RESUMO
PURPOSE/BACKGROUND: The aim of this study was to evaluate pencil beam scanning (PBS) proton therapy (PT) in deep inspiration breath-hold (DIBH) for mediastinal lymphoma patients, by retrospectively evaluating plan robustness to the clinical target volume (CTV) and organs at risk (OARs) on repeated CT images acquired throughout treatment. Methods: Sixteen mediastinal lymphoma patients treated with PBS-PT in DIBH were included. Treatment plans (TPs) were robustly optimized on the CTV (7 mm/4.5%). Repeated verification CTs (vCT) were acquired during the treatment course, resulting in 52 images for the entire patient cohort. The CTV and OARs were transferred from the planning CT to the vCTs with deformable image registration and the TPs were recalculated on the vCTs. Target coverage and OAR doses at the vCTs were compared to the nominal plan. Deviation in lung volume was also calculated. RESULTS: The TPs demonstrated high robust target coverage throughout treatment with D98%,CTV deviations within 2% for 14 patients and above the desired requirement of 95% for 49/52 vCTs. However, two patients did not achieve a robust dose to CTV due to poor DIBH reproducibility, with D98%,CTV at 78 and 93% respectively, and replanning was performed for one patient. Adequate OAR sparing was achieved for all patients. Total lung volume variation was below 10% for 39/52 vCTs. CONCLUSION: PBS PT in DIBH is generally a robust technique for treatment of mediastinal lymphomas. However, closely monitoring the DIBH-reproducibility during treatment is important to avoid underdosing CTV and achieve sufficient dose-sparing of the OARs.
Assuntos
Linfoma , Neoplasias do Mediastino , Terapia com Prótons , Humanos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Neoplasias do Mediastino/diagnóstico por imagem , Neoplasias do Mediastino/radioterapia , Linfoma/diagnóstico por imagem , Linfoma/radioterapiaRESUMO
OBJECTIVES: MicroRNAs (miRs) are non-translated RNA sequences that elicit negative control over protein expression. The adipose tissue (AT) is considered the major producer of miRs and inflammatory interleukin 6 (IL-6). This study aims to investigate the relationship between production of IL-6 and miRs in AT. METHODS: IL-6 gene expression was analysed in RNA extracts from subcutaneous AT of 75 patients with rheumatoid arthritis (RA), with qPCR. Genome-wide profile of human miRs (2565 miRs, 96.6%) was analysed in 35 AT samples on 3D microarray. The miR-processing proteins Dicer, Drosha and DGCR8 were analysed with qPCR. In silico prediction of protein targets for the differentially expressed (DE) miRs (p<0.05; log2FC >±0.5) was conducted by DIANA software. Seven AT samples were stimulated in vitro with IL-6 or IL-6+IL-6R antibody tocilizumab and analysed for the miR processing proteins. RESULTS: We identified 30 DE miRs between AT with high and low IL-6 mRNA, of which 26 miRs were inversely related with IL-6 levels. DE miRs were predicted to interfere in oestrogen (p=0.001), FoxO (p=0.006) and insulin (p=0.03) signalling pathways. High expression of IL-6 in AT was associated with significantly higher expression of Dicer (p=0.04) and Drosha (p=0.04), while inhibition of IL-6 signalling with tocilizumab decreased the levels of total miRs processing enzymes (p=0.003). CONCLUSIONS: IL-6 mRNA production in AT has a negative effect on the miRs expression profile and it increases miR-production capacity.
