RESUMO
BACKGROUND: Data are lacking to describe gene expression-based breast cancer intrinsic subtype patterns for population-based patient groups. METHODS: We studied a diverse cohort of women with breast cancer from the Life After Cancer Epidemiology and Pathways studies. RNA was extracted from 1 mm punches from fixed tumor tissue. Quantitative reverse-transcriptase PCR was conducted for the 50 genes that comprise the PAM50 intrinsic subtype classifier. RESULTS: In a subcohort of 1,319 women, the overall subtype distribution based on PAM50 was 53.1% luminal A, 20.5% luminal B, 13.0% HER2-enriched, 9.8% basal-like, and 3.6% normal-like. Among low-risk endocrine-positive tumors (i.e., estrogen and progesterone receptor positive by immunohistochemistry, HER2 negative, and low histologic grade), only 76.5% were categorized as luminal A by PAM50. Continuous-scale luminal A, luminal B, HER2-enriched, and normal-like scores from PAM50 were mutually positively correlated. Basal-like score was inversely correlated with other subtypes. The proportion with non-luminal A subtype decreased with older age at diagnosis, P Trend < 0.0001. Compared with non-Hispanic Whites, African American women were more likely to have basal-like tumors, age-adjusted OR = 4.4 [95% confidence intervals (CI), 2.3-8.4], whereas Asian and Pacific Islander women had reduced odds of basal-like subtype, OR = 0.5 (95% CI, 0.3-0.9). CONCLUSIONS: Our data indicate that over 50% of breast cancers treated in the community have luminal A subtype. Gene expression-based classification shifted some tumors categorized as low risk by surrogate clinicopathologic criteria to higher-risk subtypes. IMPACT: Subtyping in a population-based cohort revealed distinct profiles by age and race.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Carcinoma Basocelular/genética , Regulação Neoplásica da Expressão Gênica , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Carcinoma Basocelular/patologia , Estudos de Coortes , Receptor alfa de Estrogênio/metabolismo , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Grupos Raciais , Receptor ErbB-2/metabolismo , Receptores de Progesterona/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
An index that assesses the multidimensional components of the diet across the lifecycle is useful in describing diet quality. The purpose of this study was to use the Healthy Eating Index-2005, a measure of diet quality in terms of conformance to the 2005 Dietary Guidelines for Americans, to describe the diet quality of Americans by varying sociodemographic characteristics in order to provide insight as to where diets need to improve. The Healthy Eating Index-2005 scores were estimated using 1 day of dietary intake data provided by participants in the 2003-2004 National Health and Nutrition Examination Survey. Mean daily intakes of foods and nutrients, expressed per 1,000 kilocalories, were estimated using the population ratio method and compared with standards that reflect the 2005 Dietary Guidelines for Americans. Participants included 3,286 children (2 to 17 years), 3,690 young and middle-aged adults (18 to 64 years), and 1,296 older adults (65+ years). Results are reported as percentages of maximum scores and tested for significant differences (P ≤ 0.05) by age, sex, race/ethnicity, income, and education levels. Children and older adults had better-quality diets than younger and middle-aged adults; women had better-quality diets than men; Hispanics had better-quality diets than blacks and whites; and diet quality of adults, but not children, generally improved with income level, except for sodium. The diets of Americans, regardless of socioeconomic status, are far from optimal. Problematic dietary patterns were found among all sociodemographic groups. Major improvements in the nutritional health of the American public can be made by improving eating patterns.
