes, including inhibiting expression of the pro-apoptotic gene BIK. FOXO activation in FOXP1-knockdown cells resulted in increased cell death, demonstrating that FOXP1 prevents FOXO-induced apoptosis. We therefore propose that FOXP1 represents an important modulator of FOXO-induced transcription, promoting cellular survival.

Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3ß (GSK3ß). KLC2 phosphorylation by GSK3ß induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3ß on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-ß (TGFß) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFß-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

Duloxetine in patients with central neuropathic pain caused by spinal cord injury or stroke: a randomized, double-blind, placebo-controlled trial.

The mechanisms underlying central neuropathic pain are poorly understood. Pain inhibitory mechanisms including sertononergic and norepinephrine systems may be dysfunctional. In this randomized, double-blinded, placebo-controlled trial we evaluated the effects of duloxetine on pain relief (spontaneous pain and evoked pain), tolerability, health status, and quality of life in patients with central pain related to cerebrovascular lesions or spinal cord lesions. At baseline and eight weeks following start of treatment subjects were evaluated with standard measures of efficacy: pain intensity (primary efficacy variable), quantitative sensory testing, health status and quality of life (secondary efficacy variables). Forty-eight patients received escalating doses of either duloxetine (60 and 120mg/day) or matching placebo capsules. In both groups, patients started with 1 capsule per day. If pain relief was insufficient, patients were titrated to a higher dose. A trend towards a decrease in mean pain score after eight weeks was observed for duloxetine treatment (p=0.056). Duloxetine alleviated dynamic (p=0.035) and cold allodynia (p<0.001) significantly better than placebo. Tactile pain and pressure pain thresholds did not improve significantly. The duloxetine group showed a significant improvement for the bodily pain domain of the SF36 (p=0.035). No significant differences were observed in the other domains of the SF36, the Pain Disability Index, and the EQ-5D. While this trial showed no significant effect on pain intensity, duloxetine revealed a biologic effect. It would be worthwhile to suspend our judgement and to perform more studies to evaluate the role of duloxetine in modulation of the symptoms of central neuropathic pain.

Decrease in immunoglobulin free light chains in patients with rheumatoid arthritis upon rituximab (anti-CD20) treatment correlates with decrease in disease activity.

OBJECTIVES: Immunoglobulin (Ig) free light chains (FLCs) are short-lived B cell products that contribute to inflammation in several experimental disease models. In this study, FLC concentrations in inflamed joints of patients with rheumatoid arthritis (RA) as compared to patients with osteoarthritis were investigated. In addition, the relationship of FLCs and disease activity upon B cell depletion (rituximab) in patients with RA was studied. METHODS: Synovial fluid (SF) and tissue from patients with RA were analysed for local presence of FLCs using ELISA and immunohistochemistry. In addition, FLC concentrations were measured (at baseline, 3 and 6 months after treatment) in 50 patients with RA with active disease who were treated with rituximab. Changes in FLCs were correlated to changes in disease activity and compared to alterations in IgM, IgG, IgA, IgM-rheumatoid factor (RF) and IgG-anti-citrullinated protein antibody (ACPA) concentrations. RESULTS: FLCs were detected in synovial tissue from patients with RA, and high FLC concentrations were found in SF from inflamed joints, which positively correlate with serum FLC concentrations. Serum FLC concentrations significantly correlated with disease activity score using 28 joint counts, erythrocyte sedimentation rate (ESR) and C reactive protein, and changes in FLC correlated with clinical improvement after rituximab treatment. Moreover, effect of treatment on FLC concentrations discriminated clinical responders from non-responders, whereas IgM-RF and IgG-ACPA significantly decreased in both patient groups. CONCLUSIONS: FLCs are abundantly present in inflamed joints and FLC levels correlate with disease activity. The correlation of FLC concentrations and disease activity indicates that FLCs may be relevant biomarkers for treatment response to rituximab in patients with RA and suggests that targeting FLC may be of importance in the therapy of RA.

Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

Clinical response, pharmacokinetics, development of human anti-chimaeric antibodies, and synovial tissue response to rituximab treatment in patients with rheumatoid arthritis.

