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Background Intratumoral heterogeneity is crucially involved in metastasis, resistance to therapy, and cancer relapse. Amplifications of the proto-oncogene MYC display notable heterogeneity at the single-cell level and are associated with a particularly dismal prognosis in high-risk medulloblastomas (MBs). The aim of this study was to establish the relevance of interclonal cross-talk between MYC-driven and non-MYC-driven MB cells. Methods We used fluorescence in situ hybridization, single-cell transcriptomics, and immunohistochemistry, in vitro isogenic cell models, non-targeted proteomics, mass spectrometry-based metabolite quantification, HUVECs tube formation assay, and orthotopic in vivo experiments to investigate interclonal cross-talk in MB. Results We found that the release of lactate dehydrogenase A (LDHA) from MYC-driven cells facilitates metastatic seeding and outgrowth, while secretion of dickkopf WNT signaling pathway inhibitor 3 from non-MYC-driven cells promotes tumor angiogenesis. This tumor-supporting interaction between both subclones was abrogated by targeting the secretome through pharmacological and genetic inhibition of LDHA, which significantly suppressed tumor cell migration. Conclusion Our study reveals the functional relevance of clonal diversity and highlights the therapeutic potential of targeting the secretome to interrupt interclonal communication and progression in high-risk MB.

Whole-genome sequencing (WGS) of human cancers has revealed that structural variation, which refers to the rearrangement of the genome leading to the deletion, amplification of reshuffling of DNA segments ranging from a few hundred bp to entire chromosomes, is a key mutational process in cancer evolution. Notably, pan-cancer analyses have revealed that both simple and complex forms of structural variation are pervasive across diverse human cancers, and often underpin drug resistance and metastasis. To date, the study of cancer genomes has relied on the analysis of short-read WGS on the dominant Illumina platform, which generates short, highly-accurate reads of 100-300bp that allow the study of point mutations at high resolution. However, detection of structural variants (SVs) using short reads is limited, as breakpoints falling in repetitive regions cannot be reliably mapped to the human genome. As a result, our understanding of the patterns and mechanisms underpinning structural variation in cancer genomes remains incomplete. In contrast to short-read sequencing, long-read sequencing technologies, such as Oxford Nanopore and PacBio, permit continuous reading of individual DNA molecules over 10 kilobases, thus providing unparalleled information to resolve SVs in repetitive regions and complex genome rearrangements. However, novel bioinformatics methods that account for the higher error rate of long-read methods are needed to take advantage of their capabilities for cancer genome analysis. Here, we present SAVANA, a novel structural variant caller for long-read sequencing data specifically designed for the analysis of cancer genomes. To identify both somatic and germline SVs, SAVANA takes as input long-read WGS data from a tumor and normal sample pair. SAVANA scans sequencing reads to detect split reads and gapped alignments, which are then clustered to define putative SVs. Next, SAVANA applies a machine learning-informed set of heuristics to remove false positives arising from mapping errors and sequencing artifacts. Extensively validated against a multi-platform truthset, we show that SAVANA identifies a range of somatic rearrangements with high recall and precision, outperforming existing tools while maintaining a lower execution time than competing methods. In patient samples, SAVANA identifies clinically relevant alterations, such as oncogenic gene fusions, with high accuracy. Additionally, SAVANA permits the reconstruction of double minutes, multi-chromosomal chromothripsis events, and SVs mapping to highly repetitive regions, including centromeres. In sum, SAVANA permits the characterization of complex structural variants and can uncover clinically relevant mutations across diverse cancer types with high accuracy. Citation Format: Hillary Elrick, Jose Espejo Valle-Inclan, Katherine E. Trevers, Francesc Muyas, Rita Cascão, Angela Afonso, Cláudia C. Faria, Adrienne M. Flanagan, Isidro Cortés-Ciriano. SAVANA: a computational method to characterize structural variation in human cancer genomes using nanopore sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB080.

Telomere fusions (TFs) can trigger the accumulation of diverse genomic rearrangements and the acquisition of oncogenic alterations leading to malignant transformation and resistance to chemotherapy. Despite their relevance in tumour evolution, our understanding of the patterns and consequences of TFs in human cancer remains limited. Here, we have characterized the rates and spectrum of somatic TFs across >30 cancer types using whole-genome sequencing data. TFs are pervasive in human tumours with rates varying markedly across and within cancer types. In addition to end-to-end fusions, we find novel patterns of TFs that we mechanistically link to the activity of the alternative lengthening of telomeres (ALT) pathway. We show that TFs can be detected in the blood of cancer patients, which enables cancer detection with high specificity and sensitivity even for early-stage tumours and cancer types for which early detection remains a high unmet clinical need, such as pancreatic cancer and brain tumours. Overall, we report a novel genomic footprint that enables characterization of the telomere maintenance mechanism of tumours and liquid biopsy analysis, which has implications for early detection, prognosis, and treatment selection.

Brain metastases (BMs) are a devastating complication of advanced cancers associated with poor prognosis. Contrarily to the current improvement in systemic therapies, BMs are still incurable and one of the main causes of death in cancer patients. We analyzed BMs from thirty patients with various primary tumor origins by RNA sequencing and identified the upregulation of UBE2C, a gene involved in the correct transition from metaphase to anaphase. Using an independent cohort of patients with BMs, we demonstrated that high protein expression of UBE2C was associated with worse survival. UBE2C-driven cancer cells promoted migration and invasion in vitro and induced an aggressive phenotype and decreased survival in mouse orthotopic xenografts. PI3K/mTOR inhibition effectively blocked cancer cell signaling and prevented the development of leptomeningeal metastases. Therefore, we have identified UBE2C as a molecular marker of worse outcome in BMs patients and pre-clinically validated an effective therapy against UBE2C-driven brain metastatic disease.