Assuntos
Dieta , Escolaridade , Etnicidade/estatística & dados numéricos , Política Nutricional , Adolescente , Adulto , Distribuição por Idade , Idoso , Criança , Pré-Escolar , Estudos Transversais , Dieta/economia , Dieta/etnologia , Dieta/normas , Feminino , Indicadores Básicos de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Inquéritos Nutricionais , Distribuição por Sexo , Fatores Socioeconômicos , Estados Unidos , Adulto JovemRESUMO
BACKGROUND: This work tests the hypothesis that bladder instillation with vascular endothelial growth factor (VEGF) modulates sensory and motor nerve plasticity, and, consequently, bladder function and visceral sensitivity.In addition to C57BL/6J, ChAT-cre mice were used for visualization of bladder cholinergic nerves. The direct effect of VEGF on the density of sensory nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) and cholinergic nerves (ChAT) was studied one week after one or two intravesical instillations of the growth factor.To study the effects of VEGF on bladder function, mice were intravesically instilled with VEGF and urodynamic evaluation was assessed. VEGF-induced alteration in bladder dorsal root ganglion (DRG) neurons was performed on retrogradly labeled urinary bladder afferents by patch-clamp recording of voltage gated Na+ currents. Determination of VEGF-induced changes in sensitivity to abdominal mechanostimulation was performed by application of von Frey filaments. RESULTS: In addition to an overwhelming increase in TRPV1 immunoreactivity, VEGF instillation resulted in an increase in ChAT-directed expression of a fluorescent protein in several layers of the urinary bladder. Intravesical VEGF caused a profound change in the function of the urinary bladder: acute VEGF (1 week post VEGF treatment) reduced micturition pressure and longer treatment (2 weeks post-VEGF instillation) caused a substantial reduction in inter-micturition interval. In addition, intravesical VEGF resulted in an up-regulation of voltage gated Na(+) channels (VGSC) in bladder DRG neurons and enhanced abdominal sensitivity to mechanical stimulation. CONCLUSIONS: For the first time, evidence is presented indicating that VEGF instillation into the mouse bladder promotes a significant increase in peripheral nerve density together with alterations in bladder function and visceral sensitivity. The VEGF pathway is being proposed as a key modulator of neural plasticity in the pelvis and enhanced VEGF content may be associated with visceral hyperalgesia, abdominal discomfort, and/or pelvic pain.
Assuntos
Neurônios Motores/fisiologia , Plasticidade Neuronal/fisiologia , Nervos Periféricos/fisiologia , Células Receptoras Sensoriais/fisiologia , Bexiga Urinária/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vísceras/fisiologia , Administração Intravesical , Animais , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/fisiologia , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/metabolismo , Nervos Periféricos/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/metabolismo , Bexiga Urinária/inervação , Bexiga Urinária/metabolismo , Micção/fisiologia , Vísceras/inervação , Vísceras/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
BACKGROUND: Many methodologies have been used in research to identify the "intrinsic" subtypes of breast cancer commonly known as Luminal A, Luminal B, HER2-Enriched (HER2-E) and Basal-like. The PAM50 gene set is often used for gene expression-based subtyping; however, surrogate subtyping using panels of immunohistochemical (IHC) markers are still widely used clinically. Discrepancies between these methods may lead to different treatment decisions. METHODS: We used the PAM50 RT-qPCR assay to expression profile 814 tumors from the GEICAM/9906 phase III clinical trial that enrolled women with locally advanced primary invasive breast cancer. All samples were scored at a single site by IHC for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu (HER2) protein expression. Equivocal HER2 cases were confirmed by chromogenic in situ hybridization (CISH). Single gene scores by IHC/CISH were compared with RT-qPCR continuous gene expression values and "intrinsic" subtype assignment by