RESUMO
Crohn's disease is a pathological condition of the gastro-intestinal tract, causing severe transmural inflammation in the ileum and/or colon. Cigarette smoking is one of the best known environmental risk factors for the development of Crohn's disease. Nevertheless, very little is known about the effect of prolonged cigarette smoke exposure on inflammatory modulators in the gut. We examined the effect of cigarette smoke on cytokine profiles in the healthy and inflamed gut of human subjects and in the trinitrobenzene sulphonic acid mouse model, which mimics distal Crohn-like colitis. In addition, the effect of cigarette smoke on epithelial expression of transient receptor potential channels and their concurrent increase with cigarette smoke-augmented cytokine production was investigated. Active smoking was associated with increased IL-8 transcription in ileum of controls (p < 0,001; n = 18-20/group). In the ileum, TRPV1 mRNA levels were decreased in never smoking Crohn's disease patients compared to healthy subjects (p <0,001; n = 20/group). In the colon, TRPV1 mRNA levels were decreased (p = 0,046) in smoking healthy controls (n = 20/group). Likewise, healthy mice chronically exposed to cigarette smoke (n = 10/group) showed elevated ileal Cxcl2 (p = 0,0075) and colonic Kc mRNA levels (p = 0,0186), whereas TRPV1 mRNA and protein levels were elevated in the ileum (p = 0,0315). Although cigarette smoke exposure prior to trinitrobenzene sulphonic acid administration did not alter disease activity, increased pro-inflammatory cytokine production was observed in the distal colon (Kc: p = 0,0273; Cxcl2: p = 0,104; Il1-ß: p = 0,0796), in parallel with the increase of Trpv1 mRNA (p < 0,001). We infer that CS affects pro-inflammatory cytokine expression in healthy and inflamed gut, and that the simultaneous modulation of TRPV1 may point to a potential involvement of TRPV1 in cigarette smoke-induced production of inflammatory mediators.
Assuntos
Colo/metabolismo , Doença de Crohn/metabolismo , Íleo/metabolismo , Canais de Cátion TRPV/metabolismo , Fumar Tabaco/efeitos adversos , Adulto , Idoso , Animais , Células CACO-2 , Colo/patologia , Doença de Crohn/induzido quimicamente , Doença de Crohn/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Células HT29 , Humanos , Íleo/patologia , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Pesquisa Translacional Biomédica , Ácido TrinitrobenzenossulfônicoRESUMO
Inflammatory bowel disease (IBD) is characterized by severe gastrointestinal inflammation and results from a complex interplay between genetic and environmental factors. IBD includes two prominent subtypes: Crohn's disease (CD) and ulcerative colitis (UC). One of the main risk factors for the development of CD is cigarette smoking, while UC is rather a disease of ex-smokers. To date, many of the mechanisms underlying the immune imbalance in IBD and the involvement of cigarette smoke (CS) are incompletely understood. Transient receptor potential (TRP) proteins are non-selective cation channels that, upon activation, lead to plasma membrane depolarization and, in general, to Ca2+ influx. TRP channels of the ankyrin and vanilloid family, expressed by sensory neurons in the central and enteric nervous systems, have been extensively studied in the context of intestinal inflammation. Moreover, recent advances made on the role of non-neuronal expressed TRP channels shed light on the involvement of epithelial cells in inflammatory processes. This review focuses on how CS may impact TRP channel function in intestinal inflammation. Firstly, we discuss the current knowledge on neuronal TRP channels, known to be linked to IBD, in health, immune homeostasis and intestinal inflammation. Subsequently, we address how TRP channels are activated by CS and its components in other organ systems and also hypothesize on the potential implications for CS-mediated TRP channel activation in gut inflammation.