OBJECTIVES: To analyse whether persistence of synovial B lineage cells and lack of clinical response to rituximab treatment in patients with rheumatoid arthritis (RA) are associated with low rituximab serum levels and anti-rituximab antibody (ARA) formation. METHODS: Fifty-eight patients with RA were treated with rituximab. The clinical response was determined 24 weeks after each treatment course using the Disease Activity Score evaluated in 28 joints (DAS28) and EULAR response criteria. Rituximab serum levels, ARAs and synovial B lineage cell numbers were determined before and after treatment. RESULTS: Four weeks after treatment rituximab serum levels were highly variable. Low rituximab levels were associated with ARA formation (in five patients (8.6%)) and high baseline erythrocyte sedimentation rate. Interestingly, serum rituximab levels were not related to persistence of synovial B lineage cells or clinical response. Furthermore, response to treatment and re-treatment was similar in ARA-positive and ARA-negative patients. CONCLUSION: There is clear variability in serum levels after rituximab treatment, but rituximab levels are not lower in patients with persistence of synovial B lineage cells or lack of clinical response. The current treatment schedule suffices to induce and maintain a clinical response, even when ARAs are formed.

DAS-driven therapy versus routine care in patients with recent-onset active rheumatoid arthritis.

OBJECTIVES: To compare the efficacy of Disease Activity Score (DAS)-driven therapy and routine care in patients with recent-onset rheumatoid arthritis. METHODS: Patients with recent-onset rheumatoid arthritis receiving traditional antirheumatic therapy from either the BeSt study, a randomised controlled trial comparing different treatment strategies (group A), or two Early Arthritis Clinics (group B) were included. In group A, systematic DAS-driven treatment adjustments aimed to achieve low disease activity (DAS < or =2.4). In group B, treatment was left to the discretion of the treating doctor. Functional ability (Health Assessment Questionnaire (HAQ)), Disease Activity Score in 28 joints (DAS28) and Sharp/van der Heijde radiographic score (SHS) were evaluated. RESULTS: At baseline, patients in group A (n = 234) and group B (n = 201) had comparable demographic characteristics and a mean HAQ of 1.4. Group A had a longer median disease duration than group B (0.5 vs 0.4 years, p = 0.016), a higher mean DAS28 (6.1 vs 5.7, p<0.001), more rheumatoid factor-positive patients (66% vs 42%, p<0.001) and more patients with erosions (71% vs 53%, p<0.001). After 1 year, the HAQ improvement was 0.7 vs 0.5 (p = 0.029), and the percentage in remission (DAS28 <2.6) 31% vs 18% (p<0.005) in groups A and B, respectively. In group A, the median SHS progression was 2.0 (expected progression 7.0), in group B, the SHS progression was 1.0 (expected progression 4.4). CONCLUSIONS: In patients with recent-onset rheumatoid arthritis receiving traditional treatment, systematic DAS-driven therapy results in significantly better clinical improvement and possibly improves the suppression of joint damage progression.

AGC kinases regulate phosphorylation and activation of eukaryotic translation initiation factor 4B.

Eukaryotic translation initiation factor 4B (eIF4B) plays a critical role during the initiation of protein synthesis and its activity can be regulated by multiple phosphorylation events. In a search for novel protein kinase B (PKB/c-akt) substrates, we identified eIF4B as a potential target. Using an in vitro kinase assay, we found that PKB can directly phosphorylate eIF4B on serine 422 (ser422). Activation of a conditional PKB mutant, interleukin-3 (IL-3) or insulin stimulation resulted in PKB-dependent phosphorylation of this residue in vivo. This was prevented by pretreatment of cells with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or pharmacological inhibition of PKB. Pretreatment of cells with rapamycin, inhibiting mTOR or U0126 to inhibit MEK, had little effect on eIF4B ser422 phosphorylation. In contrast, following amino-acid refeeding, eIF4B ser422 phosphorylation was found to be mammalian target of rapamycin (mTOR)-dependent. We further identified eIF4B ser406 as a novel mitogen-regulated phosphorylation site. Insulin-induced phosphorylation of eIF4B ser406 was dependent on both MEK and mTOR activity. Utilizing a novel translational control luciferase assay, we could further demonstrate that phosphorylation of ser406 or ser422 is essential for optimal translational activity of eIF4B. These data provide novel insights into complex multikinase regulation of eIF4B phosphorylation and reveal an important mechanism by which PKB can regulate translation, potentially critical for the transforming capacity of this AGC kinase family member.