the PAM50. High, medium, and low expression for ESR1, PGR, ERBB2, and proliferation were selected using quartile cut-points from the continuous RT-qPCR data across the PAM50 subtype assignments. RESULTS: ESR1, PGR, and ERBB2 gene expression had high agreement with established binary IHC cut-points (area under the curve (AUC) ≥ 0.9). Estrogen receptor positivity by IHC was strongly associated with Luminal (A and B) subtypes (92%), but only 75% of ER negative tumors were classified into the HER2-E and Basal-like subtypes. Luminal A tumors more frequently expressed PR than Luminal B (94% vs 74%) and Luminal A tumors were less likely to have high proliferation (11% vs 77%). Seventy-seven percent (30/39) of ER-/HER2+ tumors by IHC were classified as the HER2-E subtype. Triple negative tumors were mainly comprised of Basal-like (57%) and HER2-E (30%) subtypes. Single gene scoring for ESR1, PGR, and ERBB2 was more prognostic than the corresponding IHC markers as shown in a multivariate analysis. CONCLUSIONS: The standard immunohistochemical panel for breast cancer (ER, PR, and HER2) does not adequately identify the PAM50 gene expression subtypes. Although there is high agreement between biomarker scoring by protein immunohistochemistry and gene expression, the gene expression determinations for ESR1 and ERBB2 status was more prognostic.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Biomarcadores Tumorais/metabolismo , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Ensaios Clínicos como Assunto , Análise por Conglomerados , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Limite de Detecção , Análise Multivariada , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Curva ROC , Receptor ErbB-2/metabolismo , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
BACKGROUND: This work tests the hypothesis that increased levels of vascular endothelial growth factor (VEGF) observed during bladder inflammation modulates nerve plasticity. METHODS: Chronic inflammation was induced by intravesical instillations of Bacillus Calmette-Guérin (BCG) into the urinary bladder and the density of nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) or pan-neuronal marker PGP9.5 was used to quantify alterations in peripheral nerve plasticity. Some mice were treated with B20, a VEGF neutralizing antibody to reduce the participation of VEGF. Additional mice were treated systemically with antibodies engineered to specifically block the binding of VEGF to NRP1 (anti-NRP1B) and NRP2 (NRP2B), or the binding of semaphorins to NRP1 (anti-NRP1 A) to diminish activity of axon guidance molecules such as neuropilins (NRPs) and semaphorins (SEMAs). To confirm that VEGF is capable of inducing inflammation and neuronal plasticity, another group of mice was instilled with recombinant VEGF165 or VEGF121 into the urinary bladder. RESULTS: The major finding of this work was that chronic BCG instillation resulted in inflammation and an overwhelming increase in both PGP9.5 and TRPV1 immunoreactivity, primarily in the sub-urothelium of the urinary bladder. Treatment of mice with anti-VEGF neutralizing antibody (B20) abolished the effect of BCG on inflammation and nerve density.NRP1A and NRP1B antibodies, known to reduce BCG-induced inflammation, failed to block BCG-induced increase in nerve fibers. However, the NRP2B antibody dramatically potentiated the effects of BCG in increasing PGP9.5-, TRPV1-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactivity (IR). Finally, instillation of VEGF121 or VEGF165 into the mouse bladder recapitulated the effects of BCG and resulted in a significant inflammation and increase in nerve density. CONCLUSIONS: For the first time, evidence is being presented supporting that chronic BCG instillation into the mouse bladder promotes a significant increase in peripheral nerve density that was mimicked by VEGF instillation. Effects of BCG were abolished by pre-treatment with neutralizing VEGF antibody. The present results implicate the VEGF pathway as a key modulator of inflammation and nerve plasticity, introduces a new animal model for investigation of VEGF-induced nerve plasticity, and suggests putative mechanisms underlying this phenomenon.
