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BACKGROUND & AIMS: Liver fibrosis is the major driver for hepatocellular carcinoma and liver disease related death. Approved anti-fibrotic therapies are absent and compounds in development have limited efficacy. Increased TGF-ß signaling drives collagen deposition by hepatic stellate cells (HSC)/myofibroblasts. Here, we aimed to dissect the role of the circadian clock (CC) in controlling TGF-ß signaling and liver fibrosis. METHODS: Using CC-mutant mice, enriched HSCs and myofibroblasts obtained from healthy and fibrotic mice in different CC-phases and loss-of-function studies in human hepatocytes and myofibroblasts, we investigated the relationship between CC and TGF-ß signaling. We explored hepatocyte-myofibroblast communication through bioinformatic analyses of single-nuclei transcriptomes and validation in cell-based models. Using mouse models for MASH fibrosis and spheroids from patients with liver disease, we performed proof-of-concept studies to validate pharmacological targetability and clinical translatability. RESULTS: We discovered that the CC-oscillator temporally gates TGF-ß signaling and this regulation is broken in fibrosis. We demonstrate that HSCs and myofibroblasts contain a functional CC with rhythmic expression of numerous genes, including fibrogenic genes. Perturbation studies in hepatocytes and myofibroblasts revealed a reciprocal relationship between TGF-ß-activation and CC perturbation, which was confirmed in patient-derived ex vivo and in vivo models. Pharmacological modulation of CC-TGF-ß signaling inhibited fibrosis in mouse models in vivo as well as patient-derived liver spheroids. CONCLUSION: The CC regulates TGF-ß signaling, and the breakdown of this control is associated with liver fibrosis in patients. Pharmacological proof-of-concept studies across different models uncover the CC as a therapeutic candidate target for liver fibrosis - a rising global unmet medical need. IMPACT AND IMPLICATIONS: Liver fibrosis due to metabolic diseases is a global health challenge. Many liver functions are rhythmic throughout the day being controlled by the circadian clock (CC). Here we demonstrate that the regulation of the CC is perturbed upon chronic liver injury and this perturbation contributes to fibrotic disease. By showing that a compound targeting the CC improves liver fibrosis in patient-derived models, this study provides a novel therapeutic candidate strategy to treat fibrosis in patients. Additional studies will be needed for clinical translation. Since the findings uncovers a previously undiscovered profibrotic mechanism and therapeutic target, the study is of interest for scientists investigating liver disease, clinical hepatologists and drug developers.
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BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Animais , Camundongos , Carcinoma Hepatocelular/genética , Claudina-1/genética , Neoplasias Hepáticas/genética , Carcinógenos , Microambiente Tumoral , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular TumoralRESUMO
BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is an important risk factor for hepatocellular carcinoma (HCC). Despite effective antiviral therapies, the risk for HCC is decreased but not eliminated after a sustained virologic response (SVR) to direct-acting antiviral (DAA) agents, and the risk is higher in patients with advanced fibrosis. We investigated HCV-induced epigenetic alterations that might affect risk for HCC after DAA treatment in patients and mice with humanized livers. METHODS: We performed genome-wide ChIPmentation-based ChIP-Seq and RNA-seq analyses of liver tissues from 6 patients without HCV infection (controls), 18 patients with chronic HCV infection, 8 patients with chronic HCV infection cured by DAA treatment, 13 patients with chronic HCV infection cured by interferon therapy, 4 patients with chronic hepatitis B virus infection, and 7 patients with nonalcoholic steatohepatitis in Europe and Japan. HCV-induced epigenetic modifications were mapped by comparative analyses with modifications associated with other liver disease etiologies. uPA/SCID mice were engrafted with human hepatocytes to create mice with humanized livers and given injections of HCV-infected serum samples from patients; mice were given DAAs to eradicate the virus. Pathways associated with HCC risk were identified by integrative pathway analyses and validated in analyses of paired HCC tissues from 8 patients with an SVR to DAA treatment of HCV infection. RESULTS: We found chronic HCV infection to induce specific genome-wide changes in H3K27ac, which correlated with changes in expression of mRNAs and proteins. These changes persisted after an SVR to DAAs or interferon-based therapies. Integrative pathway analyses of liver tissues from patients and mice with humanized livers demonstrated that HCV-induced epigenetic alterations were associated with liver cancer risk. Computational analyses associated increased expression of SPHK1 with HCC risk. We validated these findings in an independent cohort of patients with HCV-related cirrhosis (n = 216), a subset of which (n = 21) achieved viral clearance. CONCLUSIONS: In an analysis of liver tissues from patients with and without an SVR to DAA therapy, we identified epigenetic and gene expression alterations associated with risk for HCC. These alterations might be targeted to prevent liver cancer in patients treated for HCV infection.