Assuntos
Artrite Reumatoide , MicroRNAs , Humanos , MicroRNAs/genética , Interleucina-6/metabolismo , Proteínas de Ligação a RNA , Artrite Reumatoide/genética , Tecido Adiposo/metabolismoRESUMO
IL-17-producing Th17 cells are implicated in the pathogenesis of rheumatoid arthritis (RA) and TNF-α, a proinflammatory cytokine in the rheumatoid joint, facilitates Th17 differentiation. Anti-TNF therapy ameliorates disease in many patients with rheumatoid arthritis (RA). However, a significant proportion of patients do not respond to this therapy. The impact of anti-TNF therapy on Th17 responses in RA is not well understood. We conducted high-throughput gene expression analysis of Th17-enriched CCR6+CXCR3-CD45RA- CD4+ T (CCR6+ T) cells isolated from anti-TNF-treated RA patients classified as responders or nonresponders to therapy. CCR6+ T cells from responders and nonresponders had distinct gene expression profiles. Proinflammatory signaling was elevated in the CCR6+ T cells of nonresponders, and pathogenic Th17 signature genes were up-regulated in these cells. Gene set enrichment analysis on these signature genes identified transcription factor USF2 as their upstream regulator, which was also increased in nonresponders. Importantly, short hairpin RNA targeting USF2 in pathogenic Th17 cells led to reduced expression of proinflammatory cytokines IL-17A, IFN-γ, IL-22, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as transcription factor T-bet. Together, our results revealed inadequate suppression of Th17 responses by anti-TNF in nonresponders, and direct targeting of the USF2-signaling pathway may be a potential therapeutic approach in the anti-TNF refractory RA.
Assuntos
Artrite Reumatoide/etiologia , Artrite Reumatoide/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Fatores Estimuladores Upstream/genética , Antirreumáticos/farmacologia , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Biomarcadores , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , RNA Interferente Pequeno/genética , Receptores CCR6/metabolismo , Receptores CXCR3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+GLCs). Spleen and joint draining lymph nodes (dLN) CD4+GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4+GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXP3+ regulatory T cells (Tregs) of thymic origin. Activation of the ß-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype.
Assuntos
Artrite , Timo , Proteínas rho de Ligação ao GTP , Animais , Fatores de Transcrição Forkhead/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Linfócitos T Reguladores , Timo/imunologia , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Rheumatoid arthritis (RA) is an inflammatory joint disease with a neurological component including depression, cognitive deficits, and pain, which substantially affect patients' quality of daily life. Insulin-like growth factor 1 receptor (IGF1R) signaling is one of the factors in RA pathogenesis as well as a known regulator of adult neurogenesis. The purpose of this study was to investigate the association between IGF1R signaling and the neurological symptoms in RA. In experimental RA, we demonstrated that arthritis induced enrichment of IBA1+ microglia in the hippocampus. This coincided with inhibitory phosphorylation of insulin receptor substrate 1 (IRS1) and up-regulation of IGF1R in the pyramidal cell layer of the cornus ammoni and in the dentate gyrus, reproducing the molecular features of the IGF1/insulin resistance. The aberrant IGF1R signaling was associated with reduced hippocampal neurogenesis, smaller hippocampus, and increased immobility of RA mice. Inhibition of IGF1R in experimental RA led to a reduction of IRS1 inhibition and partial improvement of neurogenesis. Evaluation of physical functioning and brain imaging in RA patients revealed that enhanced functional disability is linked with smaller hippocampus volume and aberrant IGF1R/IRS1 signaling. These results point to abnormal IGF1R signaling in the brain as a mediator of neurological sequelae in RA and provide support for the potentially reversible nature of hippocampal changes.
Assuntos
Artrite Reumatoide/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Inflamação/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Animais , Artrite Reumatoide/tratamento farmacológico , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Giro Denteado/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina , Masculino , Camundongos , Pessoa de Meia-Idade , Neurogênese/efeitos dos fármacos , Dor , Medição da Dor , Fosforilação , Receptores de Somatomedina/antagonistas & inibidores , Receptores de Somatomedina/metabolismo , Regulação para Cima , Adulto JovemRESUMO
OBJECTIVES: Since low insulin-like growth factor (IGF) 1 is often linked to inflammation, we analyze whether serum levels of IGF1 are associated with cardiovascular disease (CVD) in rheumatoid arthritis (RA) in a longitudinal observational study. METHODS: A CVD risk was estimated (eCVR) in 184 female RA patients (mean age 52 years) and in 132 female patients after ischemic stroke (mean age 56 years) with no rheumatic disease, using the Framingham algorithm. The median level of IGF1 divided the cohorts in IGF1high and IGF1low groups. A 5-year prospective follow-up for new CVD events was completed in all RA patients. The Mantel-Cox analysis and event-free survival curves were prepared. Unsupervised clustering of proteins within the IGF1 signaling pathway was employed to identify their association with eCVR. RESULTS: Low IGF1 resulted in a higher eCVR in RA patients (7.2% and 3.3%, p = 0.0063) and in stroke (9.3% and 7.1%, p = 0.033). RA had higher rate for new CVD events at prospective follow-up (OR 4.96, p = 0.028). Hypertension was the major risk factor associated with low IGF1 in RA and stroke. In hypertension, IGF1 was no longer responsible for intracellular activation and lost its correlation to IRS1/2 adaptor proteins. The clustering analysis confirmed that combination of low IGF1 and IRS1/2 with high IL6, insulin, and glucose predisposed to high eCVR and emphasized the functional role of serum IGF1. CONCLUSIONS: Low serum IGF1 precedes and predicts development of early CVD events in female RA patients. Hypertension and aberrant IGF1 receptor signaling are highlighted as the important contributors to IGF1-related CVD events.