Assuntos
Doenças Inflamatórias Intestinais/etiologia , Fumar/efeitos adversos , Canais de Potencial de Receptor Transitório/fisiologia , Colite Ulcerativa/etiologia , Colite Ulcerativa/fisiopatologia , Doença de Crohn/etiologia , Doença de Crohn/fisiopatologia , Células Epiteliais/patologia , Humanos , Inflamação/etiologia , Inflamação/fisiopatologia , Doenças Inflamatórias Intestinais/fisiopatologiaRESUMO
Fragmented RNA from formalin-fixed paraffin-embedded (FFPE) tissue is a known obstacle to gene expression analysis. In this study, the impact of RNA integrity, gene-specific reverse transcription and targeted cDNA preamplification was quantified in terms of reverse transcription polymerase chain reaction (RT-qPCR) sensitivity by measuring 48 protein coding genes on eight duplicate cultured cancer cell pellet FFPE samples and twenty cancer tissue FFPE samples. More intact RNA modestly increased gene detection sensitivity by 1.6 fold (earlier detection by 0.7 PCR cycles, 95% CI = 0.593-0.850). Application of gene-specific priming instead of whole transcriptome priming during reverse transcription further improved RT-qPCR sensitivity by a considerable 4.0 fold increase (earlier detection by 2.0 PCR cycles, 95% CI = 1.73-2.32). Targeted cDNA preamplification resulted in the strongest increase of RT-qPCR sensitivity and enabled earlier detection by an average of 172.4 fold (7.43 PCR cycles, 95% CI = 6.83-7.05). We conclude that gene-specific reverse transcription and targeted cDNA preamplification are adequate methods for accurate and sensitive RT-qPCR based gene expression analysis of FFPE material. The presented methods do not involve expensive or complex procedures and can be easily implemented in any routine RT-qPCR practice.
Assuntos
DNA Complementar/genética , Biossíntese de Proteínas/genética , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA Complementar/isolamento & purificação , Formaldeído , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Inclusão em Parafina , RNA/isolamento & purificação , Fixação de Tecidos/métodosRESUMO
Inflammatory bowel diseases (IBD) are complex multifactorial diseases characterized by an inappropriate host response to an altered commensal microbiome and dysfunctional mucus barrier. Cigarette smoking is the best known environmental risk factor in IBD. Here, we studied the influence of chronic smoke exposure on the gut microbiome, mucus layer composition and immune factors in conventional mice. We compared smoke-exposed with air-exposed mice (n = 12) after a smoke exposure of 24 weeks. Both Illumina sequencing (n = 6) and denaturing gradient gel electrophoresis (n = 12) showed that bacterial activity and community structure were significantly altered in the colon due to smoke exposure. Interestingly, an increase of Lachnospiraceae sp. activity in the colon was observed. Also, the mRNA expression of Muc2 and Muc3 increased in the ileum, whereas Muc4 increased in the distal colon of smoke-exposed mice (n = 6). Furthermore, we observed increased Cxcl2 and decreased Ifn-γ in the ileum, and increased Il-6 and decreased Tgf-ß in the proximal colon. Tight junction gene expression remained unchanged. We infer that the modulating role of chronic smoke exposure as a latently present risk factor in the gut may be driven by the altered epithelial mucus profiles and changes in microbiome composition and immune factors.
Assuntos
Microbioma Gastrointestinal , Mediadores da Inflamação/metabolismo , Mucinas/metabolismo , Fumar , Animais , Bactérias/isolamento & purificação , Colo/metabolismo , Colo/microbiologia , Exposição Ambiental , Trato Gastrointestinal/microbiologia , Expressão Gênica , Íleo/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mucinas/genética , Produtos do TabacoRESUMO
The inflammatory cytokine TNF-α is a central mediator in many immune-mediated diseases, such as Crohn's disease (CD), spondyloarthritis (SpA) and chronic obstructive pulmonary disease (COPD). Epidemiologic studies have shown that cigarette smoking (CS) is a prominent common risk factor in these TNF-dependent diseases. We exposed TNFΔARE mice; in which a systemic TNF-α overexpression leads to the development of inflammation; to 2 or 4 weeks of air or CS. We investigated the effect of deregulated TNF expression on CS-induced pulmonary inflammation and the effect of CS exposure on the initiation and progression of gut and joint inflammation. Upon 2 weeks of CS exposure, inflammation in lungs of TNFΔARE mice was significantly aggravated. However, upon 4 weeks of CS-exposure, this aggravation was no longer observed. TNFΔARE mice have no increases in CD4+ and CD8+ T cells and a diminished neutrophil response in the lungs after 4 weeks of CS exposure. In the gut and joints of TNFΔARE mice, 2 or 4 weeks of CS exposure did not modulate the development of inflammation. In conclusion, CS exposure does not modulate gut and joint inflammation in TNFΔARE mice. The lung responses towards CS in TNFΔARE mice however depend on the duration of CS exposure.