Dissemination of cancer cells from primary tumors to the brain occurs in many cancer patients, increasing morbidity and death. There is an unmet medical need to develop translational platforms to evaluate therapeutic responses. Toward this goal, we established a library of 23 patient-derived xenografts (PDXs) of brain metastases (BMs) from eight distinct primary tumors. In vivo tumor formation correlates with patients' poor survival. Mouse subcutaneous xenografts develop spontaneous metastases and intracardiac PDXs increase dissemination to the CNS, both models mimicking the dissemination pattern of the donor patient. We test the FDA-approved drugs buparlisib (pan-PI3K inhibitor) and everolimus (mTOR inhibitor) and show their efficacy in treating our models. Finally, we show by RNA sequencing that human BMs and their matched PDXs have similar transcriptional profiles. Overall, these models of BMs recapitulate the biology of human metastatic disease and can be valuable translational platforms for precision medicine.

PurposeDissemination of cancer cells from primary tumors to the brain is observed in the great majority of cancer patients, contributing to increased morbidity and being the main cause of death. Most mechanistic and preclinical studies have relied on aggressive cancer cell lines, which fail to represent tumor heterogeneity and are unsuitable to validate therapies due to fast cancer progression in vivo.Experimental designWe established a unique library of subcutaneous and intracardiac patient-derived xenografts (PDXs) of brain metastases (BMs) from eight distinct primary tumor origins. Cancer progression in mice was compared to the matched patient clinical outcome, metastatic dissemination pattern and histopathological features. Preclinical studies with FDA approved drugs were performed.ResultsIn vivo tumor formation of flank-implanted BMs correlated with patients’ poor survival and serial passaging increased tumor aggressiveness. Subcutaneous xenografts originated spontaneous metastases in 61% of the cases, including in the leptomeningeal space (21%). The intracardiac model increased the tropism to the brain and leptomeninges (46%). Strikingly, 62% of intracardiac PDXs shared metastatic sites with the donor patients, including the primary cancer organ and the central nervous system (CNS). Of therapeutic relevance, PDX-derived cultures and corresponding mouse xenografts can be effectively treated with targeted anticancer drugs.ConclusionsPatient-derived models of BMs recapitulate the biology of human metastatic disease and can be a valuable translational platform for precision medicine.TRANSLATIONAL RELEVANCESubcutaneous and intracardiac mouse xenografts of human brain metastases exhibit a spontaneous dissemination pattern that resembles patients’ metastatic disease. The preclinical testing of targeted anticancer drugs using patient-derived cultures and patient-derived xenografts of brain metastasis showed an effective therapeutic response. These translational models represent an outstanding tool to advance the understanding of the biology of brain metastases and to foster the rapid discovery of novel therapeutics.

The dissemination of cancer cells to the brain parenchyma (brain metastases - BMs) and to the leptomeninges (leptomeningeal dissemination – LD) are a late-stage complication of systemic cancers, with a poor survival. Despite advances in radiation and chemotherapy, including intrathecal administration of anticancer agents, these forms of advanced cancer are incurable. Therefore, there is an unmet clinical need for novel effective therapies that target both parenchymal and leptomeningeal disease. We analysed the transcriptomic profile of BMs from patients with diverse primary tumors, treated at CHULN, to identify genetic drivers of cancer cell dissemination to the brain and potential novel targets for therapy. The most differentially expressed gene codifies a ubiquitin conjugating enzyme (UCE). UCE levels were evaluated in tissue microarrays of BMs from an independent cohort of patients and correlated with clinical data. UCE functional role was assessed in vitro and in vivo using modulated lung and breast cancer cell lines. A high-throughput drug screening was performed to find UCE-targeting compounds. High protein levels of the UCE were associated with decreased survival in patients with BMs, independently of the primary tumor origin. High levels of UCE led to increased migration and invasion abilities in cancer cell lines in vitro, with no effect in proliferation. In vivo, high levels of UCE increased leptomeningeal dissemination and decreased survival in orthotopic models of breast cancer BMs. Leptomeningeal disease promoted by UCE was prevented by oral administration of inhibitor A, identified in our high-throughput drug screening. In conclusion, we have identified UCE as a prognostic marker in patients with BMs from different primary tumors. In orthotopic mouse models of the disease, UCE led to a worse survival and promoted leptomeningeal dissemination. Strikingly, this aggressive disease phenotype was prevented by oral therapy with inhibitor A.

Background: Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease, which causes local and systemic bone damage. Objectives: The main goal of this work was to analyze, how treatment intervention with Ab501 (certolizumab mice equivalent) prevents the disturbances on bone structure and mechanics induced by arthritis. Methods: Thirty one DBA/1 collagen-induced arthritis (CIA) mice were randomly housed in experimental groups, as follows: arthritic untreated (N=10), preventive intervention with ab501 2 days before arthritis induction (N=10) and treatment intervention upon arthritis onset with ab501 (N=11). A non-induced group (N=5) was used as a control. Mice were monitored during 70 days after disease induction for the inflammatory score, ankle perimeter and body weight. Non-induced mice were used as controls. After 70 days of disease progression mice were sacrificed and bone samples were collected for histology, micro-computed tomography (μCT) and 3-point bending analysis. In addition, blood samples were also collected for bone turnover markers quantification. Statistical differences were determined with Mann–Whitney tests using GraphPad Prism (GraphPad, California, USA). Data were expressed as median with interquartile range. Differences were considered statistically significant for p Results: Results showed that Ab501 administration was able to control and abrogate disease development both in preventive and early therapeutic intervention. μCT results revealed that ab501 was able to preserve bone structure when delivered before arthritis induction. Conclusion: Ab501 preventive administration was able to control inflammation and prevent the degradative effects of arthritis on trabecular bone structure. Acknowledgement: This work was supported by UCB in the context of an Investigator Initiated Study where UCB provided financial and product support. Disclosure of Interests: Bruno Vidal Grant/research support from: Aspire Pfizer 2013, Mikko A. Finnila: None declared, Ines Lopes: None declared, Rita Cascao: None declared, Joao Eurico Fonseca: None declared