Assuntos
Artrite Reumatoide/sangue , Artrite Reumatoide/complicações , Doenças Cardiovasculares/diagnóstico , Hipertensão/sangue , Hipertensão/complicações , Fator de Crescimento Insulin-Like I/metabolismo , Adulto , Idoso , Artrite Reumatoide/diagnóstico , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/complicações , Estudos de Coortes , Feminino , Humanos , Hipertensão/diagnóstico , Fator de Crescimento Insulin-Like I/análise , Estudos Longitudinais , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Fatores de Risco , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/diagnósticoRESUMO
Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6(+) germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Switching de Imunoglobulina , Imunoglobulina G/imunologia , Tirosina Quinase 3 Semelhante a fms/fisiologia , Animais , Apoptose , Regulação da Expressão Gênica , Imunoglobulina M/imunologia , Ligantes , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmócitos/imunologia , Receptores de Interleucina-4/metabolismo , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
BACKGROUND: Signalling through insulin-like growth factor 1 receptor (IGF-1R) is essential for cell survival, but may turn pathogenic in uncontrolled tissue growth in tumours. In rheumatoid arthritis (RA), the IGF-1R signalling is activated and supports expansion of the inflamed synovia. AIM: In the present study, we assess if disruption of IGF-1R signalling resolves arthritis. MATERIAL AND METHODS: Clinical associations of IGF-1R expression in leukocytes of the peripheral blood were studied in 84 RA patients. Consequences of the IGF-1R signalling inhibition for arthritis were studied in mBSA immunised Balb/c mice treated with NT157 compound promoting degradation of insulin receptor substrates. RESULTS: In RA patients, high expression of IGF-1R in leukocytes was associated with systemic inflammation as verified by higher expression of NF-kB, serum levels of IL6 and erythrocyte sedimentation rate, and higher pain perception. Additionally, phosphorylated IGF-1R and STAT3 enriched T cells infiltrate in RA synovia. Treatment with NT157 inhibited the phosphorylation of IGF-1R and STAT3 in synovia, and alleviated arthritis and joint damage in mice. It also reduced expression of IGF-1R and despaired ERK and Akt signalling in spleen T cells. This limited IL-6 production, changed RoRgt/FoxP3 balance and IL17 levels. CONCLUSION: IGF-1R signalling contributes to T cell dependent inflammation in arthritis. Inhibition of IGF-1R on the level of insulin receptor substrates alleviates arthritis by restricting IL6-dependent formation of Th17 cells and may open for new treatment strategies in RA.
