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The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated.
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Anticuerpos Monoclonales/inmunología , Técnica del Anticuerpo Fluorescente , Formaldehído/química , Macrófagos/citología , Macrófagos/inmunología , Adhesión en Parafina , Fijación del Tejido , Animales , Humanos , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Coloración y EtiquetadoRESUMEN
BACKGROUND: Triple-negative breast cancer (TBNC) is an aggressive breast cancer subtype with a poor prognosis. Shugoshin-1 (SGO1) protects chromatids from early separation. Previous studies from our group have demonstrated that transient SGO1 downregulation suppresses early stages of metastasis (the epithelial-to-mesenchymal transition, or EMT, cell invasion, and cell migration) in TNBC cells. Thus, the inhibition of SGO1 activity may represent a potential therapeutic intervention against cancers that progress to metastasis. Therefore, we aimed to investigate the effects of sustained shRNA-mediated SGO1 downregulation on tumor growth and metastasis in TBNC. To that end, female NOD-SCID Gamma (NSG) mice were injected with 2.5 × 106 shRNA Control (n = 10) or shRNA SGO1 (n = 10) MDA-MB-231 cells. After eight weeks, the number of mice with metastasis to the lymph nodes was calculated. Primary and metastatic tumors, as well as lung and liver tissue, were harvested, measured, sectioned, and stained with hematoxylin and eosin (H&E) stain. RESULTS: Tumor growth and metastasis to the lymph nodes and lungs were significantly reduced in the shRNA SGO1-treated mice group, while metastasis to the liver tends to be lower in cells with downregulated SGO1, but it did not reach statistical significance. Furthermore, sustained SGO1 downregulation significantly reduced cell proliferation, cell migration, and invasion which correlated with lower levels of Snail, Slug, MMP2, MMP3, and MMP9. CONCLUSION: The supression of SGO1 activity in TNBC harboring dysregulated expression of SGO1 may be a potential target for preventing breast cancer growth and metastasis.
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Evidence supports involvement of microflora in the transition of chronic inflammation to neoplasia. We investigated the protective efficacy of the probiotic VSL#3 in a model of colitis-associated colorectal cancer. Chronic colitis was induced in Sprague-Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS), followed 6 wk later by systemic reactivation. To induce colitis-associated dysplasia and cancer, the animals received TNBS (intravenously) twice a week for 10 wk. One group received VSL#3 in drinking water from 1 wk before colitis induction until death. The colons were examined for damage and presence of dysplasia or cancer. Samples were analyzed for cell proliferation and apoptosis, vitamin D receptor (VDR) expression, angiogenic factors, and presence of alkaline sphingomyelinase or phosphatase. Microbial community composition was evaluated by terminal restriction fragment-length polymorphism analysis of the bacterial 16S rRNA gene. None of the probiotic-treated animals developed carcinoma, and no high-grade dysplasia was found in either the proximal or mid colon. In contrast, 29% of the animals in the control group developed carcinoma in one or more regions of the colon. VSL#3-treated animals had significantly less damage than the vehicle treated-controls in all areas of the colon, and this correlated with decreased richness and diversity of the mucosally adherent microbiota. Treatment with the probiotic increased the antiangiogenic factor angiostatin, VDR expression, and alkaline sphingomyelinase. We concluded that pretreatment with the probiotic VSL#3 can attenuate various inflammatory-associated parameters, delaying transition to dysplasia and cancer, thus offering its potential therapeutic use in patients with long-standing colitis.