Assuntos
Vacina BCG/farmacologia , Inflamação/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Vacina BCG/imunologia , Calcitonina/imunologia , Calcitonina/metabolismo , Feminino , Inflamação/induzido quimicamente , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/imunologia , Neuropilina-1/imunologia , Neuropilina-1/metabolismo , Neuropilina-2/imunologia , Neuropilina-2/metabolismo , Neuropilinas/efeitos dos fármacos , Neuropilinas/imunologia , Neuropilinas/metabolismo , Precursores de Proteínas/imunologia , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/farmacologia , Semaforinas/imunologia , Semaforinas/metabolismo , Transdução de Sinais , Substância P/imunologia , Substância P/metabolismo , Canais de Cátion TRPV/imunologia , Canais de Cátion TRPV/metabolismo , Ubiquitina Tiolesterase/imunologia , Ubiquitina Tiolesterase/metabolismo , Bexiga Urinária/imunologia , Bexiga Urinária/patologia , Urotélio/efeitos dos fármacos , Urotélio/imunologia , Urotélio/metabolismoRESUMO
Recent findings indicate that VEGF receptors and coreceptors (neuropilins; NRP) are expressed on nonendothelial cells in human bladder urothelium, in one human bladder cancer cell line (J82), and in the mouse bladder urothelium. In addition, VEGFR1, VEGFR2, NRP1, and NRP2 expressions were upregulated in animal models of chronic bladder inflammation induced by four weekly instillations of protease-activated receptors (PAR)-activating peptides or bacillus Calmette-Guérin (BCG) into the mouse bladder. Here, we used four weekly instillations of BCG as a model for chronic bladder inflammation to further investigate whether VEGF receptors and NRPs play a role in the migration of inflammatory cells and inflammation-induced lymphangiogenesis and angiogenesis. For this purpose, we used neutralizing antibodies that were engineered to specifically block the binding of VEGF to NRP (anti-NRP1(B)) and the binding of semaphorins to NRP (anti-NRP1(A)). C57BL/6 mice received intraperitoneal injections of PBS, anti-NRP1(A)- or anti-NRP1(B)-neutralizing antibodies and then were challenged chronically with intravesical PBS or BCG. At the end of chronic challenge period, a fluorescent internalizable tracer, scVEGF/Cy5.5, was administered to all mice and near-infrared fluorescence images were obtained in vivo and in real time. BCG increased the overall accumulation of scVEGF/Cy5.5 in the urinary bladder urothelium and inflammatory cells. In addition, BCG increased the density of blood and lymphatic vessels concomitantly with an upregulation of NRP2 expression in lymphatic vessels. Treatment of the mice with NRP1-neutralizing antibodies dramatically reduced scVEGF/Cy5.5 uptake, polymorphonuclear (myeloperoxidase-positive cells) and dendritic cell (CD11c-positive cells) infiltration, and decreased the overall density of BCG-induced blood and lymphatic vessels. These results implicate NRPs as critical in vivo regulators of the vascular and inflammatory responses to the intravesical administration of BCG.
Assuntos
Cistite/fisiopatologia , Neuropilina-1/fisiologia , Neuropilinas/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Transdução de Sinais/fisiologia , Animais , Vacina BCG , Movimento Celular/imunologia , Cistite/induzido quimicamente , Feminino , Humanos , Linfangiogênese/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/fisiopatologia , Neuropilina-1/imunologia , Bexiga Urinária/irrigação sanguínea , Bexiga Urinária/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Interstitial cystitis (IC) is a chronic and painful bladder syndrome of unknown cause with no reliable biological marker or effective therapy. Vascular endothelial growth factor (VEGF), which plays a key role in bladder inflammation, is closely associated with the vascular alterations observed in patients with IC. However, our recent findings of VEGF receptors (VEGF-Rs) and VEGF coreceptors on nonendothelial cells in human and mouse urothelium suggest that additional VEGF targets and functions are possible in IC bladders. We report here that VEGF-Rs and coreceptors (neuropilins; NRP) are strongly expressed in both the human bladder urothelium and in the human bladder cancer cell line (J82) and that the expression of NRP2 and VEGF-R1 is significantly downregulated in IC compared with control subjects. In addition, treatment of J82 cells with bacillus Calmette-Guérin (BCG), a novel treatment strategy for IC, upregulates the messages for NRPs and VEGF-Rs. Furthermore, intravesical instillation of an internalizable VEGF fluorescent tracer (scVEGF/Cy5.5) into mouse urinary bladders results in a marked ligand accumulation in the urothelium and bladder parenchyma, indicating that urothelial VEGF-Rs are functionally active and capable of ligand interaction and internalization. Our results suggest that the VEGF pathway is altered in IC, that urinary VEGF may gain access to the bladder wall via these receptors, and that BCG treatment may replenish the missing VEGF-Rs/NRP receptors. Together, these results suggest that levels of NRPs, VEGF-Rs, and VEGF are new putative markers for the diagnosis of IC and that modulating these receptors can be exploited as therapeutic strategies.