INTRODUCTION: Medulloblastoma is the most common malignant brain tumor of childhood and is a genetically heterogeneous disease. Amongst all medulloblastoma subgroups, MYC-amplified medulloblastomas are associated with a particularly poor prognosis as they are commonly metastatic and resistant to standard therapy. However, MYC amplification is restricted to a cellular subpopulation within MYC-amplified tumors. The underlying mechanisms mediating this cellular heterogeneity remain poorly understood. HYPOTHESIS: We hypothesize that a secretome-dependent crosstalk between MYC-amplified and non-MYC-amplified cells shapes an intratumoral microenvironment that promotes medulloblastoma aggressiveness. METHODS AND RESULTS: We first demonstrate that MYC overexpressing (high-MYC) cells induce proliferation and invasive growth patterns in their isogenic counterparts with low MYC expression (low-MYC) using contacting and non-contacting co-culture systems in three different medulloblastoma cell lines (ONS76, DAOY and UW228-3). Secondly, we demonstrate that exosomes from high-MYC cells promote the migratory propensity of low-MYC cells in vitro. Importantly, we confirmed the pro-tumorigenic interaction of high-MYC and low-MYC cells using two orthotopic medulloblastoma xenograft models. We further demonstrate that low-MYC cells promote tumor angiogenesis in vivo. Using an unbiased proteomic discovery approach and targeted validation by Western blotting, we identified and verified secreted proteins that may mediate this interaction. Rescue experiments validated the relevance of these candidates using functional genomics approaches in vitro, with in vivo validation being currently ongoing. CONCLUSION: We demonstrate that the crosstalk between cellular subclones in MYC-driven medulloblastoma may promote tumor growth, metastasis and angiogenesis. Understanding the underlying secretory signaling networks may elucidate novel therapeutic vulnerabilities in this prognostically poor subgroup of medulloblastoma.

A correction has been published: Rheumatology, Volume 58, Issue 2, February 2019, Page 371, https://doi.org/10.1093/rheumatology/key377. Rheumatoid arthritis (RA) causes immune mediated local and systemic bone damage. Objectives - The main goal of this work was to analyze, how treatment intervention with tofacitinib prevents the early disturbances on bone structure and mechanics in adjuvant induced arthritis rat model. This is the first study to access the impact of tofacitinib on the systemic bone effects of inflammation. Methods - Fifty Wistar adjuvant-induced arthritis (AIA) rats were randomly housed in experimental groups, as follows: non-arthritic healthy group (N=20), arthritic non-treated (N=20) and 10 animals under tofacitinib treatment. Rats were monitored during 22 days after disease induction for the inflammatory score, ankle perimeter and body weight. Healthy non-arthritic rats were used as controls for comparison. After 22 days of disease progression rats were sacrificed and bone samples were collected for histology, micro computed tomography (micro-CT), 3-point bending and nanoindentation analysis. Blood samples were also collected for bone turnover markers and systemic cytokine quantification. Results - At tissue level, measured by nanoindentation, tofacitinib increased bone cortical and trabecular hardness. However, micro-CT and 3-point bending tests revealed that tofacitinib did not revert the effects of arthritis on cortical and trabecular bone structure and on mechanical properties. Conclusion - Possible reasons for these observations might be related with the mechanism of action of tofacitinib, which leads to direct interactions with bone metabolism, and/or with kinetics of its bone effects that might need longer exposure.

Subgroups of ependymoma, especially RELA fusion-positive and posterior fossa type A tumors, are associated with poor prognosis. Curative therapeutic strategies have not yet been identified. We set up a high-throughput drug screening (HTS) pipeline to evaluate clinically established compounds (n=196) in primary ependymoma cultures (n=12). As culturing ependymoma is challenging, assay miniaturization to 1536-well microplates emerged as a key feature to process HTS despite smallest cell numbers. DNA methylation profiling showed that entity and subgroup affiliation from primary diagnosis was maintained in primary cultures, as assessed through molecular neuropathology 2.0 based classification (MNP 2.0, Capper, D. et al., Nature, 2018). A comparison of HTS data of ependymoma and other pediatric brain tumor models (n=48) revealed a remarkable chemoresistance in vitro. However, we identified Neratinib, an irreversible ERBB2 inhibitor, as the most prominent candidate which was preferentially active in a subset of the investigated ependymoma cultures (n=5). Combinatory treatment with Copanlisib, a PI3K inhibitor, was able to overcome resistance to single agent treatment using Neratinib in established cell lines of ependymoma (n=3) and 2/4 primary cultures for which combinatory treatment could be tested. Finally, we validated efficacy of Neratinib combined with Copanlisib in mice bearing ependymoma xenografts which revealed significantly reduced tumor size compared to vehicle-treated animals. In summary, our study demonstrates that HTS may reveal targeted therapies for pediatric brain tumors. Specifically, we found a synergistic interaction of Neratinib and Copanlisib for treatment of ependymoma, thereby providing a novel therapeutic approach in an otherwise largely chemoresistant entity.