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Colitis/terapia , Neoplasias Colorrectales/prevención & control , Lesiones Precancerosas/terapia , Probióticos/farmacología , Fosfatasa Alcalina/metabolismo , Angiostatinas/metabolismo , Animales , Bifidobacterium , Enfermedad Crónica , Colitis/inmunología , Colitis/microbiología , Colon/metabolismo , Colon/microbiología , Colon/patología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/microbiología , Modelos Animales de Enfermedad , Endostatinas/metabolismo , Lactobacillus , Masculino , Metagenoma , Lesiones Precancerosas/inmunología , Lesiones Precancerosas/microbiología , Ratas , Ratas Sprague-Dawley , Receptores de Calcitriol/metabolismo , Streptococcus , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
E2F3 is a transcription factor that may initiate tumorigenesis if overexpressed. Previously, we demonstrated that E2F3 mRNA is overexpressed in breast cancer and that E2F3 overexpression results in centrosome amplification and unregulated mitosis, which can promote aneuploidy and chromosome instability to initiate and sustain tumors. Further, we demonstrated that E2F3 leads to overexpression of the mitotic regulator Shugoshin-1, which until recently had unknown roles in cancer. This study aims to evaluate the roles of E2F3 and Shugoshin-1 in breast cancer metastatic potential. Here we demonstrated that E2F3 and Shugoshin-1 silencing leads to reduced cell invasion and migration in two mesenchymal triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and Hs578t). Moreover, E2F3 and Shugoshin-1 modulate the expression of epithelial-to-mesenchymal transition-associated genes such as Snail, E-Cadherin, and multiple matrix metalloproteinases. Furthermore, E2F3 depletion leads to reductions in tumor growth and metastasis in NOD-scid Gamma mice. Results from this study suggest a key role for E2F3 and a novel role for Shugoshin-1 in metastatic progression. These results can further help in the improvement of TNBC targeted therapies by interfering with pathways that intersect with the E2F3 and Shugoshin-1 signaling pathways.
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Movimiento Celular/genética , Factor de Transcripción E2F3/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Masculino , Ratones , Ratones SCID , Transducción de Señal/genéticaRESUMEN
AIMS: This study investigated the protective effect of vitamin D analog paricalcitol combined with angiotensin-converting enzyme inhibitor (enalapril) on aortic oxidative injury in atherosclerotic mice. METHODS: Female mice were treated for 16 weeks as follows: (1) ApoE deficient + vehicle, (2) ApoE deficient + paricalcitol (200 ng 3 times a week), (3) ApoE deficient + enalapril (30 mg/l in drinking water), (4) ApoE deficient + paricalcitol + enalapril, and (5) wild-type controls. RESULTS: ApoE-deficient mice developed hypertension which was prevented by enalapril or enalapril + paricalcitol treatment but not by paricalcitol treatment. Histology showed atherosclerotic plaque in the aorta of ApoE-deficient mice which was prevented by paricalcitol, enalapril, and paricalcitol + enalapril treatments. Aortic malondialdehyde levels, NADPH oxidase subunit p22(phox), manganese-superoxide dismutase (Mn-SOD), inducible nitric oxide synthase, monocyte chemoattaractant protein-1, tumor necrosis factor (TNF)-α, and cyclooxygenase-2 protein expressions increased, whereas glutathione levels, CuZn-SOD, and endothelial protein expressions decreased in ApoE-deficient mice compared to controls. Treatment with paricalcitol and enalapril alone or in combination protected the inflammatory and oxidative endothelial injury of the aorta in atherosclerotic mice. CONCLUSION: Combination therapy affords greater protection against aortic inflammatory and oxidative injury in atherosclerosis than monotherapy.
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Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Aorta/fisiopatología , Aterosclerosis/prevención & control , Enalapril/uso terapéutico , Ergocalciferoles/uso terapéutico , Vitaminas/uso terapéutico , Análisis de Varianza , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Aorta/metabolismo , Aorta/patología , Apolipoproteínas E/deficiencia , Presión Sanguínea/efectos de los fármacos , Quimiocina CCL2/metabolismo , Ciclooxigenasa 2/metabolismo , Enalapril/farmacología , Endotelio/metabolismo , Ergocalciferoles/farmacología , Femenino , Glutatión/metabolismo , Malondialdehído/metabolismo , Ratones , Ratones Noqueados , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Estrés Oxidativo/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Vitaminas/farmacologíaRESUMEN
Even though HIV-1 replication can be suppressed by combination antiretroviral therapy (cART) inflammatory processes still occur, contributing to comorbidities. Comorbidities are attributed to variety of factors, including HIV-1 mediated inflammation. Several HIV-1 proteins mediate central nervous system (CNS) inflammation, including Nef. Nef is an early HIV-1 protein, toxic to neurons and glia and is sufficient to cause learning impairment similar to some deficits observed in HIV-1 associated neurocognitive disorders. To determine whether hippocampal Nef expression by astrocytes contributes to comorbidities, specifically peripheral inflammation, we infused Sprague Dawley rats with GFP- (control) or Nef-transfected astrocytes into the right hippocampus. Brain, lung, and ileum were collected postmortem for the measurement of inflammatory markers. Increased blood-brain-barrier permeability and serum IL-1ß levels were detected in the Nef-treated rats. The lungs of Nef-treated rats demonstrated leukocyte infiltration, macrophage upregulation, and enhanced vascular permeability. Ileal tissue showed reactive follicular lymphoid hyperplasia, increased permeability and macrophage infiltration. The intracerebroventricular application of IL-1 receptor antagonist reduced infiltration of immune cells into ileum and lung, indicating the important role of IL-1ß in mediating the spread of inflammation from the brain to other tissues. This suggests that localized expression of a single viral protein, HIV-1 Nef, can contribute to a broader inflammatory response by upregulation of IL-1ß. Further, these results suggest that Nef contributes to the chronic inflammation seen in HIV patients, even in those whose viremia is controlled by cART.