Assuntos
Artrite/patologia , Doenças Inflamatórias Intestinais/patologia , Pneumonia/patologia , Fumar/efeitos adversos , Fator de Necrose Tumoral alfa/genética , Proteínas de Fase Aguda/metabolismo , Animais , Artrite/genética , Artrite/imunologia , Citocinas/sangue , Modelos Animais de Doenças , Fezes/química , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Lipocalina-2 , Lipocalinas/metabolismo , Camundongos , Proteínas Oncogênicas/metabolismo , Pneumonia/genética , Pneumonia/imunologia , Deleção de Sequência , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Neoadjuvant radio(chemo)therapy is increasingly used in rectal cancer and induces a number of morphologic changes that affect prognostication after curative surgery, thereby creating new challenges for surgical pathologists, particularly in evaluating morphologic changes and tumour response to preoperative treatment. Surgical pathologists play an important role in determining the many facets of rectal carcinoma patient care after neoadjuvant treatment. These range from proper handling of macroscopic specimens to accurate microscopic evaluation of pathological features associated with patients' prognosis. This review presents the well-established pathological prognostic indicators and discusses challenging features in order to provide both surgical pathologists and treating physicians with a checklist that is useful in a neoadjuvant setting.
Assuntos
Neoplasias Retais/diagnóstico , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/radioterapia , Diferenciação Celular , Quimiorradioterapia , Humanos , Inflamação , Linfonodos/patologia , Mucinas/metabolismo , Terapia Neoadjuvante , Estadiamento de Neoplasias , Prognóstico , Resultado do TratamentoRESUMO
Testosterone (T) levels are decreased in obese men, but the underlying causes are incompletely understood. Our objective was to explore the relation between low (free) T levels and male obesity, by evaluating metabolic parameters, subcutaneous adipose tissue (SAT) aromatase expression, and parameters of the hypothalamic-pituitary-gonadal axis. We recruited 57 morbidly obese men [33 had type 2 diabetes (DM2)] and 25 normal-weight men undergoing abdominal surgery. Fourteen obese men also attended a follow-up, 2 years after gastric bypass surgery (GBS). Circulating T levels were quantified by LC-MS/MS, whereas free T levels were measured using serum equilibrium dialysis and sex hormone-binding globulin, luteinizing hormone, and follicle-stimulating hormone by immunoassay. SAT biopsies were used to determine adipocyte cell size and aromatase expression by real-time PCR. Total and free T levels were decreased in obese males versus controls, with a further decrease in obese men with DM2 versus obese men without DM2. There were no differences in aromatase expression among the study groups, and sex steroids did not correlate with aromatase expression. Pearson analysis revealed an inverse association between (free) T and SAT cell size, triglycerides, and HOMA-IR. Multivariate analysis confirmed the inverse association between (free) T and SAT cell size (ß = -0.321, P = 0.037 and ß = -0.441, P = 0.011, respectively), independent of age, triglycerides, HOMA-IR, obesity, or diabetes. T levels were normalized 2 years after GBS. These data suggest that SAT cell size rather than SAT aromatase expression or parameters of the hypothalamic-pituitary-gonadal axis is related to low T in male obesity, which points to adipose cell size-related metabolic changes as a major trigger in decreased T levels.
Assuntos
Aromatase/metabolismo , Diabetes Mellitus Tipo 2/sangue , Obesidade/sangue , Gordura Subcutânea/metabolismo , Testosterona/sangue , Adulto , Comorbidade , Diabetes Mellitus Tipo 2/epidemiologia , Derivação Gástrica , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/epidemiologia , Obesidade Mórbida/sangue , Obesidade Mórbida/epidemiologia , Gordura Subcutânea/citologiaRESUMO
Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self-constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell-specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut-associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL-17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life.