Introduction Arthritis induces joint erosions and skeletal bone fragility. Objectives The main goal of this work was to analyze the early arthritis induced events at bone architecture and mechanical properties at tissue level. Methods Eighty-eight Wistar rats were randomly housed in experimental groups, as follows: adjuvant induced arthritis (AIA) (N = 47) and a control healthy group (N = 41). Rats were monitored during 22 days for the inflammatory score, ankle perimeter and body weight and sacrificed at different time points (11 and 22 days post disease induction). Bone samples were collected for histology, micro computed tomography (micro-CT), 3-point bending and nanoindentation. Blood samples were also collected for bone turnover markers and systemic cytokine quantification. Results At bone tissue level, measured by nanoindentation, there was a reduction of hardness in the arthritic group, associated with an increase of the ratio of bone concentric to parallel lamellae and of the area of the osteocyte lacuna. In addition, increased bone turnover and changes in the microstructure and mechanical properties were observed in arthritic animals, since the early phase of arthritis, when compared with healthy controls. Conclusion We have shown in an AIA rat model that arthritis induces very early changes at bone turnover, structural degradation and mechanical weakness. Bone tissue level is also affected since the early phase of arthritis, characterized by decreased tissue hardness associated with changes in bone lamella organization and osteocyte lacuna surface. These observations highlight the pertinence of immediate control of inflammation in the initial stages of arthritis.

Background Celastrol, a pentacyclic-triterpene isolated from Tripterigium wilfordii roots, has shown great therapeutic potential for the treatment of several inflammatory diseases, including rheumatoid arthritis (RA). We have previously demonstrated that celastrol has significant anti-inflammatory and bone protective effects in the adjuvant-induced rat model of arthritis (AIA), when administered via intraperitoneal route. For further preclinical evaluation of celastrol as a candidate compound for RA treatment, an effective and safe oral administration is crucial. Objectives In this work we aimed to study the dose range for both therapeutic and toxic effects for oral administration of pure celastrol using the AIA rat model. Methods Celastrol (1, 2.5, 5, 7.5, 12.5 and 25μg/g/day, N=5/group) was administrated orally in female AIA rats after 8 days of disease induction (therapeutic model) for a period of 14-days. A group of healthy (N=8) and untreated arthritic (vehicle, N=15) gender and age-matched Wistar rats were used as control. During the period of treatment, the inflammatory score, ankle perimeter and body weight were measured. At the end of the treatment, animals were sacrificed, blood was collected for clinical pathology, and necropsy was performed, with collection of internal organs for histopathological analysis and of paw samples for disease scoring. Results Oral administration of pure celastrol at 2.5, 5 and 7.5μ g/g/day reduced the inflammatory score and ankle swelling, preserved articular joint structure with a reduction in synovial inflammatory infiltrates and proliferation, halted articular bone destruction, and diminished the number of synovial CD68+ macrophages (a biomarker of response to anti-arthritic treatment). This compound also reduced the number of osteoclasts and osteoblasts present in joints. Bone resorption and turnover was also reduced at both 5 and 7.5μg/g/day, with a significant decrease in serum levels of TRACP-5b, P1NP and CTX-I. Of note, no significant variation in body weight, evidence of nephro-, hepato- or cardiotoxic effects, nor alterations in blood cell counts were observed at these concentrations. However, the dose of 7.5μ g/g/day was already associated with thymic and hepatotoxic changes, and higher doses showed toxicity signs. The lethal dose (LD) and LD50 were defined as 25μg/g/day and 12.5μg/g/day, respectively. Of note, oral celastrol at 1μg/g/day had no effect in arthritis progression. Conclusions Our results clearly show that 2.5μg/g/day is the lowest and 5μg/g/day is the highest effective and safe oral doses of celastrol in the setting of AIA rat model. These findings suggest that while celastrol is potentially very effective to treat RA, it has a narrow therapeutic window. Acknowledgements The authors would like to acknowledge Bruno Vidal and Ines Lopes for technical assistance. Disclosure of Interest None declared

Background Arthritis induces joint erosions and skeletal bone fragility. Objectives The main goal of this work was to analyze the early arthritis induced events at bone tissue level. Methods Eighty-eight Wistar rats were randomly housed in experimental groups, as follows: adjuvant induced arthritis (N=47) and a control healthy group (N=41). Rats were monitored during 22 days for the inflammatory score, ankle perimeter and body weight and sacrificed at different time points (11 and 22 days post disease induction). Bone samples were collected for histology, micro-CT, 3-point bending, nanoindentation and Fourier transformed infrared spectroscopy (FTIR) analysis. Blood samples were also collected for bone turnover markers and systemic cytokine quantification. Results At bone tissue level, measured by FTIR analysis and nanoindentation, there was a reduction of the mineral and collagen content and of hardness in the arthritic group, associated with an increase of the ratio of bone concentric to parallel lamellae and of the area of the osteocyte lacuna. In addition, increased bone turnover and changes in the microstructure and mechanical properties were observed in arthritic animals, since the early phase of arthritis, when compared with healthy controls. Conclusions Arthritis induces very early changes at bone tissue level characterized by decreased tissue hardness and of collagen and mineral content. These observations highlight the pertinence of immediate control of inflammation in the initial stages of arthritis. Disclosure of Interest None declared

Background Rheumatoid arthritis (RA) causes immune mediated local and systemic bone damage. Objectives The main goal of this work was to analyze, how treatment intervention with tofacitinib prevents the early disturbances on bone structure and mechanics in adjuvant induced arthritis rat model. This is the first study to access the impact of tofacitinib on the systemic bone effects of inflammation. Methods Fifty Wistar adjuvant-induced arthritis (AIA) rats were randomly housed in experimental groups, as follows: non-arthritic healthy group (N=20), arthritic non-treated (N=20) and 10 animals under tofacitinib treatment. Rats were monitored during 22 days after disease induction for the inflammatory score, ankle perimeter and body weight. Healthy non-arthritic rats were used as controls for comparison. After 22 days of disease progression rats were sacrificed and bone samples were collected for histology, micro-CT, 3-point bending and nanoindentation analysis. Blood samples were also collected for bone turnover markers and systemic cytokine quantification. Results At tissue level, measured by nanoindentation, tofacitinib increased bone cortical and trabecular hardness. However, micro-CT and 3-point bending tests revealed that tofacitinib did not revert the effects of arthritis on cortical and trabecular bone structure and on mechanical properties. Conclusions Possible reasons for these observations might be related with the mechanism of action of tofacitinib, which leads to direct interactions with bone metabolism, and/or with kinetics of its bone effects that might need longer exposure. Disclosure of Interest None declared