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Astrocitos/trasplante , Barrera Hematoencefálica/patología , Hipocampo/patología , Enfermedades Pulmonares Intersticiales/etiología , Neuronas/patología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Astrocitos/metabolismo , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Hipocampo/metabolismo , Interleucina-1beta/metabolismo , Enfermedades Pulmonares Intersticiales/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Prediction of lung cancer metastasis relies on post-resection assessment of tumor histology, which is a severe limitation since only a minority of lung cancer patients are diagnosed with resectable disease. Therefore, characterization of metastasis-predicting biomarkers in pre-resection small biopsy specimens is urgently needed. Here we report a biomarker consisting of the phosphorylation of the retinoblastoma protein (Rb) on serine 249 combined with elevated p39 expression. This biomarker correlates with epithelial-to-mesenchymal transition traits in non-small cell lung carcinoma (NSCLC) cells. Immunohistochemistry staining of NSCLC tumor microarrays showed that strong phospho-Rb S249 staining positively correlated with tumor grade specifically in the squamous cell carcinoma (SCC) subtype. Strong immunoreactivity for p39 positively correlated with tumor stage, lymph node invasion, and distant metastases, also in SCC. Linear regression analyses showed that the combined scoring for phospho-Rb S249, p39 and E-cadherin in SCC is even more accurate at predicting tumor staging, relative to each score individually. We propose that combined immunohistochemistry staining of NSCLC samples for Rb phosphorylation on S249, p39, and E-cadherin protein expression could aid in the assessment of tumor staging and metastatic potential when tested in small primary tumor biopsies. The intense staining for phospho-Rb S249 that we observed in high grade SCC could also aid in the precise sub-classification of poorly differentiated SCCs.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas del Citoesqueleto/biosíntesis , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Proteína de Retinoblastoma/metabolismo , Biomarcadores de Tumor/genética , Cadherinas/biosíntesis , Cadherinas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Adhesión Celular/genética , Línea Celular Tumoral , Proteínas del Citoesqueleto/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Clasificación del Tumor , Metástasis de la Neoplasia , Fosforilación , Proteína de Retinoblastoma/genéticaRESUMEN
The probiotic mixture VSL#3 attenuates colitis in patients with Inflammatory Bowel Disease (IBD) and in animal models of this condition, but the mechanisms involved are incompletely understood. VSL#3 alters macrophage morphology and secretory profile in vitro in a polarization-dependent manner. We examined the effect of VSL#3 on macrophages in acute trinitrobenzene sulfonic acid-induced colitis. Rats were randomized to normal, colitis, or colitis+VSL#3 groups. After sacrifice, the colons were evaluated for macroscopic and microscopic damage. Serum cytokine levels were measured, and microbiome analysis undertaken. Total and M1 colonic macrophages, and total and proliferating hepatic macrophages were assessed by double immunofluorescence staining. Colitis+VSL#3 rats had lower macroscopic damage, with less microscopic damage in the proximal colon, compared with colitis alone. Colitis significantly increased colonic macrophage infiltration, which was significantly reduced by VSL#3 treatment. VSL#3 did not decrease the colitis-induced surge of colonic M1 macrophages or hepatic macrophages. VSL#3 reduced colitis-induced serum cytokine levels, and induced restoration of colonic transcript levels for pro-inflammatory, anti-inflammatory, and barrier proteins to, or past, normal levels. Fecal bacteria distribution changed between groups. In summary, the probiotic VSL#3 reduces colitis severity, colonic macrophage infiltration, and serum cytokine levels, but does not dampen the pro-inflammatory phenotype of M1 macrophages.