Assuntos
Artrite Reumatoide/imunologia , Interleucina-6/imunologia , Receptor IGF Tipo 1/imunologia , Transdução de Sinais/imunologia , Membrana Sinovial/imunologia , Células Th17/imunologia , Adulto , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Receptor IGF Tipo 1/metabolismo , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Células Th17/metabolismo , Células Th17/patologiaRESUMO
MicroRNAs (miRs) represent a part of epigenetic control of autoimmunity gaining increasing attention in rheumatoid arthritis (RA). Since cigarette smoking plays important role in RA pathogenesis and reprograms transcriptional profile of miRNAs, we ask if the onco-protein survivin, a novel biomarker of RA, may provide a link between smoking and miRNA. Studying survivin expression in leukocytes of 144 female RA patients we observed that smoking patients had higher survivin transcription and a remarkable spreading of survivin isoforms. This was associated with restricted pattern and low production of miRs. Additionally, miRNA processing enzymes Dicer and DGRC8 were decreased in the patients with survivin isoform spreading. The direct contribution of survivin in miRs biogenesis was confirmed by a massive increase of miRs production following inhibition of survivin in leukocyte cultures. Dicer is shown to mediate these effects of survivin. Chromatin immunoprecipitation analysis demonstrated binding of survivin to the Dicer promoter region. Dicer expression increased 5-folds following survivin inhibition. Taken together, this study presents experimental evidence of a novel cellular function of survivin, control of miRs biogenesis. Up-regulation of survivin in smokers suggests its role as effector of the adverse epigenetic control in RA.
Assuntos
Artrite Reumatoide/genética , Fumar Cigarros/genética , Proteínas Inibidoras de Apoptose/genética , MicroRNAs/genética , Ativação Transcricional , Transcriptoma , Adulto , Idoso , Artrite Reumatoide/etiologia , Fumar Cigarros/efeitos adversos , Feminino , Humanos , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Fumantes , SurvivinaRESUMO
CD8+ T cells have an emerging role in RA. Resent research indicates a causal relationship between the non-exhausted state of CD8+ T cells, defined by lost function of PD-1, and development of arthritis. We investigated how smoking contributes to the non-exhausted phenotype of CD8+ T cells and cause survivin release to serum. We compared serum survivin levels between smokers and non-smokers in 252 RA and 168 healthy subjects. Nicotine effects on CD8+ T cells were studied in peripheral blood of smoking women, bone marrow of nicotine treated mice and in sorted CD8 spleen cells in vitro using flow cytometry and quantitative PCR. Smoking increased the frequency of survivin release in serum of healthy women (OR 3.64, p = 0.025) and in RA patients (OR 1.98, p = 0.039). CD8+ T cells of smokers gained a non-exhausted PD-1 deficient phenotype. Expression of the cytotoxic marker CD107 correlated to survivin levels in serum. In the experimental setting, nicotine exposure led to an accumulation of non-exhausted PD-1-IL-7R+ CD8+ T cells in the bone marrow that is abundant with survivin producing cells. The production of the cytolytic protein perforin in bone marrow correlated to serum survivin levels. In vitro stimulation of nicotinic receptors on murine CD8+ T cells induced repressive transcription factors T-bet and Blimp-1 in support of the non-exhausted phenotype. We conclude that nicotine contributes to autoimmunity by supporting the non-exhausted state of CD8+ T cells resulting in the release of survivin. This presents a new mechanism by which smoking may contribute to the pathogenesis of RA.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Proteínas Inibidoras de Apoptose/biossíntese , Ativação Linfocitária/imunologia , Fumar , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Artrite Reumatoide/genética , Biomarcadores , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Humanos , Imunofenotipagem , Proteínas Inibidoras de Apoptose/sangue , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Nicotina/farmacologia , Fenótipo , Receptor de Morte Celular Programada 1/deficiência , Receptor de Morte Celular Programada 1/metabolismo , Survivina , Linfócitos T Citotóxicos/efeitos dos fármacos , Adulto JovemRESUMO
Despite the predominance of female patients and uncommon obesity, rheumatoid arthritis (RA) is tightly connected to increased cardiovascular morbidity. The aim of this study was to investigate transcriptional activity in the subcutaneous white adipose tissue (WAT) with respect to this disproportionate cardiovascular risk (CVR) in RA. CVR was estimated in 182 female patients, using the modified Systematic Coronary Risk Evaluation scale, and identified 93 patients with increased CVR. The overall transcriptional activity in WAT was significantly higher in patients with CVR and was presented by higher serum levels of WAT products leptin, resistin and IL-6 (all, p < 0.001). CVR was associated with high WAT-specific transcription of the signal transducer and activator of transcription 3 (STAT3) and the nuclear factor NF-kappa-B p65 subunit (RELA), and with high transcription of serine-threonine kinase B (AKT1) in leukocytes. These findings suggest Interleukin 6 (IL-6) and leptin take part in WAT-specific activation of STAT3. The binary logistic regression analysis confirmed an independent association of CVR with IL-6 in serum, and with STAT3 in WAT. The study shows an association of CVR with transcriptional activity in WAT in female RA patients. It also emphasizes the importance of STAT3 regulatory circuits for WAT-related CVR in RA.