Assuntos
Cistite Intersticial/fisiopatologia , Neuropilinas/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , Linhagem Celular , Cistite Intersticial/genética , Feminino , Humanos , Inflamação/fisiopatologia , Masculino , Camundongos , Reação em Cadeia da Polimerase , Bexiga Urinária/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Recent evidence supports a role for vascular endothelium growth factor (VEGF) signaling in bladder inflammation. However, it is not clear what bladder cells are targeted by VEGF. Therefore, we determined the nature of cells responding to VEGF in normal and inflamed bladders by tagging such cells in vivo with a targeted fluorescent tracer, scVEGF/Cy, an engineered single-chain VEGF labeled with Cy5.5 dye, which identifies cells with accessible and functionally active VEGF receptors. Inflammation was induced by intravesical instillation of PAR-activating peptides or BCG. In vivo NIRF imaging with intravenously injected scVEGF/Cy revealed accumulation of the tracer in the control mouse bladder and established that inflammation increased the steady-state levels of tracer uptake. Ex vivo colocalization of Cy5.5 dye revealed that in normal and at a higher level in inflamed bladder, accumulation of scVEGF/Cy occurs in both urothelial and ganglial cells, expressing VEGF receptors VEGFR-1 and VEGFR-2, as well as VEGF coreceptors neuropilins (NRP) NRP1 and NRP2. PCR results indicate that the messages for VEGF-Rs and NRPs are present in the bladder mucosa and ChIP/QPCR analysis indicated that inflammation induced upregulation of genes encoding VEGFRs and NRPs. Our results strongly suggest new and blossoming VEGF-driven processes in bladder urothelial cells and ganglia in the course of inflammation. We expect that molecular imaging of the VEGF pathway in the urinary tract by receptor-mediated cell tagging in vivo will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of bladder-targeting drugs and treatments.
Assuntos
Cistite/metabolismo , Neuropilinas/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular/biossíntese , Bexiga Urinária/metabolismo , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neurônios/metabolismo , Neuropilina-1/biossíntese , Neuropilina-2/biossíntese , Espectroscopia de Luz Próxima ao Infravermelho , Bexiga Urinária/citologia , Urotélio/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
BACKGROUND: The lymphatics form a second circulatory system that drains the extracellular fluid and proteins from the tumor microenvironment, and provides an exclusive environment in which immune cells interact and respond to foreign antigen. Both cancer and inflammation are known to induce lymphangiogenesis. However, little is known about bladder lymphatic vessels and their involvement in cancer formation and progression. METHODS: A double transgenic mouse model was generated by crossing a bladder cancer-induced transgenic, in which SV40 large T antigen was under the control of uroplakin II promoter, with another transgenic mouse harboring a lacZ reporter gene under the control of an NF-kappaB-responsive promoter (kappaB-lacZ) exhibiting constitutive activity of beta-galactosidase in lymphatic endothelial cells. In this new mouse model (SV40-lacZ), we examined the lymphatic vessel density (LVD) and function (LVF) during bladder cancer progression. LVD was performed in bladder whole mounts and cross-sections by fluorescent immunohistochemistry (IHC) using LYVE-1 antibody. LVF was assessed by real-time in vivo imaging techniques using a contrast agent (biotin-BSA-Gd-DTPA-Cy5.5; Gd-Cy5.5) suitable for both magnetic resonance imaging (MRI) and near infrared fluorescence (NIRF). In addition, IHC of Cy5.5 was used for time-course