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Colon/inmunología , Colon/microbiología , Enfermedades Inflamatorias del Intestino/terapia , Macrófagos/inmunología , Probióticos/uso terapéutico , Enfermedad Aguda , Animales , Enfermedad Crónica , Colon/patología , Citocinas/metabolismo , Heces/microbiología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/microbiología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratas Sprague-Dawley , Proteínas de Uniones Estrechas/metabolismo , Ácido TrinitrobencenosulfónicoRESUMEN
AIM: To determine serum vitamin D levels and colonic vitamin D receptor (VDR) expression in inflammatory bowel disease (IBD) and non-IBD patients and correlate these with histopathology. METHODS: Puerto Rican IBD (n = 10) and non-IBD (n = 10) patients ≥ 21 years old scheduled for colonoscopy were recruited. Each patient completed a questionnaire and provided a serum sample and a colonic biopsy of normal-appearing mucosa. For IBD patients, an additional biopsy was collected from visually diseased mucosa. Serum vitamin D levels were measured by ultra-performance liquid chromatography and mass spectrometry. Hematoxylin and eosin stained tissue sections from colonic biopsies were classified histologically as normal or colitis (active/inactive), and scored for the degree of inflammation present (0-3, inactive/absent to severe). Tissue sections from colonic biopsies were also stained by immunohistochemistry for VDR, for which representative diagnostic areas were photographed and scored for staining intensity using a 4-point scale. RESULTS: The IBD cohort was significantly younger (40.40 ± 5.27, P < 0.05) than the non-IBD cohort (56.70 ± 1.64) with a higher prevalence of vitamin D deficiency (40% vs 20%, respectively) and insufficiency (70% vs 50%, respectively). Histologic inflammation was significantly higher in visually diseased mucosa from IBD patients (1.95 ± 0.25) than in normal-appearing mucosa from control patients (0.25 ± 0.08, P < 0.01) and from IBD patients (0.65 ± 0.36, P < 0.05) and correlated inversely with VDR expression in visually diseased colonic tissue from IBD patients (r = -0.44, P < 0.05) and from IBD patients with Crohn's disease (r = -0.69, P < 0.05), but not in normal-appearing colonic tissue from control patients or IBD patients. Control and IBD patient serum vitamin D levels correlated positively with VDR expression in normal colon from control and IBD patients (r = 0.38, P < 0.05) and with patient age (r = 0.54, P < 0.01). CONCLUSION: Levels of serum vitamin D correlate positively with colonic VDR expression in visually normal mucosa whereas inflammation correlates negatively with colonic VDR expression in visually diseased mucosa in Puerto Rican patients.
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Colitis Ulcerosa/sangre , Colon/química , Enfermedad de Crohn/sangre , Mucosa Intestinal/química , Receptores de Calcitriol/análisis , Deficiencia de Vitamina D/sangre , Vitamina D/análogos & derivados , Adulto , Anciano , Biomarcadores/sangre , Biopsia , Estudios de Casos y Controles , Cromatografía Liquida , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/epidemiología , Colon/patología , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/epidemiología , Femenino , Humanos , Inmunohistoquímica , Mucosa Intestinal/patología , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Prevalencia , Puerto Rico/epidemiología , Vitamina D/sangre , Deficiencia de Vitamina D/diagnóstico , Deficiencia de Vitamina D/epidemiología , Adulto JovenRESUMEN
AIM: To investigate the protective effect of paricalcitol and enalapril on renal inflammation and oxidative stress in ApoE-knock out mice. METHODS: Animals treated for 4 mo as group (1) ApoE-knock out plus vehicle, group (2) ApoE-knock out plus paricalcitol (200 ng thrice a week), (3) ApoE-knock out plus enalapril (30 mg/L), (4) ApoE-knock out plus paricalcitol plus enalapril and (5) normal. Blood pressure (BP) was recorded using tail cuff method. The kidneys were isolated for biochemical assays using spectrophotometer and Western blot analyses. RESULTS: ApoE-deficient mice developed high BP (127 ± 3 mmHg) and it was ameliorated by enalapril and enalapril plus paricalcitol treatments but not with paricalcitol alone. Renal malondialdehyde concentrations, p22(phox), manganese-superoxide dismutase, inducible nitric oxide synthase (NOS), monocyte chemoattractant protein-1, tumor necrosis factor-alpha and transforming growth factor-ß1 levels significantly elevated but reduced glutathione, CuZn-SOD and eNOS levels significantly depleted in ApoE-knock out animals compared to normal. Administration of paricalcitol, enalapril and combined together ameliorated the renal inflammation and oxidative stress in ApoE-knock out animals. CONCLUSION: Paricalcitol and enalapril combo treatment ameliorates renal inflammation as well as oxidative stress in atherosclerotic animals.