Assuntos
Artrite Reumatoide/metabolismo , Doenças Cardiovasculares/metabolismo , Fator de Transcrição STAT3/metabolismo , Gordura Subcutânea/metabolismo , Adulto , Artrite Reumatoide/epidemiologia , Biomarcadores/metabolismo , Doenças Cardiovasculares/epidemiologia , Estudos de Casos e Controles , Feminino , Humanos , Interleucina-6/sangue , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/genéticaRESUMO
T-helper cells producing interleukin (IL)-17A and IL-17F cytokines (Th17 cells) are considered the source of autoimmunity in rheumatoid arthritis (RA). In this study, we characterized specific pathogenic features of Th17 cells in RA. By using nano-string technology, we analyzed transcription of 419 genes in the peripheral blood CCR6(+)CXCR3(-) CD4(+) cells of 14 RA patients and 6 healthy controls and identified 109 genes discriminating Th17 cells of RA patients from the controls. Th17 cells of RA patients had an aggressive pathogenic profile and in addition to signature cytokines IL-17, IL-23 and IL-21, and transcriptional regulators RAR-related orphan receptor gamma of T cells (RORγt) and Janus kinase 2 (JAK2), they produced high levels of IL-23R, C-C chemokine ligand type 20 (CCL20), granulocyte-monocyte colony-stimulating factor (GM-CSF ) and transcription factor Tbet required for synovial homing. We showed that Th17 cells are enriched with Helios-producing Foxp3- and IL2RA-deficient cells, indicating altered regulatory profile. The follicular T-helper (Tfh) cells presented a functional profile of adaptor molecules, transcriptional regulator Bcl-6 and B-cell activating cytokines IL-21, IL-31 and leukemia inhibitory factor (LIF ). We observed that anti-tumor necrosis factor (TNF) treatment had a limited effect on the transcription signature of Th17 cells. Patients in remission retained the machinery of receptors (IL-23R and IL-1R1), proinflammatory cytokines (IL-17F, IL-23, IL-21 and TNF ) and adaptor molecules (C-X-C chemokine receptor 5 [CXCR5] and cytotoxic T-lymphocyte-associated protein 4 [CTLA-4]), essential for efficient transdifferentiation and accumulation of Th17 cells. This study convincingly shows that the peripheral blood CCR6(+)CXCR3(-) CD4(+) cells of RA patients harbor pathogenic subsets of Th17 and Tfh cells, which may transdifferentiate from Tregs and contribute to perpetuation of the disease.
Assuntos
Artrite Reumatoide/genética , Transdiferenciação Celular/genética , Células Th17/imunologia , Fator de Necrose Tumoral alfa/administração & dosagem , Adulto , Idoso , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Feminino , Humanos , Interleucina-17/imunologia , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Células Th17/patologia , Transcriptoma/genética , Transcriptoma/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: S100A4 is a Ca-binding protein that regulates cell growth, survival, and motility. The abundant expression of S100A4 in rheumatiod arthritis contributes to the invasive growth of joint tissue and to bone damage. In the present study, we analysed the role of S100A4 in bone homeostasis. METHODS: Peripheral quantitative computed tomography and histomorphometric analysis were performed in mice lacking the entire S100A4 protein (S100A4KO) and in wild-type (WT) counterparts treated with shRNA-lentiviral constructs targeting S100A4 (S100A4-shRNA). Control groups consisted of sex-matched WT counterparts and WT mice treated with a non-targeting RNA construct. RESULTS: S100A4 deficiency was associated with higher trabecular and cortical bone mass, increased number and thickness of trabeculi combined with larger periosteal circumference and higher predicted bone strength. S100A4 inhibition by shRNA led to an increase in cortical bone in WT mice. S100A4-deficieny was associated with a reduced number of functional osteoclasts. S100A4KO and S100A4-shRNA-treated bone marrow progenitors gave rise to a large number of small TRAP+ cells with few nuclei and few pseudopodial processes. Poor osteoclastogenesis and the low resorptive capacity in S100A4Ko mice may be linked to low levels of surface integrins, impaired adhesion capacity, and poor multinucleation in S100A4-deficient osteoclasts, as well as a low content of proteolytic enzymes cathepsin K and MMP3 and MMP9 to break down the organic matrix. CONCLUSION: S100A4 emerges as a negative regulator of bone metabolism potentially responsible for the excessive bone turnover in conditions marked by high levels of S100A4 protein, such as inflammation and rheumatoid arthritis.