A prospective, randomised, placebo-controlled study to identify biomarkers associated with active treatment in psoriatic arthritis: effects of adalimumab treatment on synovial tissue.

OBJECTIVE: To determine which of the changes in synovial tissue correlates best with clinical response associated with effective therapy (adalimumab) to facilitate the planning of future studies with therapeutic agents for psoriatic arthritis (PsA). METHODS: A total of 24 patients with active PsA were randomised to receive adalimumab (n = 12) or placebo (n = 12) for 4 weeks. Synovial biopsies were obtained before and after 4 weeks of treatment. Immunohistochemical analysis was performed to characterise the cell infiltrate, expression of cytokines and matrix metalloproteinases (MMPs) and vascularity. Sections were analysed by digital image analysis. Statistical analysis was performed using covariance analysis. RESULTS: The mean Disease Activity Score in 28 joints (DAS28) after 4 weeks was 1.92 units lower (95% confidence interval (CI) 1.07 to 2.77) after adalimumab therapy compared with placebo. Paired pretreatment and post-treatment synovial samples were available from 19 patients. Many cell types were reduced after adalimumab treatment compared to placebo. After applying a ranked analysis of covariance (ANCOVA) model to correct for baseline imbalances, a significant effect of treatment was observed on CD3-positive cells: there was a median reduction of 248 cells/mm(2) after adalimumab versus placebo treatment (p = 0.035). In addition, the expression of MMP13 was significantly reduced after active treatment: the integrated optical density (IOD)/mm(2) was 18 190 lower after adalimumab treatment as compared to placebo (p = 0.033). CONCLUSION: Adalimumab therapy in PsA is associated with a marked reduction in T cell infiltration and MMP13 expression in synovial tissue, suggesting that these parameters could be used as biomarkers that are sensitive to change after active treatment in small proof of concept studies in PsA.

FOXO-binding partners: it takes two to tango.

Modulation FOXO transcription factor activities can lead to a variety of cellular outputs resulting in changes in proliferation, apoptosis, differentiation and metabolic responses. Although FOXO proteins all contain an identical DNA-binding domain their cellular functions appear to be distinct, as exemplified by differences in the phenotype of Foxo1, Foxo3 and Foxo4 null mutant mice. While some of these differences may be attributable to the differential expression patterns of these transcription factors, many cells and tissues express several FOXO isoforms. Recently it has become clear that FOXO proteins can regulate transcriptional responses independently of direct DNA-binding. It has been demonstrated that FOXOs can associate with a variety of unrelated transcription factors, regulating activation or repression of diverse target genes. The complement of transcription factors expressed in a particular cell type is thus critical in determining the functional end point of FOXO activity. These interactions greatly expand the possibilities for FOXO-mediated regulation of transcriptional programmes. This review details currently described FOXO-binding partners and examines the role of these interactions in regulating cell fate decisions.

The clinical response to infliximab in rheumatoid arthritis is in part dependent on pretreatment tumour necrosis factor alpha expression in the synovium.

OBJECTIVE: To determine whether the heterogeneous clinical response to tumour necrosis factor (TNF)alpha blocking therapy in rheumatoid arthritis (RA) can be predicted by TNFalpha expression in the synovium before initiation of treatment. METHODS: Prior to initiation of infliximab treatment, arthroscopic synovial tissue biopsies were obtained from 143 patients with active RA. At week 16, clinical response was evaluated using the 28-joint Disease Activity Score (DAS28). Immunohistochemistry was used to analyse the cell infiltrate as well as the expression of various cytokines, adhesion molecules and growth factors. Stained sections were evaluated by digital image analysis. Student t tests were used to compare responders (decrease in DAS28 > or =1.2) with non-responders (decrease in DAS28 <1.2) and multivariable regression was used to identify the independent predictors of clinical response. RESULTS: Synovial tissue analysis confirmed our hypothesis that the baseline level of TNFalpha expression is a significant predictor of response to TNFalpha blocking therapy. TNFalpha expression in the intimal lining layer and synovial sublining were significantly higher in responders than in non-responders (p = 0.047 and p = 0.008, respectively). The numbers of macrophages, macrophage subsets and T cells (all able to produce TNFalpha) were also significantly higher in responders than in non-responders. The expression of interleukin (IL)1beta, IL6, IL18, IL10, E-selectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was not associated with response to anti-TNFalpha treatment. CONCLUSION: The effects of TNFalpha blockade are in part dependent on synovial TNFalpha expression and infiltration by TNFalpha producing inflammatory cells. Clinical response cannot be predicted completely, indicating involvement of other as yet unknown mechanisms.