Objective Rheumatoid arthritis (RA) is characterised by chronic inflammation leading to articular bone and cartilage damage. Despite recent progress in RA management, adverse effects, lack of efficacy and economic barriers to treatment access still limit therapeutic success. Therefore, safer and less expensive treatments that control inflammation and bone resorption are needed. We have previously shown that celastrol is a candidate for RA treatment. We have observed that it inhibits both interleukin (IL)-1β and tumor necrosis factor (TNF) in vitro, and that it has anti-inflammatory properties and ability to decrease synovial CD68+ macrophages in vivo. Herein our goal was to evaluate the effect of celastrol in local and systemic bone loss. Methods Celastrol was administrated intraperitoneally at a dose of 1 µg/g/day to female Wistar adjuvant-induced arthritic rats. Rats were sacrificed after 22 days of disease progression, and blood, femurs, tibiae and paw samples were collected for bone remodelling markers quantification, 3-point bending test, micro-CT analysis, nanoindentation and Fourier transform infrared spectroscopy measurements, and immunohistochemical evaluation. Results We have observed that celastrol preserved articular structures and decreased the number of osteoclasts and osteoblasts present in arthritic joints. Moreover, celastrol reduced tartrate-resistant acid phosphatase 5b, procollagen type 1 amino-terminal propeptide and C terminal crosslinked telopeptide of type II collagen serum levels. Importantly, celastrol prevented bone loss and bone microarchitecture degradation. Celastrol also preserved bone nanoproperties and mineral content. Additionally, animals treated with celastrol had less fragile bones, as depicted by an increase in maximum load and yield displacement. Conclusions These results suggest that celastrol reduces both bone resorption and cartilage degradation, and preserves bone structural properties., published version, peerReviewed

Copyright: © 2015 Cascão et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, Background: Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by cellular infiltration into the joints, hyperproliferation of synovial cells and bone damage. Available treatments for RA only induce remission in around 30% of the patients, have important adverse effects and its use is limited by their high cost. Therefore, compounds that can control arthritis, with an acceptable safety profile and low production costs are still an unmet need. We have shown, in vitro, that celastrol inhibits both IL-1β and TNF, which play an important role in RA, and, in vivo, that celastrol has significant anti-inflammatory properties. Our main goal in this work was to test the effect of celastrol in the number of sublining CD68 macrophages (a biomarker of therapeutic response for novel RA treatments) and on the overall synovial tissue cellularity and joint structure in the adjuvant-induced rat model of arthritis (AIA). Methods: Celastrol was administered to AIA rats both in the early (4 days after disease induction) and late (11 days after disease induction) phases of arthritis development. The inflammatory score, ankle perimeter and body weight were evaluated during treatment period. Rats were sacrificed after 22 days of disease progression and blood, internal organs and paw samples were collected for toxicological blood parameters and serum proinflammatory cytokine quantification, as well as histopathological and immunohistochemical evaluation, respectively. Results: Here we report that celastrol significantly decreases the number of sublining CD68 macrophages and the overall synovial inflammatory cellularity, and halted joint destruction without side effects. Conclusions: Our results validate celastrol as a promising compound for the treatment of arthritis., RC was supported with a fellowship from Fundação para a Ciência e a Tecnologia (FCT, SFRH/BPD/92860/2013).

© 2010 Elsevier B.V. All rights reserved., Rheumatoid arthritis is the most common inflammatory joint disease. The etiopathogenesis of this condition has been classically explained by a T cell-driven process. However, recent studies have highlighted the possible contribution of neutrophils for the early phases of RA physiopathology. These cells are phagocytic leukocytes that play crucial roles in the acute defense against pathogens while modulating the function of other immune cells and contributing to the perpetuation of an initial inflammatory response. The herein article reviews recent progresses in the understanding of the immunopathology of RA with a special emphasis on the role of neutrophils., This work was supported by a fellowship from Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/40513/2007 and partially supported by a grant from Sociedade Portuguesa de Reumatologia and FCT grant PTDC/SAU-OSM/73449/2006.

Copyright: © 2015 Vidal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, Background: We have previously found in the chronic SKG mouse model of arthritis that long standing (5 and 8 months) inflammation directly leads to high collagen bone turnover, disorganization of the collagen network, disturbed bone microstructure and degradation of bone biomechanical properties. The main goal of the present work was to study the effects of the first days of the inflammatory process on the microarchitecture and mechanical properties of bone. Methods: Twenty eight Wistar adjuvant-induced arthritis (AIA) rats were monitored during 22 days after disease induction for the inflammatory score, ankle perimeter and body weight. Healthy non-arthritic rats were used as controls for compar-ison. After 22 days of disease progression rats were sacrificed and bone samples were collected for histomorphometrical, energy dispersive X-ray spectroscopical analysis and 3-point bending. Blood samples were also collected for bone turnover markers. Results: AIA rats had an increased bone turnover (as inferred from increased P1NP and CTX1, p = 0.0010 and p = 0.0002, respectively) and this was paralleled by a decreased mineral content (calcium p = 0.0046 and phos-phorus p = 0.0046). Histomorphometry showed a lower trabecular thickness (p = 0.0002) and bone volume (p = 0.0003) and higher trabecular sepa-ration (p = 0.0009) in the arthritic group as compared with controls. In addition, bone mechanical tests showed evidence of fragility as depicted by diminished values of yield stress and ultimate fracture point (p = 0.0061 and p = 0.0279, re-spectively) in the arthritic group. Conclusions: We have shown in an AIA rat model that arthritis induc-es early bone high turnover, structural degradation, mineral loss and mechanical weak-ness., This work was supported by a grant SFRH/BD/81527/2011 from Fundação para a Ciência e a Tecnologia (FCT) and grant ECTS/Amgen Bone Biology Fellowship 2014 from European Calcified Tissue Society.