Assuntos
Autoimunidade/imunologia , Microbiota/imunologia , Animais , Anticorpos Antinucleares/genética , Anticorpos Antinucleares/imunologia , Autoimunidade/genética , Feminino , Citometria de Fluxo , Linfotoxina-alfa/genética , Linfotoxina-alfa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
BACKGROUND: Inflammatory patterns of nasal polyps (NPs) may vary. Changes over time have not been investigated so far. This study was designed to evaluate the inflammatory patterns of NPs in Thailand at two time points 12 years apart, explore differences in Staphylococcus aureus (SA) mucosal carriage rates over time, and the latter's relationship with the inflammatory patterns. METHODS: Formalin-fixed nasal tissue was obtained from 89 (47 in 1999 and 42 in 2011) patients suffering from chronic rhinosinusitis with NPs (CRSwNPs). Tissues were evaluated for eosinophils, neutrophils, IgE(+) cells, IgE and macrophage mannose receptors, interleukin (IL)-5 and IL-17 cytokine profile, and the presence of SA, using automated immunohistochemistry and peptide nucleic acid-fluorescence in situ hybridization. RESULTS: We found a significant increase in the absolute values of eosinophils and IgE(+) cells in the 2011 CRSwNP tissue series compared with 1999 and a significant but smaller increase in neutrophils. Semiquantitative evaluation revealed significantly higher mean values of positive cells for all studied inflammatory markers in the 2011 group of patients, except for the high-affinity IgE receptor. This "eosinophilic shift" of inflammation was accompanied by higher SA carriage, as well as higher frequencies of SA invasion (54.8% versus 10.6%; p < 0.001) in the 2011 compared with 1999 subjects. Patients with asthma were more likely to have higher SA carriage rates compared with nonasthmatic patients. CONCLUSION: There was a shift from predominantly neutrophilic to eosinophilic CRSwNPs in Thai patients within 12 years, with an increase in various inflammatory markers including IgE, which is associated with an increase in intramucosal presence of SA.
Assuntos
Eosinófilos/imunologia , Pólipos Nasais/imunologia , Rinite/imunologia , Sinusite/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Adulto , Carga Bacteriana , Doença Crônica , Feminino , Seguimentos , Humanos , Imunoglobulina E/metabolismo , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Interleucina-17/metabolismo , Interleucina-5/metabolismo , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/complicações , Neutrófilos/imunologia , Rinite/complicações , Sinusite/complicações , Infecções Estafilocócicas/complicações , Staphylococcus aureus/crescimento & desenvolvimento , TailândiaRESUMO
BACKGROUND: Composition changes of extracellular matrix (ECM) can lead to functional disorders of the upper airways (UA). The aim of this study was to systematically measure both the association patterns and the correlation degree between tissue composition parameters in UA inflammatory diseases. METHODOLOGY: Nasal samples were obtained from patients with chronic rhinosinusitis with (CRS+NP), without nasal polyps (CRS), with post-operative adhesions (S) and normal nasal mucosa (NM). A reproducible semi-quantitative method, which takes epithelial and lamina propria damages into account was applied for haematoxylin and eosin, alpha-smooth muscle actin, reticulin, elastin, laminin and collagen type IV stainings. RESULTS: The most severe cases of epithelial shedding have been found in a significant higher amount in CRS+NP when compared with NM. The most severe cases of inflammatory reaction were mainly found in CRS+NP. CRS+NP had significantly more severe cases of oedema than NM. Excluding elastin, networks in other ECM proteins were found modified in fibrotic fields but to a lesser extend in oedematous regions in all conditions. CONCLUSION: Although non specific, oedema in the lamina propria is a key-feature of CRS+NP, while fibrosis, massively present in CRS and S, affects profoundly the distribution of ECM proteins in these areas.
Assuntos
Pólipos Nasais/metabolismo , Rinite/metabolismo , Sinusite/metabolismo , Actinas/metabolismo , Adolescente , Adulto , Doença Crônica , Colágeno Tipo IV/metabolismo , Tecido Conjuntivo/metabolismo , Edema/metabolismo , Elastina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrose , Humanos , Imuno-Histoquímica , Laminina/metabolismo , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Reticulina/metabolismo , Adulto JovemRESUMO
Epidemiological evidence demonstrates that smoking is the most important environmental risk factor in Crohn's disease while it positively interferes with the disease course of ulcerative colitis. However, the underlying mechanisms through which smoking exerts this divergent effect and affects pathogenesis of inflammatory bowel disease are largely unknown. Animal smoke models are good models to investigate the impact of cigarette smoke on intestinal physiology and inflammation. They enable one to explore the interaction of smoke components and the gut on cellular and molecular level, clarifying how smoking interferes with normal gut function and with disease course in inflammatory conditions. This review describes the currently used animal models for studying the impact of cigarette smoke on the intestinal tract. We first discuss the different methods for simulation of smoking. Furthermore, we focus on the effect of smoke exposure on normal gut physiology and immunology, on experimental (entero)colitis, and on inflammation-induced neoplasia. Based on this current knowledge, a hypothesis is formulated about the mechanisms through which cigarette smoke interferes with the gut in normal and pathological conditions.