Assuntos
Reabsorção Óssea/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Proteínas S100/metabolismo , Animais , Remodelação Óssea , Reabsorção Óssea/complicações , Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Membrana Celular/metabolismo , Forma Celular , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Tamanho do Órgão , Osteólise/complicações , Osteólise/patologia , Osteólise/fisiopatologia , Fenótipo , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/deficiênciaRESUMO
Metal objects in the body such as hip prostheses cause artifacts in CT images. When CT images degraded by artifacts are used for treatment planning of radiotherapy, the artifacts can yield inaccurate dose calculations and, for particle beams, erroneous penetration depths. A metal artifact reduction software (O-MAR) installed on a Philips Brilliance Big Bore CT has been tested for applications in treatment planning of proton radiotherapy. Hip prostheses mounted in a water phantom were used as test objects. Images without metal objects were acquired and used as reference data for the analysis of artifact-affected regions outside of the metal objects in both the O-MAR corrected and the uncorrected images. Water equivalent thicknesses (WET) based on proton stopping power data were calculated to quantify differences in the calculated proton beam penetration for the different image sets. The WET to a selected point of interest between the hip prostheses was calculated for several beam directions of clinical relevance. The results show that the calculated differences in WET relative to the reference case were decreased when the O-MAR algorithm was applied. WET differences up to 2.0 cm were seen in the uncorrected case while, for the O-MAR corrected case, the maximum difference was decreased to 0.4 cm. The O-MAR algorithm can significantly improve the accuracy in proton range calculations. However, there are some residual effects, and the use of proton beam directions along artifact streaks should only be used with caution and appropriate margins.
Assuntos
Algoritmos , Artefatos , Prótese de Quadril , Metais , Intensificação de Imagem Radiográfica/métodos , Planejamento da Radioterapia Assistida por Computador/métodos , Tomografia Computadorizada por Raios X/métodos , Humanos , Imagens de Fantasmas , Terapia com Prótons , Radioterapia de Alta Energia/métodos , Radioterapia Guiada por Imagem/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Background and Purpose: Hippocampal-sparing (HS) is a method that can potentially reduce late cognitive complications for pediatric medulloblastoma (MB) patients treated with craniospinal proton therapy (PT). The aim of this study was to investigate robustness and dosimetric plan verification of pencil beam scanning HS PT. Materials and Methods: HS and non-HS PT plans for the whole brain part of craniospinal treatment were created for 15 pediatric MB patients. A robust evaluation of the plans was performed. Plans were recalculated in a water phantom and measured field-by-field using an ion chamber detector at depths corresponding to the central part of hippocampi. All HS and non-HS fields were measured with the standard resolution of the detector and in addition 16 HS fields were measured with high resolution. Measured and planned dose distributions were compared using gamma evaluation. Results: The median mean hippocampus dose was reduced from 22.9 Gy (RBE) to 8.9 Gy (RBE), while keeping CTV V95% above 95 % for all nominal HS plans. HS plans were relatively robust regarding hippocampus mean dose, however, less robust regarding target coverage and maximum dose compared to non-HS plans. For standard resolution measurements, median pass rates were 99.7 % for HS and 99.5 % for non-HS plans (p < 0.001). For high-resolution measurements, median pass rates were 100 % in the hippocampus region and 98.2 % in the surrounding region. Conclusions: A substantial reduction of dose in the hippocampus region appeared feasible. Dosimetric accuracy of HS plans was comparable to non-HS plans and agreed well with planned dose distribution in the hippocampus region.