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AIM: To determine the expression of neurokinin-1 receptor (NK-1R), phosphorylated epidermal growth factor receptor (pEGFR), cyclooxygenase-2 (Cox-2), and vitamin D receptor (VDR) in normal, inflammatory bowel disease (IBD), and colorectal neoplasia tissues from Puerto Ricans. METHODS: Tissues from patients with IBD, colitis-associated colorectal cancer (CAC), sporadic dysplasia, and sporadic colorectal cancer (CRC), as well as normal controls, were identified at several centers in Puerto Rico. Archival formalin-fixed, paraffin-embedded tissues were de-identified and processed by immunohistochemistry for NK-1R, pEGFR, Cox-2, and VDR. Pictures of representative areas of each tissues diagnosis were taken and scored by three observers using a 4-point scale that assessed intensity of staining. Tissues with CAC were further analyzed by photographing representative areas of IBD and the different grades of dysplasia, in addition to the areas of cancer, within each tissue. Differences in the average age between the five patient groups were assessed with one-way analysis of variance and Tukey-Kramer multiple comparisons test. The mean scores for normal tissues and tissues with IBD, dysplasia, CRC, and CAC were calculated and statistically compared using one-way analysis of variance and Dunnett's multiple comparisons test. Correlations between protein expression patterns were analyzed with the Pearson's product-moment correlation coefficient. Data are presented as mean ± SE. RESULTS: On average, patients with IBD were younger (34.60 ± 5.81) than normal (63.20 ± 6.13, P < 0.01), sporadic dysplasia (68.80 ± 4.42, P < 0.01), sporadic cancer (74.80 ± 4.91, P < 0.001), and CAC (57.50 ± 5.11, P < 0.05) patients. NK-1R in cancer tissue (sporadic CRC, 1.73 ± 0.34; CAC, 1.57 ± 0.53) and sporadic dysplasia (2.00 ± 0.45) were higher than in normal tissues (0.73 ± 0.19). pEGFR was significantly increased in sporadic CRC (1.53 ± 0.43) and CAC (2.25 ± 0.47) when compared to normal tissue (0.07 ± 0.25, P < 0.05, P < 0.001, respectively). Cox-2 was significantly increased in sporadic colorectal cancer (2.20 ± 0.23 vs 0.80 ± 0.37 for normal tissues, P < 0.05). In comparison to normal (2.80 ± 0.13) and CAC (2.50 ± 0.33) tissues, VDR was significantly decreased in sporadic dysplasia (0.00 ± 0.00, P < 0.001 vs normal, P < 0.001 vs CAC) and sporadic CRC (0.47 ± 0.23, P < 0.001 vs normal, P < 0.001 vs CAC). VDR levels negatively correlated with NK-1R (r = -0.48) and pEGFR (r = -0.56) in normal, IBD, sporadic dysplasia and sporadic CRC tissue, but not in CAC. CONCLUSION: Immunohistochemical NK-1R and pEGFR positivity with VDR negativity can be used to identify areas of sporadic colorectal neoplasia. VDR immunoreactivity can distinguish CAC from sporadic cancer.