analysis of co-localization of Gd-Cy5.5 with LYVE-1-positive lymphatics and CD31-positive blood vessels. RESULTS: SV40-lacZ mice develop bladder cancer and permitted visualization of lymphatics. A significant increase in LVD was found concomitantly with bladder cancer progression. Double labeling of the bladder cross-sections with LYVE-1 and Ki-67 antibodies indicated cancer-induced lymphangiogenesis. MRI detected mouse bladder cancer, as early as 4 months, and permitted to follow tumor sizes during cancer progression. Using Gd-Cy5.5 as a contrast agent for MRI-guided lymphangiography, we determined a possible reduction of lymphatic flow within the tumoral area. In addition, NIRF studies of Gd-Cy5.5 confirmed its temporal distribution between CD31-positive blood vessels and LYVE-1 positive lymphatic vessels. CONCLUSION: SV40-lacZ mice permit the visualization of lymphatics during bladder cancer progression. Gd-Cy5.5, as a double contrast agent for NIRF and MRI, permits to quantify delivery, transport rates, and volumes of macromolecular fluid flow through the interstitial-lymphatic continuum. Our results open the path for the study of lymphatic activity in vivo and in real time, and support the role of lymphangiogenesis during bladder cancer progression.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma in Situ/diagnóstico , Carcinoma in Situ/patologia , Vasos Linfáticos/diagnóstico por imagem , Vasos Linfáticos/patologia , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/patologia , Animais , Carcinoma in Situ/imunologia , Modelos Animais de Doenças , Glicoproteínas/análise , Glicoproteínas/imunologia , Imuno-Histoquímica , Linfangiogênese , Vasos Linfáticos/metabolismo , Linfografia/métodos , Imageamento por Ressonância Magnética , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Espectroscopia de Luz Próxima ao Infravermelho , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/imunologia , Uroplaquina IIRESUMO
BACKGROUND: Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following repeated intravesical BCG therapy. METHODS: Mice were transurethrally instilled with BCG or pyrogen-free on days 1, 7, 14, and 21. Seven days after the last instillation, urothelia along with the submucosa was removed and amplified ds-DNA was prepared from control- and BCG-treated bladder mucosa and used to generate suppression subtractive hybridization (SSH). Plasmids from control- and BCG-specific differentially expressed clones and confirmed by Virtual Northern were then purified and the inserts were sequenced and annotated. Finally, chromatin immune precipitation combined with real-time polymerase chain reaction assay (ChIP/Q-PCR) was used to validate SSH-selected transcripts. RESULTS: Repeated intravesical BCG treatment induced an up regulation of genes associated with antigen presentation (B2M, HLA-A, HLA-DQA1, HLA-DQB2, HLA-E, HLA-G, IGHG, and IGH) and representatives of two IFNgamma-induced small GTPase families: the GBPs (GBP1, GBP2, and GBP5) and the p47GTPases (IIGTP1, IIGTP2, and TGTP). Genes expressed in saline-treated bladders but down-regulated by BCG included: the single-spanning uroplakins (UPK3a and UPK2), SPRR2G, GSTM5, and RSP 19. CONCLUSION: Here we introduced a hypothesis-generator approach to determine key genes involved in the urothelium/sumbmucosa responses to BCG therapy. Urinary bladder responds to repeated BCG treatment by up-regulating not only antigen presentation-related genes, but also GBP and p47 small GTPases, both potentially serving to mount a resistance to the replication of the Mycobacterium. It will be of tremendous future interest to determine whether these immune response cascades play a role in the anti-cancer effects exerted by BCG.