Background. We have previously reported a continuous activation of caspase-1 and increased interleukin (IL)-1βlevels in early rheumatoid arthritis (RA). These observations raised the hypothesis that drugs targeting the IL-1βpathway, in addition to tumour necrosis factor (TNF), may be particularly effective for early RA treatment. We have recently identified gambogic acid as a promising therapeutic candidate to simultaneously block IL-1βand TNF secretion. Our main goal here was to investigate whether gambogic acid administration was able to attenuate inflammation in antigen-induced arthritis (AIA) rats.Methods. Gambogic acid was administered to AIA rats in the early and late phases of arthritis. The inflammatory score, ankle perimeter, and body weight were evaluated during the period of treatment. Rats were sacrificed after 19 days of disease progression and paw samples were collected for histological and immunohistochemical evaluation.Results. We found that inflammation in joints was significantly suppressed following gambogic acid administration. Histological and immunohistochemical evaluation of treated rats revealed normal joint structures with complete abrogation of the inflammatory infiltrate and cellular proliferation.Conclusions. Our results suggest that gambogic acid has significant anti-inflammatory properties and can possibly constitute a prototype anti-inflammatory drug with therapeutic efficacy in the treatment of inflammatory diseases such as RA.

Background We have previously found in the chronic SKG mouse model of arthritis that long standing (5 and 8 months) inflammation directly leads to high collagen bone turnover, disorganization of the collagen network, disturbed bone microstructure and ultimately declining of bone biomechanical properties [1]. Objectives Our main goal was to study the effects of the inflammatory process on the microarchitecture and mechanical properties of bone in the early stages of arthritis development. Methods Fifty Wistar adjuvant-induced arthritis (AIA) rats were monitored throughout arthritis development and sacrificed after 4, 11 and 22 days of disease induction. Thirty healthy non-arthritic rats, age and sex-matched, were sacrificed at the end of the experiment and used as controls for comparison. The inflammatory score, ankle perimeter and body weight were measured over the experimental period. At the time of sacrifice, bone and serum samples were collected for micro-CT and 3-point bending analysis as well as bone turnover markers (CTX-I and P1NP), respectively. All experiments were approved by the Animal User and Ethical Committees at the Instituto de Medicina Molecular (Lisbon University), according to the Portuguese law and the European recommendations. Results We have observed that bone turnover markers, CTX-I and P1NP, increased soon after arthritis onset (p Conclusions The inflammatory process induced bone loss, and reduces bone strength since the very early phase of arthritis. References Caetano-Lopes J, Nery AM, Canhao H, Duarte J, Cascao R, et al. (2010) Chronic arthritis leads to disturbances in the bone collagen network. Arthritis Res Ther 12: R9. Disclosure of Interest None declared

Our goal was to test the performance of the new American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria for the classification of rheumatoid arthritis (RA) in a cohort of patients with very recent onset polyarthritis.Untreated polyarthritis patients with less than 6 weeks of duration were enrolled. All patients were followed-up in order to establish a definitive diagnosis.Thirty-seven patients were included. During the follow up 57% of the patients evolved to RA. The median age of the RA-group patients was simi­lar to the median age of the non-RA group [median (IQR) 47 (31-58.5) vs 43 (34-69) years, p=0.74]. At the initial visit the DAS 28 in the RA group was significantly higher than in the non-RA group, as well as the visual analogue scale (VAS), the HAQ and the number of swollen joints. Among the 21 RA patients, 43% presented RF and 28.6% presented anti-citrullinated protein antibody (ACPA) in the first visit. RF and ACPA were not detectable in any of the patients who did not evolve to RA. According to the new ACR/EULAR criteria, the mean total score of the RA group at baseline was significantly higher than the non-RA group [median (IQR) 6 (4.5-8) vs 4.5 (2.2-6), p=0.007].In our cohort high DAS28, swollen joint count, VAS and HAQ and the presence of RF or ACPA were eventually associated with the evolution into RA. The new ACR/EULAR criteria for the classification of RA seem to perform well in very early RA.

We have previously reported an increase in interleukin (IL)-1β and IL-17 levels, and a continuous activation of caspase-1 in early rheumatoid arthritis (RA) patients. These results suggest that drugs targeting IL-1β regulatory pathways, in addition to tumor necrosis factor (TNF), may constitute promising therapeutic agents in early RA. We have recently used a THP-1 macrophage-like cell line to screen 2320 compounds for those that down-regulate both IL-1β and TNF secretion. Celastrol was one of the most promising therapeutic candidates identified in that study. Our main goal in the present work was to investigate whether administration of celastrol is able to attenuate inflammation in a rat model of adjuvant-induced arthritis (AIA). Moreover, since IL-1β is known to play a role in the polarization of Th17 cells, we also investigate whether administration of digoxin, a specific inhibitor of Th17 cells polarization, is able to attenuate inflammation in the same rat model. We found that celastrol administration significantly suppressed joint inflammation. The histological and immunohistochemical evaluation revealed that celastrol-treated rats had a normal joint structure with complete abrogation of the inflammatory infiltrate and cellular proliferation. In contrast, we observed that digoxin administration significantly ameliorated inflammation but only if administrated in the early phase of disease course (after 4 days of disease induction), and it was not efficient at inhibiting the infiltration of immune cells within the joint and in preventing damage. Thus, our results suggest that celastrol has significant anti-inflammatory and anti-proliferative properties and can constitute a potential anti-inflammatory drug with therapeutic efficacy in the treatment of immune-mediated inflammatory diseases such as RA. Furthermore, we find that early inhibition of Th17 cells polarization ameliorates arthritis but it is not as effective as celastrol.