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Antivirais/uso terapêutico , Carcinoma Hepatocelular/virologia , Hepatite C Crônica/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Adulto , Animais , Carcinoma Hepatocelular/genética , Estudos de Casos e Controles , Estudos de Coortes , Modelos Animais de Doenças , Epigênese Genética , Europa (Continente) , Feminino , Regulação Neoplásica da Expressão Gênica , Hepatite C Crônica/complicações , Hepatite C Crônica/tratamento farmacológico , Humanos , Japão , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos SCID , Distribuição Aleatória , Resposta Viral SustentadaRESUMO
BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a chronic inflammatory disease often accompanied by impairment of sense of smell. This symptom has been somewhat overlooked, and its relationship to inflammatory cytokines, tissue compression, neuronal loss, and neurogenesis is still unclear. METHODS: In order to elucidate potential mechanisms leading to CRS in humans, we have established a type 2/T helper type 2 cell (Th2)-mediated allergic CRS mouse model, based on house dust mite (HDM) and Staphylococcus aureus enterotoxin B (SEB) sensitization. The inflammatory status of the olfactory epithelium (OE) was assessed using histology, biochemistry, and transcriptomics. The sense of smell was evaluated by studying olfactory behavior and recording electro-olfactograms (EOGs). RESULTS: After 22 weeks, a typical type 2/Th2-mediated inflammatory profile was obtained, as demonstrated by increased interleukin (IL)-4, IL-5, and IL-13 in the OE. The number of mast cells and eosinophils was increased, and infiltration of these cells into the olfactory mucosa was also observed. In parallel, transcriptomic and histology analyses indicated a decreased number of immature olfactory neurons, possibly due to decreased renewal. However, the number of mature sensory neurons was not affected and neither the EOG nor olfactory behavior was impaired. CONCLUSION: Our mouse model of CRS displayed an allergic response to HDM + SEB administration, including the type 2/Th2 inflammatory profile characteristic of human eosinophilic CRSwNP. Although the sense of smell did not appear to be altered in these conditions, the data reveal the influence of chronic inflammation on olfactory neurogenesis, suggesting that factors unique to humans may be involved in CRSwNP-associated anosmia.
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Neurogênese , Mucosa Olfatória/metabolismo , Rinite/etiologia , Rinite/metabolismo , Sinusite/etiologia , Sinusite/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Animais , Biomarcadores , Doença Crônica , Modelos Animais de Doenças , Camundongos , Neurogênese/genética , Neurogênese/imunologia , Mucosa Olfatória/fisiopatologia , Neurônios Receptores Olfatórios/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Rinite/fisiopatologia , Sinusite/fisiopatologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Chronic pain is a major global public health issue causing a severe impact on both the quality of life for sufferers and the wider economy. Despite the significant clinical burden, little progress has been made in terms of therapeutic development. A unique approach to identifying new human-validated analgesic drug targets is to study rare families with inherited pain insensitivity. Here we have analysed an otherwise normal family where six affected individuals display a pain insensitive phenotype that is characterized by hyposensitivity to noxious heat and painless bone fractures. This autosomal dominant disorder is found in three generations and is not associated with a peripheral neuropathy. A novel point mutation in ZFHX2, encoding a putative transcription factor expressed in small diameter sensory neurons, was identified by whole exome sequencing that segregates with the pain insensitivity. The mutation is predicted to change an evolutionarily highly conserved arginine residue 1913 to a lysine within a homeodomain. Bacterial artificial chromosome (BAC) transgenic mice bearing the orthologous murine p.R1907K mutation, as well as Zfhx2 null mutant mice, have significant deficits in pain sensitivity. Gene expression analyses in dorsal root ganglia from mutant and wild-type mice show altered expression of genes implicated in peripheral pain mechanisms. The ZFHX2 variant and downstream regulated genes associated with a human pain-insensitive phenotype are therefore potential novel targets for the development of new analgesic drugs.awx326media15680039660001.