Assuntos
Doenças Inflamatórias Intestinais/fisiopatologia , Intestinos/efeitos dos fármacos , Modelos Animais , Nicotiana/efeitos adversos , Nicotina/efeitos adversos , Fumar/efeitos adversos , Animais , Humanos , Doenças Inflamatórias Intestinais/imunologia , Neoplasias Intestinais/fisiopatologia , Intestinos/patologia , Intestinos/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fatores de RiscoRESUMO
Cigarette smoke (CS) exposure is associated with increased autophagy in several cell types, such as bronchial epithelial cells. Smoking is also an environmental risk factor in Crohn's disease, in which impairment of the autophagy-mediated anti-bacterial pathway has been implicated. So far, it is unknown whether CS induces autophagy in the gut. Here, we examined the effect of chronic CS exposure on autophagy in the follicle-associated epithelium (FAE) of murine Peyer's patches. Transmission electron microscopy revealed that the proportion of cell area occupied by autophagic vesicles significantly increased in the FAE after CS exposure. An increased number of autophagic vesicles was observed in the FAE, whereas the vesicle size remained unaltered. Besides enterocytes, also M-cells contain more autophagic vesicles upon CS exposure. In addition, the mRNA level of the autophagy-related protein Atg7 in the underlying Peyer's patches is increased after CS exposure, which indicates that the autophagy-inducing effect of CS is not limited to the FAE. In conclusion, our results demonstrate that CS exposure induces autophagy in murine FAE and in the underlying immune cells of Peyer's patches, suggesting that CS exposure increases the risk for Crohn's disease by causing epithelial oxidative damage, which needs to be repaired by autophagy.
Assuntos
Autofagia/fisiologia , Íleo/patologia , Mucosa Intestinal/patologia , Nódulos Linfáticos Agregados/patologia , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 5 Relacionada à Autofagia , Proteína 7 Relacionada à Autofagia , Proteína Beclina-1 , Doença Crônica , Doença de Crohn/epidemiologia , Doença de Crohn/patologia , Íleo/ultraestrutura , Mucosa Intestinal/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Nódulos Linfáticos Agregados/ultraestrutura , RNA Mensageiro/metabolismo , Fatores de RiscoRESUMO
Smokers have a twofold increased risk to develop Crohn's disease (CD). However, little is known about the mechanisms through which smoking affects CD pathogenesis. Especially Crohn's ileitis is negatively influenced by smoking. Interestingly, the ileum and, more in particular, the Peyer's patches in the terminal ileum are also the sites where the first CD lesions are found. Several chemokines are implicated in the pathogenesis, among which is the CCL20-CCR6 pathway. Here, we studied the gut-associated lymphoid tissue in C57BL/6 wild-type mice and in CCR6-deficient mice after exposure to air or cigarette smoke for 24 weeks. Apoptotic index of the follicle-associated epithelium overlying the Peyer's patches was evaluated. We found that chronic smoke exposure induced apoptosis in the follicle-associated epithelium. Furthermore, immune cell numbers and differentiation along with chemokine expression were determined in Peyer's patches. Important changes in immune cell composition were observed: total dendritic cells, CD4+ T cells (including regulatory T cells) and CD8+ T cells increased significantly after smoke exposure. The CD11b+ dendritic cell subset almost doubled. Interestingly, these changes were accompanied by an upregulated mRNA expression of the chemokines CCL9 and CCL20. However, no differences in the increase of dendritic cells were observed between wild-type and CCR6-deficient mice. Our results show that cigarette smoke exposure increases apoptosis in the follicle-associated epithelium and is associated with immune cell accumulation in Peyer's patches.