We have previously reported an increase in interleukin (IL)-1β levels and a continuous activation of caspase-1 in rheumatoid arthritis (RA) patients in the early phase of the disease. This result suggests that drugs targeting IL-1β regulatory pathways, in addition to tumor necrosis factor (TNF), may constitute promising therapeutic agents in early RA. We have recently used a THP-1 macrophage-like cell line to screen 2320 compounds for those which down-regulate IL-1β and TNF secretion. Gambogic acid, celastrol and pristimerin were three of the most promising therapeutic candidates identified in that study.

Involvement of GJB2 noncoding regions in hearing loss (HL) has not been extensively investigated. However, three noncoding mutations, c.-259C>T, c.-23G>T, and c.-23+1G>A, were reported. Also, c.-684_-675del, of uncertain pathogenicity, was found upstream of the basal promoter. We performed a detailed analysis of GJB2 noncoding regions in Portuguese HL patients (previously screened for GJB2 coding mutations and the common GJB6 deletions) and in control subjects, by sequencing the basal promoter and flanking upstream region, exon 1, and 3'UTR. All individuals were genotyped for c.-684_-675del and 14 SNPs. Novel variants (c.-731C>T, c.-26G>T, c.*45G>A, and c.*985A>T) were found in controls. A hearing individual homozygous for c.-684_-675del was for the first time identified, supporting the nonpathogenicity of this deletion. Our data indicate linkage disequilibrium (LD) between SNPs rs55704559 (c.*168A>G) and rs5030700 (c.*931C>T) and suggest the association of c.[*168G;*931T] allele with HL. The c.*168A>G change, predicted to alter mRNA folding, might be involved in HL.

© The Author 2010. Published by Oxford University Press on behalf of the British Society for Rheumatology., Objectives. B cells play an important role in the perpetuation of RA, particularly as autoantibody-producing cells. The ICs that further develop deposit in the joints and aggravate the inflammatory process. However, B-cell contribution in the very early stage of the disease remains unknown. The main goal of this work was to determine the concentration of cytokines potentially relevant for B-cell activation in serum from very early polyarthritis patients, with, Sociedade Portuguesa de Reumatologia/Schering-Plough 2005. R.A.M. and R.C. were funded by Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/30247/2006 and SFRH/BD/40513/2007, respectively. M.M.S.-C. was funded by Marie Curie Intra-European Fellowship PERG-2008-239422 and an EULAR Young Investigator Award.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint destruction. We have previously reported an increase in interleukin (IL)-1β levels in the serum of very early RA and in the synovial fluid (SF) of RA patients. Therefore, the molecular mechanisms that regulate IL-1β activation, such as caspase-1, might play a relevant role and have potential as therapeutic targets in RA.

Tese de mestrado, Bioestatística, Faculdade de Ciências, Universidade de Lisboa, 2009 Submitted by Teresa Boa (tdboa@fc.ul.pt) on 2012-03-19T13:55:10Z No. of bitstreams: 1 ulfc055990_tm_Ana_Torres.pdf: 1784009 bytes, checksum: 37c4b3ebd59f884f69346faa8b320b60 (MD5) Made available in DSpace on 2012-03-19T13:55:30Z (GMT). No. of bitstreams: 1 ulfc055990_tm_Ana_Torres.pdf: 1784009 bytes, checksum: 37c4b3ebd59f884f69346faa8b320b60 (MD5) Previous issue date: 2009

Background and objectives We have previously found in the chronic SKG mouse model of arthritis that long standing (5 and 8 months) inflammation directly leads to high collagen bone turnover, disorganisation of the collagen network, disturbed bone microstructure and degradation of bone biomechanical properties. The main goal of the present work was to study the effects of the first days of the inflammatory process on the microarchitecture and mechanical properties of bone. Methods Twenty eight Wistar adjuvant-induced arthritis (AIA) rats were monitored during 22 days after disease induction for the inflammatory score, ankle perimeter and body weight. Healthy non-arthritic rats were used as controls for comparison. After 22 days of disease progression rats were sacrificed and bone samples were collected for histomorphometrical, energy dispersive X-ray spectroscopical analysis and 3-point bending. Blood samples were also collected for bone turnover markers. Results AIA rats had an increased bone turnover (as inferred from increased P1NP and CTX1, p = 0.0010 and p = 0.0002, respectively) and this was paralleled by a decreased mineral content (calcium p = 0.0046 and phosphorus p = 0.0046). Histomorphometry showed a lower trabecular thickness (p = 0.0002) and bone volume (p = 0.0003) and higher trabecular separation (p = 0.0009) in the arthritic group as compared with controls. In addition, bone mechanical tests showed evidence of fragility as depicted by diminished values of yield stress and ultimate fracture point (p = 0.0061 and p = 0.0279, respectively) in the arthritic group. Conclusions We have shown in an AIA rat model that arthritis induces early bone high turnover, structural degradation, mineral loss and mechanical weakness.

Background The reasons for the phenotypic differences between spondyloarthritis (SpA) and rheumatoid arthritis (RA) are still unclear. Slight divergences in cytokine networks driving the pathologies might contribute to the distinct clinical manifestations and may represent new treatment opportunities. Objectives The main goal of this work was to compare serum and synovial cytokines in established SpA and RA patients. Materials and methods The concentration of a panel of cytokines was measured in the serum of SpA and RA patients, as well as in synovial fluid from SpA, RA and osteoarthritis patients. Results The authors found that SpA and RA patients shared a similar pattern of cytokine concentration, with the exception of higher levels of interleukin-21 in the synovial fluid of RA patients. Conclusions Our results suggest that although SpA and RA are chronic inflammatory diseases with clearly distinct clinical manifestations, both pathologies share a similar cytokine profile, including Th17-related cytokines.