Synovial tissue response to rituximab: mechanism of action and identification of biomarkers of response.

OBJECTIVE: To investigate the synovial tissue in patients with rheumatoid arthritis (RA) treated with rituximab and to identify possible predictors of clinical response. METHODS: A total of 24 patients with RA underwent synovial biopsy before, 4 and 16 weeks after initiation of rituximab treatment (without peri-infusional corticosteroids to prevent bias). Immunohistochemical analysis was performed and stained sections were analysed by digital image analysis. Linear regression analysis was used to identify predictors of clinical response. RESULTS: The 28-joint Disease Activity Score (DAS28) was unaltered at 4 weeks, but significantly reduced at 16 and 24 weeks. Serum levels of IgM-rheumatoid factor (RF) decreased significantly at 24 weeks and anti-citrullinated peptide antibody (ACPA) levels at 36 weeks. Peripheral blood B cells were depleted at 4 weeks and started to return at 24 weeks. Synovial B cells were significantly decreased at 4 weeks, but were not completely depleted in all patients; there was a further reduction at 16 weeks in some patients. We found a significant decrease in macrophages at 4 weeks, which was more pronounced at 16 weeks. At that timepoint, T cells were also significantly decreased. The reduction of plasma cells predicted clinical improvement at 24 weeks. CONCLUSIONS: The results support the view that B cells orchestrate local cellular infiltration. The kinetics of the serological as well as the tissue response in clinical responders are consistent with the notion that rituximab exerts its effects in part by an indirect effect on plasma cells associated with autoantibody production, which could help explain the delayed response after rituximab treatment.

The Rhizobium etli opt operon is required for symbiosis and stress resistance.

Rhizobium etli is a Gram-negative root-colonizing soil bacterium capable of fixing nitrogen while living in symbiosis with its leguminous host Phaseolus vulgaris. A genome-wide screening for R. etli symbiotic mutants revealed a R. etli operon encoding an oligopeptide ABC-transporter (Opt), two redA homologous genes and one redB gene. Expression analysis showed this opt operon to be transcribed both under free-living and symbiotic conditions and expression levels were demonstrated to be growth-phase-dependent. Plants nodulated by R. etli opt mutants showed a reduced symbiotic nitrogen fixation activity (approximately 50% reduction). Growth experiments with opt mutants in the presence of oligopeptides as the sole nitrogen source confirmed the involvement of the opt genes in oligopeptide uptake. Further phenotypic analysis of the opt mutants revealed them to display an enhanced resistance to the oligopeptide antibiotic bacitracin, an increased susceptibility to the beta-lactam antibiotic ampicillin and a decreased osmotolerance. In conclusion, our results demonstrate that the opt operon plays a crucial role during symbiosis and stress resistance.

[The humoral response in rheumatoid arthritis and the effect of B-cell depleting therapy]. / De humorale respons bij reumatoïde artritis en het effect van B-celdepleterende therapie.

Recent research has shown that the humoral response plays an important role in the pathogenesis of rheumatoid arthritis. B-cells produce rheumatoid factors and antibodies directed against cyclic citrullinated peptides (anti-CCP antibodies) and are able to present auto-antigens to T-cells. Furthermore, B-cells can produce cytokines and stimulate T-cells. B-cell depletion with rituximab, a monoclonal antibody directed against CD20, has a surprisingly strong and long-lasting therapeutic effect. After administration a sharp decrease of specific auto-antibody titres has been observed. Future developments in B-cell targeted therapy are expected to lead to further improvements in the treatment of rheumatoid arthritis and other autoimmune diseases.