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Insensibilidade Congênita à Dor/genética , Limiar da Dor/fisiologia , Dor/fisiopatologia , Mutação Puntual/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Adolescente , Adulto , Idoso , Animais , Cálcio/metabolismo , Capsaicina/efeitos adversos , Modelos Animais de Doenças , Feminino , Gânglios Espinais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Dor/induzido quimicamente , Insensibilidade Congênita à Dor/patologia , Insensibilidade Congênita à Dor/fisiopatologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Pele/patologia , Adulto JovemRESUMO
MicroRNAs control the differentiation and function of B cells, which are considered key elements in the pathogenesis of systemic lupus erythematosus (SLE). However, a common micro(mi)RNA signature has not emerged since published data includes patients of variable ethnic background, type of disease, and organ involvement, as well as heterogeneous cell populations. Here, we aimed at identifying a miRNA signature of purified B cells from renal and non-renal severe SLE patients of Latin American background, a population known to express severe disease. Genome-wide miRNA expression analyses were performed on naive and memory B cells and revealed two categories of miRNA signatures. The first signature represents B cell subset-specific miRNAs deregulated in SLE: 11 and six miRNAs discriminating naive and memory B cells of SLE patients from healthy controls (HC), respectively. Whether the miRNA was up or down-regulated in memory B cells as compared with naive B cells in HC, this difference was abolished in SLE patients, and vice versa. The second signature identifies six miRNAs associated with specific pathologic features affecting renal outcome, providing a further understanding for SLE pathogenesis. Overall, the present work provided promising biomarkers in molecular diagnostics for disease severity as well as potential new targets for therapeutic intervention in SLE.
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Subpopulações de Linfócitos B/metabolismo , Biomarcadores/metabolismo , Perfilação da Expressão Gênica , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , MicroRNAs/genética , Adulto , Estudos de Casos e Controles , Chile , Análise por Conglomerados , França , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Nefrite Lúpica/diagnóstico , Nefrite Lúpica/genética , MicroRNAs/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a leading cause of death among cirrhotic patients, for which chemopreventive strategies are lacking. Recently, we developed a simple human cell-based system modeling a clinical prognostic liver signature (PLS) predicting liver disease progression and HCC risk. In a previous study, we applied our cell-based system for drug discovery and identified captopril, an approved angiotensin converting enzyme (ACE) inhibitor, as a candidate compound for HCC chemoprevention. Here, we explored ACE as a therapeutic target for HCC chemoprevention. Captopril reduced liver fibrosis and effectively prevented liver disease progression toward HCC development in a diethylnitrosamine (DEN) rat cirrhosis model and a diet-based rat model for nonalcoholic steatohepatitis-induced (NASH-induced) hepatocarcinogenesis. RNA-Seq analysis of cirrhotic rat liver tissues uncovered that captopril suppressed the expression of pathways mediating fibrogenesis, inflammation, and carcinogenesis, including epidermal growth factor receptor (EGFR) signaling. Mechanistic data in liver disease models uncovered a cross-activation of the EGFR pathway by angiotensin. Corroborating the clinical translatability of the approach, captopril significantly reversed the HCC high-risk status of the PLS in liver tissues of patients with advanced fibrosis. Captopril effectively prevents fibrotic liver disease progression toward HCC development in preclinical models and is a generic and safe candidate drug for HCC chemoprevention.
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Captopril , Carcinoma Hepatocelular , Neoplasias Hepáticas , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Captopril/farmacologia , Captopril/uso terapêutico , Carcinogênese , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/prevenção & controle , Quimioprevenção , Progressão da Doença , Receptores ErbB/metabolismo , Cirrose Hepática/prevenção & controle , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/prevenção & controle , Peptidil Dipeptidase A/metabolismo , Ratos , Ativação TranscricionalRESUMO
Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.