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Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/química , Receptores ErbB/análisis , Inmunohistoquímica , Enfermedades Inflamatorias del Intestino/metabolismo , Receptores de Calcitriol/análisis , Receptores de Neuroquinina-1/análisis , Adulto , Anciano , Biopsia , Estudios de Casos y Controles , Neoplasias Colorrectales/etnología , Neoplasias Colorrectales/patología , Ciclooxigenasa 2/análisis , Femenino , Hispánicos o Latinos , Humanos , Enfermedades Inflamatorias del Intestino/etnología , Enfermedades Inflamatorias del Intestino/patología , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Fosforilación , Puerto Rico/epidemiologíaRESUMEN
BACKGROUND: Patients with Inflammatory Bowel Disease (IBD), most commonly Crohn's disease (CD) or ulcerative colitis (UC), suffer from chronic intestinal inflammation of unknown etiology. Increased proinflammatory macrophages (M1) have been documented in tissue from patients with CD. Anti-inflammatory macrophages (M2) may play a role in UC given the preponderance of Th2 cytokines in this variant of IBD. Animal and clinical studies have shown that the probiotic VSL#3 can ameliorate signs and symptoms of IBD. Although animal data suggests a modulatory effect on macrophage phenotype, the effect of VSL#3 on human macrophages remains unknown. OBJECTIVE: To determine the effect of the probiotic VSL#3 on the phenotype of polarized (M1/M2) and unpolarized (MΦ) human macrophages. METHODS: Human monocyte-derived macrophages, generated by culturing monocytes with M-CSF, were left unpolarized or were polarized towards an M1 or an M2 phenotype by culture with LPS and IFN-γ or IL-4, respectively, and were then cultured in the presence or absence of VSL#3 for 3 days. Changes in macrophage morphology were assessed. Cytokine and chemokine levels in supernatants were determined by multiplex assay. RESULTS: VSL#3 decreased the granuloma-like aggregates of M1 macrophages, increased fibroblast-like M2 macrophages, and decreased fibroblast-like MΦ macrophages. VSL#3 increased the secretion of IL-1ß, IL-6, IL-10, and G-CSF by M1, M2, and MΦ macrophages. VSL#3 exposure maintained the proinflammatory phenotype of M1 macrophages, sustaining IL-12 secretion, increasing IL-23 secretion, and decreasing MDC secretion. Both VSL#3-treated M2 and MΦ macrophages secreted higher levels of anti-inflammatory and pro-healing factors such as IL-1Ra, IL-13, EGF, FGF-2, TGF-α, and VEGF, as well as proinflammatory cytokines, including IL-12 and TNF-α. CONCLUSION: Under our experimental conditions VSL#3 induced a mixed proinflammatory and anti-inflammatory phenotype in polarized and unpolarized macrophages. This differential effect could explain why patients with CD do not respond to probiotic therapy as well as patients with UC.
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While the mechanisms of cellular aging remain controversial, a leading hypothesis is that mitochondrial oxidative stress and mitochondrial dysfunction play a critical role in this process. Here, we provide data in aging rhesus macaques supporting the hypothesis that increased oxidative stress is a major characteristic of aging and may be responsible for the age-associated increase in mitochondrial dysfunction. We measured mitochondrial DNA (mtDNA) damage by quantitative PCR in liver and peripheral blood mononuclear cells of young, middle age, and old monkeys and show that older monkeys have increases in the number of mtDNA lesions. There was a direct correlation between the amount of mtDNA lesions and age, supporting the role of mtDNA damage in the process of aging. Liver from older monkeys showed significant increases in lipid peroxidation, protein carbonylations and reduced antioxidant enzyme activity. Similarly, peripheral blood mononuclear cells from the middle age group showed increased levels in carbonylated proteins, indicative of high levels of oxidative stress. Together, these results suggest that the aging process is associated with defective mitochondria, where increased production of reactive oxygen species results in extensive damage at the mtDNA and protein levels. This study provides valuable data based on the rhesus macaque model further validating age-related mitochondrial functional decline with increasing age and suggesting that mtDNA damage might be a good biomarker of aging.
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Envejecimiento/fisiología , Daño del ADN/fisiología , ADN Mitocondrial , Mitocondrias Hepáticas/genética , Estrés Oxidativo/fisiología , Envejecimiento/metabolismo , Animales , Catalasa/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Glutatión Peroxidasa/metabolismo , Leucocitos Mononucleares/metabolismo , Peroxidación de Lípido/fisiología , Hígado/anatomía & histología , Hígado/metabolismo , Macaca mulatta , Masculino , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/fisiología , Carbonilación Proteica/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
AIM: To investigate the role of epidermal growth factor receptor (EGFR) in colitis-associated dysplasia using the EGFR tyrosine kinase inhibitor erlotinib. METHODS: Sprague-Dawley rats received trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ethanol, ic), followed 6 wk later by reactivation with TNBS (5 mg/kg, iv) for 3 d. To induce colitis-associated dysplasia, rats then received TNBS (iv) twice a week for 10 wk. One group received erlotinib (10 mg/kg, ip) for 1 wk before the start of the reactivation of the colitis and 2 wk after (21 d); the rest received the vehicle. After rats were euthanized, the colons were removed and analyzed for damage and expression of the EGFR downstream effectors Erk1/2 and c-Myc. RESULTS: Ninety percent of the vehicle-treated animals had dysplasia in any region of the colon. Erlotinib-treated animals had a significant decrease in the incidence of dysplasia compared to vehicle-treated animals in all regions of the colon (50.00% ± 11.47% vs 90.00% ± 10.00% in proximal, P < 0.05; 15.00% ± 8.19% vs 50.00% ± 16.67% in mid, P < 0.05; and 20.00% ± 9.17% vs 70.00% ± 15.28% in distal, P < 0.01). Erlotinib-treated animals also had reduced cell proliferation, reduced active Erk1/2, and reduced c-Myc in colon epithelium compared with the vehicle-treated animals. In vitro, erlotinib treatment was shown to markedly decrease c-Myc and pErk1/2 levels in rat epithelial cells. Proliferation of rat epithelial cells was stimulated by epidermal growth factor and inhibited by erlotinib (P < 0.05). CONCLUSION: Erlotinib can decrease the development of colitis-associated dysplasia, suggesting a potential therapeutic use for erlotinib in patients with long-standing colitis.