Background The authors have previously reported an increase of interleukin (IL)-17 levels during the first weeks of rheumatoid arthritis onset. Objectives Our main goal here was to investigate whether administration of digoxin, a specific inhibitor of Th17 cells polarisation, is able to attenuate inflammation in a rat model of adjuvant-induced arthritis (AIA). Materials and methods Digoxin was administered to AIA rats in the early phase (4 days after disease induction) or in the established phase of arthritis (11 days after disease induction). The inflammatory score, paw perimeter and body weight were evaluated during the period of treatment. Rats were killed after 19 days of disease evolution and paw samples were collected for histological and immunohistochemical evaluation. Results The authors found that digoxin administration significantly suppressed joint inflammation if administrated in the early phase of disease course. The histological and immunohistochemical evaluation revealed that digoxin treatment was not efficient in inhibiting the infiltration of immune cells within the joint, but it was able to reduce local immune cells proliferation, if administrated in the early phase of arthritis. Conclusions Early inhibition of Th17 polarisation ameliorates AIA but does not inhibit immune cell infiltration into the joints.

Background Rheumatoid arthritis (RA) is a chronic inflammatory disease where the excessive secretion of pro-inflammatory cytokines plays an important role. The authors have previously reported an increase in circulating interleukin 1β (IL-1β) levels in very recent onset arthritis and in the synovial fluid of established RA patients. This observation may be explained by the continuous activation of caspase-1 that the authors observed both in early polyarthritis and in established RA patients. Therefore, pathways that regulate IL-1β, such as caspase-1 and nuclear factor-κB (NF-κB) activation, might play a relevant role in arthritis onset and drugs that target these pathways could potentially constitute promising therapeutic agents in RA. The authors have thus used a THP1 macrophage-like cell line to screen among several Food and Drug Administration approved drugs those which downregulate IL-1β and caspase-1 activation. Gambogic acid and celastrol were two of the most promising therapeutic candidates obtained by this screening. Objectives The study main goal was to investigate whether gambogic acid and celastrol administration is able to attenuate inflammation in a rat model of antigen-induced arthritis (wistar AIA rat). Materials and methods Drugs and vehicle control were administrated to arthritic wistar AIA rats after 4 days of disease induction for a period of 15 days. The inflammatory score, paw diameter and body weight were evaluated during the time of treatment. Rats were killed after 19 days of disease evolution and paw samples were collected for histology observation of erosions, pannus formation and cellular infiltration. Results The authors found that both gambogic acid and celastrol significantly suppressed inflammation in the joints in comparison with arthritic rats treated with vehicle only. Moreover, the rats treated with these drugs did not show differences in body weight or other evident side effects. The histology results revealed that gambogic acid and celastrol treated rats showed a normal joint structure with a complete abrogation of the inflammatory infiltrate. Conclusions This results suggest that both drugs have significant anti-inflammatory properties, in agreement with their reported ability to suppress NF-κB activation. Gambogic acid and celastrol might therefore constitute putative anti-inflammatory drugs with therapeutic efficacy in the treatment of inflammatory diseases, such as RA.

© 2010 Cascão et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by sustained synovitis. Recently, several studies have proposed neutrophils and Th17 cells as key players in the onset and perpetuation of this disease. The main goal of this work was to determine whether cytokines driving neutrophil and Th17 activation are dysregulated in very early rheumatoid arthritis patients with less than 6 weeks of disease duration and before treatment (VERA). Methods: Cytokines related to neutrophil and Th17 activation were quantified in the serum of VERA and established RA patients and compared with other very early arthritis (VEA) and healthy controls. Synovial fluid (SF) from RA and osteoarthritis (OA) patients was also analyzed. Results: VERA patients had increased serum levels of cytokines promoting Th17 polarization (IL-1b and IL-6), as well as IL-8 and Th17-derived cytokines (IL-17A and IL-22) known to induce neutrophil-mediated inflammation. In established RA this pattern is more evident within the SF. Early treatment with methotrexate or corticosteroids led to clinical improvement but without an impact on the cytokine pattern. Conclusions: VERA patients already display increased levels of cytokines related with Th17 polarization and neutrophil recruitment and activation, a dysregulation also found in SF of established RA. 0 Thus, our data suggest that a cytokine-milieu favoring Th17 and neutrophil activity is an early event in RA pathogenesis., This work was supported by a grant from Sociedade Portuguesa de Reumatologia/Schering-Plough 2005. RAM and RC were funded by Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/30247/2006 and SFRH/BD/40513/2007, respectively. MMS-C was funded by Marie Curie Intra-European Fellowship PERG-2008-239422 and a EULAR Young Investigator Award.

Thirty-seven patients were included. During the follow up most of the patients evolved into RA (57%). The median age of the patients of the RA-group was similar to the median age of the non-RA group (median (IQR) 47 (31-58.5) vs 43 (34-69) years, p=0.74). At the initial visit the DAS 28 in the RA group was significantly higher than in the non-RA group, as well as the VAS and the number of swollen joints. The ESR values did not differ significantly between RA and non-RA groups (Table 1).

Rheumatoid arthritis (RA) is a chronic inflammatory disease mainly characterised by synovial hyperplasia and joint destruction. Although the aetiopathology of this autoimmune disease is not completely understood, it is known that it is associated with a misregulation of both the cellular immune system and the cytokine network. Nevertheless, little is known about the blood cytokine milieu in the first few weeks of RA. To determine …

Rheumatoid arthritis (RA) is a systemic disease characterised by hyperactivation of the immune system leading to chronic inflammation and joint damage. The inflammatory environment potentiates bone resorption, modulating the balance between RANKL and osteoprotegerin (OPG). Prednisone (PDN), methotrexate (MTX), anti-tumour necrosis factor (TNF) and anti-interleukin 6 (IL6) receptor therapies reduce disease activity, but their differential biological impact …