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Anticorpos Monoclonais , Plasticidade Celular , Animais , Camundongos , Humanos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Claudina-1 , Cirrose Hepática/tratamento farmacológicoRESUMO
Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) world-wide. The molecular mechanisms of viral hepatocarcinogenesis are still partially understood. Here, we applied two complementary single-cell RNA-sequencing protocols to investigate HBV-HCC host cell interactions at the single cell level of patient-derived HCC. Computational analyses revealed a marked HCC heterogeneity with a robust and significant correlation between HBV reads and cancer cell differentiation. Viral reads significantly correlated with the expression of HBV-dependency factors such as HLF in different tumor compartments. Analyses of virus-induced host responses identified previously undiscovered pathways mediating viral carcinogenesis, such as E2F- and MYC targets as well as adipogenesis. Mapping of fused HBV-host cell transcripts allowed the characterization of integration sites in individual cancer cells. Collectively, single-cell RNA-Seq unravels heterogeneity and compartmentalization of both, virus and cancer identifying new candidate pathways for viral hepatocarcinogenesis. The perturbation of pro-carcinogenic gene expression even at low HBV levels highlights the need of HBV cure to eliminate HCC risk.
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Carcinoma Hepatocelular/etiologia , Transformação Celular Viral , Vírus da Hepatite B/fisiologia , Hepatite B/complicações , Hepatite B/virologia , Neoplasias Hepáticas/etiologia , Adulto , Idoso , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Suscetibilidade a Doenças , Feminino , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Hepatite B Crônica/complicações , Hepatite B Crônica/virologia , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , RNA Viral , Análise de Célula Única/métodos , Transcriptoma , Carga ViralRESUMO
Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2+, CLEC5Ahigh, MARCOlow liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention.
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Descoberta de Drogas , Fígado/patologia , Modelos Biológicos , Animais , Carcinogênese/patologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Quimioprevenção , Estudos de Coortes , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Hepacivirus/fisiologia , Hepatite C/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Vigilância Imunológica/efeitos dos fármacos , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Knockout , Nizatidina/farmacologia , Prognóstico , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Rationale: Monocytes play critical roles in the pathogenesis of arthritis by contributing to the inflammatory response and bone erosion. Among genes involved in regulating monocyte functions, miR-146a negatively regulates the inflammatory response and osteoclast differentiation of monocytes. It is also the only miRNA reported to differentially regulate the cytokine response of the two classical Ly6Chigh and non-classical Ly6Clow monocyte subsets upon bacterial challenge. Although miR-146a is overexpressed in many tissues of arthritic patients, its specific role in monocyte subsets under arthritic conditions remains to be explored. Methods: We analyzed the monocyte subsets during collagen-induced arthritis (CIA) development by flow cytometry. We quantified the expression of miR-146a in classical and non-classical monocytes sorted from healthy and CIA mice, as well as patients with rheumatoid arthritis (RA). We monitored arthritis features in miR-146a-/- mice and assessed in vivo the therapeutic potential of miR-146a mimics delivery to Ly6Chigh monocytes. We performed transcriptomic and pathway enrichment analyses on both monocyte subsets sorted from wild type and miR-146a-/- mice. Results: We showed that the expression of miR-146a is reduced in the Ly6Chigh subset of CIA mice and in the analogous monocyte subset (CD14+CD16-) in humans with RA as compared with healthy controls. The ablation of miR-146a in mice worsened arthritis severity, increased osteoclast differentiation in vitro and bone erosion in vivo. In vivo delivery of miR-146a to Ly6Chigh monocytes, and not to Ly6Clow monocytes, rescues bone erosion in miR-146a-/- arthritic mice and reduces osteoclast differentiation and pathogenic bone erosion in CIA joints of miR-146a+/+ mice, with no effect on inflammation. Silencing of the non-canonical NF-κB family member RelB in miR-146a-/- Ly6Chigh monocytes uncovers a role for miR-146a as a key regulator of the differentiation of Ly6Chigh, and not Ly6Clow, monocytes into osteoclasts under arthritic conditions. Conclusion: Our results show that classical monocytes play a critical role in arthritis bone erosion. They demonstrate the theranostics potential of manipulating miR-146a expression in Ly6Chigh monocytes to prevent joint destruction while sparing inflammation in arthritis.