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Colitis/complicaciones , Colitis/tratamiento farmacológico , Colitis/patología , Neoplasias del Colon/etiología , Progresión de la Enfermedad , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinas/uso terapéutico , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Colitis/inducido químicamente , Colon/efectos de los fármacos , Colon/patología , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib , Humanos , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Ratas , Ratas Sprague-Dawley , Ácido Trinitrobencenosulfónico/farmacologíaRESUMEN
AIM: The role of substance P and the neurokinin-1 receptor (NK-1R) in the transition from inflammation to dysplasia in inflammatory bowel disease is not clear. MATERIALS AND METHODS: Colitis-associated dysplasia was induced in Sprague-Dawley rats by intracolonic, then systemic, administration of trinitrobenzene sulfonic acid. One group of animals received the NK-1R antagonist SR140333; the rest received vehicle. Colons were removed and analyzed for damage and expression of NK-1R downstream components. RESULTS: The NK-1R antagonist-treated animals had significantly reduced macroscopic and microscopic damage and decreased incidence of inflammatory bowel disease. Twice as many of these animals had a normal diagnosis in any region of the colon. A decrease in proliferation index, Cox-2 expression, and active Erk1/2 was found compared with the vehicle-treated group. In Caco-2 cells, Erk1/2 was activated by substance P and prostaglandin E2. CONCLUSION: A selective NK-1R antagonist may delay the development of further colonic damage, offering a potential treatment for patients with long-standing colitis.
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Antineoplásicos/farmacología , Colitis/complicaciones , Neoplasias del Colon/prevención & control , Antagonistas del Receptor de Neuroquinina-1 , Piperidinas/farmacología , Quinuclidinas/farmacología , Animales , Western Blotting , Transformación Celular Neoplásica/efectos de los fármacos , Colitis/inducido químicamente , Colitis/patología , Neoplasias del Colon/etiología , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inmunohistoquímica , Masculino , Ratas , Ratas Sprague-Dawley , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
Inflammatory bowel disease (IBD) is a gastrointestinal disorder of unknown etiology or cure. One complication of IBD is an increased risk for development of colon cancer. The aims of this study were to use a previously established rat model of colitis to develop a new model of colitis-associated colon cancer and ascertain the involvement of three cancer-related genes: K-ras, adenomatous polyposis coli (APC), and p53. Four groups of rats were used: reactivated 1,2-dimethylhydrazine [DMH; trinitrobenzene sulfonic acid (TNBS) was used to induce colitis followed by a weekly s.c. dose of DMH], prolonged reactivation (inflammation was induced with TNBS, then maintained twice a week), saline-DMH (animals received saline instead of TNBS followed by a weekly dose of DMH), and normal (received no treatment). Animals were sacrificed at 5, 10, or 15 weeks, and colon samples were taken for pathologic analysis and gene mutation detection. No dysplasia was found in the normal group. The highest incidences of dysplasia were as follows: prolonged reactivation group at 5 weeks (60%), reactivated DMH group at 10 weeks (83%), and saline-DMH group at 15 weeks (67%). Carcinoma was found in both the prolonged reactivation and saline-DMH groups. No mutations were found in the K-ras oncogene; however 62% of the APC samples (exon 15 at nucleotide 2778) and 76% of p53 (exon 6 at nucleotide 1327) showed substitutions. The prolonged reactivation group may be considered a new model of colitis-associated colon cancer, offering the potential to study cancer prevention strategies for patients with IBD.