Assuntos
Apresentação de Antígeno/genética , Vacina BCG/farmacologia , GTP Fosfo-Hidrolases/biossíntese , Regulação Neoplásica da Expressão Gênica/imunologia , Antígenos HLA/biossíntese , Antígenos de Histocompatibilidade Classe II/biossíntese , Proteínas de Membrana/biossíntese , Bexiga Urinária/efeitos dos fármacos , Urotélio/efeitos dos fármacos , Animais , Apresentação de Antígeno/imunologia , Vacina BCG/administração & dosagem , Northern Blotting , Imunoprecipitação da Cromatina , Feminino , GTP Fosfo-Hidrolases/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos H-2/biossíntese , Antígenos H-2/genética , Antígenos de Histocompatibilidade Classe II/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Técnica de Subtração , Bexiga Urinária/metabolismoRESUMO
BACKGROUND: All four PARs are present in the urinary bladder, and their expression is altered during inflammation. In order to search for therapeutic targets other than the receptors themselves, we set forth to determine TFs downstream of PAR activation in the C57BL/6 urinary bladders. METHODS: For this purpose, we used a protein/DNA combo array containing 345 different TF consensus sequences. Next, the TF selected was validated by EMSA and IHC. As mast cells seem to play a fundamental role in bladder inflammation, we determined whether c-kit receptor deficient (Kit w/Kit w-v) mice have an abrogated response to PAR stimulation. Finally, TFEB antibody was used for CHIP/Q-PCR assay and revealed up-regulation of genes known to be downstream of TFEB. RESULTS: TFEB, a member of the MiTF family of basic helix-loop-helix leucine zipper, was the only TF commonly up-regulated by all PAR-APs. IHC results confirm a correlation between inflammation and TFEB expression in C57BL/6 mice. In contrast, Kit w/Kit w-v mice did not exhibit inflammation in response to PAR activation. EMSA results confirmed the increased TFEB binding activity in C57BL/6 but not in Kit w/Kit w-v mice. CONCLUSION: This is the first report describing the increased expression of TFEB in bladder inflammation in response to PAR activation. As TFEB belongs to a family of TFs essential for mast cell survival, our findings suggest that this molecule may influence the participation of mast cells in PAR-mediated inflammation and that targeting TFEB/MiTF activity may be a novel approach for the treatment of bladder inflammatory disorders.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cistite/metabolismo , Redes Reguladoras de Genes , Inflamação/metabolismo , Receptores Ativados por Proteinase/metabolismo , Bexiga Urinária/metabolismo , Animais , Feminino , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL/metabolismo , Camundongos Mutantes , Mucosa/citologia , Mucosa/metabolismo , Receptores Ativados por Proteinase/isolamento & purificaçãoRESUMO
BACKGROUND: In general, inflammation plays a role in most bladder pathologies and represents a defense reaction to injury that often times is two edged. In particular, bladder neurogenic inflammation involves the participation of mast cells and sensory nerves. Increased mast cell numbers and tryptase release represent one of the prevalent etiologic theories for interstitial cystitis and other urinary bladder inflammatory conditions. The activity of mast cell-derived tryptase as well as thrombin is significantly increased during inflammation. Those enzymes activate specific G-protein coupled proteinase-activated receptors (PAR)s. Four PARs have been cloned so far, and not only are all four receptors highly expressed in different cell types of the mouse urinary bladder, but their expression is altered during experimental bladder inflammation. We hypothesize that PARs may link mast cell-derived proteases to bladder inflammation and, therefore, play a fundamental role in the pathogenesis of cystitis. RESULTS: Here, we demonstrate that in addition to the mouse urinary bladder, all four PA receptors are also expressed in the J82 human urothelial cell line. Intravesical administration of PAR-activating peptides in mice leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P, and antigen was strongly attenuated by PAR1-, and to a lesser extent, by PAR2-deficiency. CONCLUSION: Our results reveal an overriding participation of PAR1 in bladder inflammation, provide a working model for the involvement of downstream signaling, and evoke testable hypotheses regarding the role of PARs in bladder inflammation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestations of cystitis.