RESUMO
RAS and RHO proteins, which contribute to tumorigenesis and metastasis, undergo posttranslational modification with an isoprenyl lipid by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase-I (GGTase-I). Inhibitors of FTase and GGTase-I were developed to block RAS-induced malignancies, but their utility has been difficult to evaluate because of off-target effects, drug resistance, and toxicity. Moreover, the impact of FTase deficiency and combined FTase/GGTase-I deficiency has not been evaluated with genetic approaches. We found that inactivation of FTase eliminated farnesylation of HDJ2 and H-RAS, prevented H-RAS targeting to the plasma membrane, and blocked proliferation of primary and K-RAS(G12D)-expressing fibroblasts. FTase inactivation in mice with K-RAS-induced lung cancer reduced tumor growth and improved survival, similar to results obtained previously with inactivation of GGTase-I. Simultaneous inactivation of FTase and GGTase-I markedly reduced lung tumors and improved survival without apparent pulmonary toxicity. These data shed light on the biochemical and therapeutic importance of FTase and suggest that simultaneous inhibition of FTase and GGTase-I could be useful in cancer therapeutics.
Assuntos
Transformação Celular Neoplásica/metabolismo , Dimetilaliltranstransferase/metabolismo , Neoplasias Pulmonares/enzimologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Alelos , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Dimetilaliltranstransferase/deficiência , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Knockout , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Objective: Activation of Rho-GTPases in macrophages causes inflammation and severe arthritis in mice. In this study, we explore if Rho-GTPases define the joint destination of pathogenic leukocytes, the mechanism by which they perpetuate rheumatoid arthritis (RA), and how JAK inhibition mitigates these effects. Methods: CD14+ cells of 136 RA patients were characterized by RNA sequencing and cytokine measurement to identify biological processes and transcriptional regulators specific for CDC42 hiCD14+ cells, which were summarized in a metabolic signature (MetSig). The effect of hypoxia and IFN-γ signaling on the metabolic signature of CD14+ cells was assessed experimentally. To investigate its connection with joint inflammation, the signature was translated into the single-cell characteristics of CDC42 hi synovial tissue macrophages. The sensitivity of MetSig to the RA disease activity and the treatment effect were assessed experimentally and clinically. Results: CDC42 hiCD14+ cells carried MetSig of genes functional in the oxidative phosphorylation and proteasome-dependent cell remodeling, which correlated with the cytokine-rich migratory phenotype and antigen-presenting capacity of these cells. Integration of CDC42 hiCD14+ and synovial macrophages marked with MetSig revealed the important role of the interferon-rich environment and immunoproteasome expression in the homeostasis of these pathogenic macrophages. The CDC42 hiCD14+ cells were targeted by JAK inhibitors and responded with the downregulation of immunoproteasome and MHC-II molecules, which disintegrated the immunological synapse, reduced cytokine production, and alleviated arthritis. Conclusion: This study shows that the CDC42-related MetSig identifies the antigen-presenting CD14+ cells that migrate to joints to coordinate autoimmunity. The accumulation of CDC42 hiCD14+ cells discloses patients perceptive to the JAKi treatment.