Assuntos
Apoptose , Mucosa Intestinal/patologia , Tecido Linfoide/patologia , Nicotiana , Fumar/patologia , Animais , Citometria de Fluxo , Interleucina-10/metabolismo , Mucosa Intestinal/imunologia , Tecido Linfoide/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fumar/imunologiaRESUMO
UNLABELLED: The aim of this study was to examine systemic and local nasal leptin and leptin receptor expression in patients with nasal polyposis and healthy controls. METHODS AND RESULTS: Serum leptin and soluble leptin receptor levels were examined by enzyme-linked immunosorbent assay (ELISA). The presence of leptin and leptin receptor mRNA was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), and tissue leptin and leptin receptor protein expression was analysed by immunohistochemistry and ELISA. Serum levels of biologically active leptin were significantly elevated in patients with nasal polyps compared with control subjects. These serum leptin levels were strongly correlated with the levels found in tissue in both study groups, although leptin was not significantly elevated in nasal polyp tissue. Using RT-PCR, we showed that both leptin and its receptors were produced in nasal mucosa. Finally, immunohistochemistry showed that leptin and leptin receptor protein were expressed in several cells of the normal and inflamed nasal mucosa. CONCLUSIONS: Leptin receptors and their biological ligand leptin are expressed in the nasal mucosa, suggesting a possible role in upper airway immunology.
Assuntos
Leptina/metabolismo , Mucosa Nasal/metabolismo , Receptores para Leptina/metabolismo , Adulto , Idoso , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Leptina/sangue , Leptina/genética , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/metabolismo , RNA Mensageiro/metabolismo , Receptores para Leptina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
BACKGROUND: Restoring p53 function by antagonizing its interaction with the negative regulator MDM2 is an appealing nongenotoxic approach to treating tumors with wild-type p53. Mutational inactivation of p53 is rare in neuroblastoma tumors at diagnosis and occurs in only a subset of multidrug-resistant neuroblastomas. METHODS: The antiproliferative and cytotoxic effect of nutlin-3, a small-molecule MDM2 antagonist, was examined in chemosensitive (UKF-NB-3) and matched chemoresistant neuroblastoma cells with wild-type p53 (UKF-NB-3(r)DOX20) or with mutant p53 (UKF-NB-3(r)VCR10). Activation of the p53 pathway was assessed by expression analysis of p53 target genes, flow cytometric cell cycle analysis, and apoptosis assays. Mice with established chemoresistant tumor xenografts were treated orally with nutlin-3 or vehicle control (n = 5-10 mice per group) and were used to evaluate effects on tumor growth, p53 pathway activity, and metastatic tumor burden. All statistical tests were two-sided. RESULTS: Nutlin-3 induced a similar activation of the p53 pathway in UKF-NB-3 and UKF-NB-3(r)DOX20 cells, as evidenced by increased expression of p53 target genes, G1 cell cycle arrest, and induction of apoptosis. No such response was observed in UKF-NB-3(r)VCR10 cells with mutant p53. Oral administration of nutlin-3 to UKF-NB-3(r)DOX20 xenograft-bearing mice led to inhibition of primary tumor growth (mean tumor volume after 3 weeks of treatment, nutlin-3- vs vehicle-treated mice: 772 vs 1661 mm3, difference = 890 mm3, 95% confidence interval = 469 to 1311 mm3, P < .001), p53 pathway activation, and reduction in the extent of metastatic disease. The growth of UKF-NB-3(r)VCR10 xenografts was unaffected by nutlin-3. CONCLUSIONS: Nutlin-3 activates the p53 pathway and suppresses tumor growth in this model system of chemoresistant neuroblastoma, provided that wild-type p53 is present.
Assuntos
Antineoplásicos/farmacologia , Imidazóis/farmacologia , Mutação , Neuroblastoma/tratamento farmacológico , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/efeitos dos fármacos , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Caspase 3/metabolismo , Caspase 7/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fragmentação do DNA , Diploide , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/administração & dosagem , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Nus , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Piperazinas/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
OBJECTIVES: Xenogenic extracellular heart valve matrices have been suggested as scaffolds for tissue engineering. However, these matrices are immunogenic and stimulate an intense cell-mediated immune response and calcification. Mitigating the immunogenicity was attempted by different doses of gamma irradiation. METHODS: Mechanical properties of gamma-irradiated porcine matrices and control matrices (nonirradiated) were examined by tensile strength testing. Irradiated matrices (1, 10, 50, and 100 gray [Gy]) and control matrices were implanted subcutaneously in Wistar rats (n = 20). After 24 h, 1, 2, 3, and 4 weeks the explants were examined by light microscopy and transmission electron microscopy. Calcium (Ca) content was determined using inductively coupled plasma-mass spectrometry. Antibody reaction against porcine tissue in the rat serum was determined. RESULTS: Tensile strength increased in irradiated matrices at the expense of elasticity. Ten gray-irradiated leaflets showed minimal lymphocytic inflammatory infiltration with preservation of ultrastructure. Ca levels after 2 weeks were as follows: control (0 Gy), 388 +/- 264 microg/mg; 1 Gy, 240 +/- 95 microg/mg; 10 Gy, 188 +/- 54 microg/mg; 50 Gy, 289 +/- 94 microg/mg; 100 Gy, 651 +/- 57 microg/mg. All implants still elicit an antibody immunoglobulin G reaction. CONCLUSIONS: Exposure to 10 Gy gamma irradiation reduces lymphocytic inflammatory infiltrates and Ca levels in acellular porcine matrices with preservation of structural integrity. This could prolong the durability of these matrices.