Assuntos
Artrite Reumatoide , Complexo de Endopeptidases do Proteassoma , Animais , Camundongos , Homeostase , Proteínas rho de Ligação ao GTP , Inflamação , CitocinasRESUMO
Objective: Insulin-like growth factor 1 receptor (IGF1R) acts at the crossroad between immunity and cancer, being an attractive therapeutic target in these areas. IGF1R is broadly expressed by antigen-presenting cells (APC). Using mice immunised with the methylated albumin from bovine serum (BSA-immunised mice) and human CD14+ APCs, we investigated the role that IGF1R plays during adaptive immune responses. Methods: The mBSA-immunised mice were treated with synthetic inhibitor NT157 or short hairpin RNA to inhibit IGF1R signalling, and spleens were analysed by immunohistology and flow cytometry. The levels of autoantibody and cytokine production were measured by microarray or conventional ELISA. The transcriptional profile of CD14+ cells from blood of 55 patients with rheumatoid arthritis (RA) was analysed with RNA-sequencing. Results: Inhibition of IGF1R resulted in perifollicular infiltration of functionally compromised S256-phosphorylated FoxO1+ APCs, and an increased frequency of IgM+CD21+ B cells, which enlarged the marginal zone (MZ). Enlargement of MHCII+CD11b+ APCs ensured favourable conditions for their communication with IgM+ B cells in the MZ. The reduced expression of ICOSL and CXCR5 by APCs after IGF1R inhibition led to impaired T cell control, which resulted in autoreactivity of extra-follicular B cells and autoantibody production. In the clinical setting, the low expression of IGF1R on CD14+ APCs was associated with an involuted FOXO pathway, non-inflammatory cell metabolism and a high IL10 production characteristic for tolerogenic macrophages. Furthermore, autoantibody positivity was associated with low IGF1R signalling in CD14+ APCs. Conclusions: In experimental model and in patient material, this study demonstrates that IGF1R plays an important role in preventing autoimmunity. The study raises awareness of that immune tolerance may be broken during therapeutic IGF1R targeting.
Assuntos
Neoplasias , Transdução de Sinais , Animais , Humanos , Tolerância Imunológica , Imunoglobulina M , Camundongos , Receptor IGF Tipo 1 , Tolerância a Antígenos PrópriosRESUMO
Proper physiological functioning of any cell type requires ordered chromatin organization. In this context, cohesin complex performs important functions preventing premature separation of sister chromatids after DNA replication. In partnership with CCCTC-binding factor, it ensures insulator activity to organize enhancers and promoters within regulatory chromatin. Homozygous mutations and dysfunction of individual cohesin proteins are embryonically lethal in humans and mice, which limits in vivo research work to embryonic stem cells and progenitors. Conditional alleles of cohesin complex proteins have been generated to investigate their functional roles in greater detail at later developmental stages. Thus, genome regulation enabled by action of cohesin proteins is potentially crucial in lineage cell development, including immune homeostasis. In this review, we provide current knowledge on the role of cohesin complex in leukocyte maturation and adaptive immunity. Conditional knockout and shRNA-mediated inhibition of individual cohesin proteins in mice demonstrated their importance in haematopoiesis, adipogenesis and inflammation. Notably, these effects occur rather through changes in transcriptional gene regulation than through expected cell cycle defects. This positions cohesin at the crossroad of immune pathways including NF-kB, IL-6, and IFNγ signaling. Cohesin proteins emerged as vital regulators at early developmental stages of thymocytes and B cells and after antigen challenge. Human genome-wide association studies are remarkably concordant with these findings and present associations between cohesin and rheumatoid arthritis, multiple sclerosis and HLA-B27 related chronic inflammatory conditions. Furthermore, bioinformatic prediction based on protein-protein interactions reveal a tight connection between the cohesin complex and immune relevant processes supporting the notion that cohesin will unearth new clues in regulation of autoimmunity.
Assuntos
Cromatina , Estudo de Associação Genômica Ampla , Animais , Autoimunidade/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Camundongos , CoesinasRESUMO
In this study, we explore the role of nuclear survivin in maintaining the effector phenotype of IFNγ-producing T cells acting through the transcriptional control of glucose utilization. High expression of survivin in CD4+T cells was associated with IFNγ-dependent phenotype and anaerobic glycolysis. Transcriptome of CD4+ cells and sequencing of survivin-bound chromatin showed that nuclear survivin had a genome-wide and motif-specific binding to regulatory regions of the genes controlling cell metabolism. Survivin coprecipitates with transcription factors IRF1 and SMAD3, which repressed the transcription of the metabolic check-point enzyme phosphofructokinase 2 gene PFKFB3 and promoted anaerobic glycolysis. Combining transcriptome analyses of CD4+ cells and functional studies in glucose metabolism, we demonstrated that the inhibition of survivin reverted PFKFB3 production, inhibited glucose uptake, and reduces interferon effects in CD4+ cells. These results present a survivin-dependent mechanism in coordinating the metabolic adaptation of CD4+T cells and propose an attractive strategy to counteract IFNγ-dependent inflammation in autoimmunity.