Assuntos
Bioprótese , Próteses Valvulares Cardíacas , Valvas Cardíacas/fisiologia , Valvas Cardíacas/efeitos da radiação , Engenharia Tecidual/métodos , Animais , Relação Dose-Resposta à Radiação , Módulo de Elasticidade/efeitos da radiação , Análise de Falha de Equipamento , Raios gama , Valvas Cardíacas/ultraestrutura , Desenho de Prótese , Doses de Radiação , Ratos , Ratos Wistar , Resistência à Tração/efeitos da radiaçãoRESUMO
The follicle-associated epithelium (FAE) overlying the follicles of mucosa-associated lymphoid tissue is a key player in the initiation of mucosal immune responses. We recently reported strong clusterin expression in the FAE of murine Peyer's patches. In this study, we examined the expression of clusterin in the human gut-associated lymphoid tissue (GALT) and Waldeyer's ring. Immunohistochemistry for clusterin in human Peyer's patches, appendix and colon lymphoid follicles revealed expression in M cells and in follicular dendritic cells (FDCs). Using cryo-immunogold electron microscopy in Peyer's patches, we observed cytosolic immunoreactivity in M cells and labeling in the ER/Golgi biosynthetic pathway in FDCs. In palatine tonsils and adenoids, we demonstrated clusterin expression in germinal centers and in the lymphoepithelium in the crypts where M cells are localized. In conclusion, clusterin is expressed in M cells and follicular dendritic cells at inductive sites of human mucosa-associated lymphoid tissue suggesting a role for this protein in innate immune responses. Moreover, the use of clusterin as a human M cell marker could prove to be a valuable tool in future M cell research.
Assuntos
Clusterina/metabolismo , Células Dendríticas Foliculares/metabolismo , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Tonsila Faríngea/metabolismo , Especificidade de Anticorpos , Western Blotting , Clusterina/biossíntese , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Tonsila Palatina/metabolismoRESUMO
PURPOSE: Radiolabeled annexin V may allow for repetitive and selective in vivo identification of apoptotic cell death without the need for invasive biopsy. This study reports on the relationship between quantitative technetium-99m- (99mTc-) 6-hydrazinonicotinic (HYNIC) radiolabeled annexin V tumor uptake, and the number of tumor apoptotic cells derived from histologic analysis. PATIENTS AND METHODS: Twenty patients (18 men, two women) suspected of primary (n = 19) or recurrent (n = 1) head and neck carcinoma were included. All patients underwent a spiral computed tomography (CT) scan, 99mTc-HYNIC annexin V tomography, and subsequent surgical resection of the suspected primary or recurrent tumor. Quantitative 99mTc-HYNIC annexin V uptake in tumor lesions divided by the tumor volume, derived from CT, was related to the number of apoptotic cells per tumor high-power field derived from terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assays performed on sectioned tumor slices. RESULTS: Diagnosis was primary head and neck tumor in 18 patients, lymph node involvement of a cancer of unknown primary origin in one patient, and the absence of recurrence in one patient. Mean percentage absolute tumor uptake of the injected dose per cubic centimeter tumor volume derived from tomographic images was 0.0003% (standard deviation [SD], 0.0004%) at 1 hour postinjection (PI) and 0.0001% (SD, 0.0000%) at 5 to 6 hours PI (P =.012). Quantitative 99mTc-HYNIC annexin V tumor uptake correlated well with the number of apoptotic cells if only tumor samples with no or minimal amounts of necrosis were considered. CONCLUSION: In the absence of necrosis, absolute 99mTc-HYNIC annexin V tumor uptake values correlate well with the number of apoptotic cells derived from TUNEL assays.
Assuntos
Anexina A5 , Apoptose , Carcinoma/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Compostos de Organotecnécio , Compostos Radiofarmacêuticos , Tomografia Computadorizada de Emissão de Fóton Único , Idoso , Carcinoma/patologia , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/diagnóstico por imagem , Recidiva Local de Neoplasia/patologia , Estudos Prospectivos , Tomografia Computadorizada EspiralRESUMO
BACKGROUND: University of Wisconsin (UW) solution (Viaspan) is currently used to preserve organs from nonheartbeating donors. Histidine-tryptophan-ketoglutarate (HTK) solution (Custodiol) is of proven efficacy in experimental pancreas preservation, but its efficacy in combined warm ischemia (WI) and cold ischemia (CI) is unknown. The viability of HTK-preserved porcine pancreatic grafts was assessed after various periods of WI and compared with grafts flushed and preserved with UW solution. METHODS: A total of 14 pigs were used: G1 (n=4, UW) and G2 (n=4, HTK) with 15-min WI and 16-hr cold storage; G3 (n=3, UW) and G4 (n=3, HTK) with 30-min WI and 16-hr cold storage. RESULTS: All animals in G1 and G2 were normoglycemic, whereas only 66% of pancreases were functioning in G3 and G4. HTK perfusion was associated with increased wet weight. Transient hyperinsulinemia was noted in all the groups on postoperative day 1 (mean range: 8.9-12.4 microU/L). Postoperative serum amylase and lipase were more pronounced in G3 and G4. However, HTK-stored grafts exhibited less evidence of biochemical pancreatitis as compared with UW-stored grafts on the first postoperative day in the group with 15-min WI. Mean K values of intravenous glucose tolerance tests on postoperative day 14 were similar in both groups. Vascular congestion was uniformly observed and was considered a typical feature of WI. CONCLUSIONS: Porcine pancreatic grafts are viable after 16-hr CI following 15-min WI in this experimental nonheartbeating donor model. HTK solution seems to provide reliable graft function in this setting and to be equivalent to UW.
Assuntos
Adenosina/farmacologia , Alopurinol/farmacologia , Glutationa/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Insulina/farmacologia , Soluções para Preservação de Órgãos/farmacologia , Transplante de Pâncreas , Rafinose/farmacologia , Traumatismo por Reperfusão/patologia , Amilases/sangue , Animais , Temperatura Baixa , Edema/sangue , Edema/patologia , Temperatura Alta , Hiperinsulinismo/sangue , Hiperinsulinismo/patologia , Hipoglicemia/sangue , Hipoglicemia/patologia , Lipase/sangue , Modelos Animais , Preservação de Órgãos , Pâncreas/patologia , Pâncreas/fisiologia , Traumatismo por Reperfusão/sangue , SuínosRESUMO
The objective of this study was to investigate CD163+ macrophages in the synovial membrane of patients with spondyloarthropathy (SpA). Immunohistochemistry was performed on synovium of 17 SpA and 18 rheumatoid arthritis (RA) patients, on colonic biopsies of 16 SpA patients and ten healthy controls, and on paired synovial biopsies of eight SpA patients, before and after anti-TNFalpha therapy. Phenotype and cytokine production were analysed by flow cytometry. CD163+ macrophages were increased in the synovial lining and sublining in SpA versus RA, as well as in colonic lamina propria in SpA versus controls. The number of CD163+ macrophages in the synovial sublining correlated with C-reactive protein levels and erythrocyte sedimentation rate. Paralleling the increase of CD163, HLA-DR was increased in the synovial lining and sublining of SpA. In contrast, the co-stimulatory molecules CD80 and CD86 and the dendritic cell markers CD1a and CD83 were scarce in SpA synovium. Flow cytometry indicated that CD163+ macrophages expressed high levels of HLA-DR and could produce in vitro tumour necrosis factor alpha (TNFalpha) but not interleukin-10 (IL-10). Finally, anti-TNFalpha therapy in vivo induced a decrease of CD163+ macrophages in the synovial lining and sublining. In conclusion, macrophages expressing the scavenger receptor CD163 are increased in synovium and in colonic mucosa in SpA, highlighting the relationship between joint and gut in this disease. The correlation with inflammatory parameters, the expression of HLA-DR, the production of TNFalpha but not IL-10, and the reduction by anti-TNFalpha therapy support a role for CD163+ macrophages in the synovial inflammation in SpA.