RESUMO
Despite rice consumption, rice bran as a byproduct of rice milling contains higher arsenic (As). The present study evaluated the metabolic potency of in vitro cultured human colon microbiota toward As from five rice bran products with 0.471-1.491 mg of As/kg. Arsenic bioaccessibility ranged from 52.8 to 78.8% in the gastric phase, and a 1.2-fold increase (66.0-95.8%) was observed upon the small intestinal phase. Subsequently, a significant decline of As bioaccessibility (11.3-63.6%) and a high methylation percentage of 18.5-79.8% were found in the colon phase. The predominant As species in the solid phase was always As(V) (49.6-63.4%), and As-thiolate complexes increased by 10% at the end of colon incubation. Human gut microbiota could induce As bioaccessibility lowering and As transformation in rice bran, which illustrated the importance of food-bound As metabolism in the human body. This will result in a better understanding of health implications associated with As exposures.
Assuntos
Arsenicais/metabolismo , Bactérias/metabolismo , Colo/metabolismo , Intestino Delgado/metabolismo , Oryza/química , Arsenicais/química , Bactérias/classificação , Bactérias/isolamento & purificação , Biotransformação , Colo/química , Colo/microbiologia , Microbioma Gastrointestinal , Humanos , Intestino Delgado/química , Intestino Delgado/microbiologia , Metilação , Oryza/metabolismo , Medição de RiscoRESUMO
BACKGROUND: The basal pattern of p53 expression, defined as its immunoreactivity confined to the basal half of the glands, is associated with early neoplastic lesions in ulcerative colitis (UC). However, their clinical utility of this finding is limited by the use of "visual estimation" (approximate immunoreactivity on the basis of scanning the stained slide, without formal counting). This study was designed to analyze the basal pattern of p53 using computer-assisted cytometry and to identify the optimal cutoff value for discriminating between UC-associated early-stage neoplasia and regenerative atypia. METHODS: The specimens were obtained from eight UC patients undergoing colectomy and were classified according to the criteria by the Research Committee of Inflammatory Bowel Disease of the Ministry of Health and Welfare in Japan. Patients with classes UC-IIa (indefinite for dysplasia, probably regenerative), UC-IIb (indefinite for dysplasia, probably dysplastic), and UC-III (definitive dysplasia) were enrolled in the study. Based on the percentage of immunoreactive cells in the basal half of the crypt with visual estimation, basal positivity of p53 was classified into three categories: grade 1 (1 - 9%), grade 2 (10 - 19%), and grade 3 (≥ 20%). Next, crypts classified as grade 3 by visual estimation were analyzed by computer-assisted image analysis. RESULTS: Using visual estimation, grade-3 p53 basal positivity was observed in 46.0% of UC-IIa crypts (128 of 278), 61.9% of UC-IIb crypts (39 of 63), and 94.2% of UC-III crypts (81 of 86). Using image analysis, the median p53 basal positivities were 30.3% in UC-IIa, 52.3% in UC-IIb, and 65.4% in UC-III (P ≤ 0.002). A receiver operating characteristics curve was generated to determine the method's diagnostic utility in differentiating UC-IIa from UC-III. In this cohort, the sensitivity was 0.78; the specificity was 0.98; the negative predictive value was 87.4%; the positive predictive value was 95.5%, and the accuracy was 90.2% with a cutoff value for p53 basal positivity of 46.1%. CONCLUSIONS: Our findings indicate that assessing p53 basal positivity by image analysis with an optimal threshold represents an alternative to visual estimation for the accurate diagnosis of UC-associated early-stage neoplasia. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3588120501252608.
Assuntos
Biomarcadores Tumorais/análise , Colite Ulcerativa/complicações , Colo/química , Neoplasias do Colo/química , Interpretação de Imagem Assistida por Computador , Imuno-Histoquímica , Proteína Supressora de Tumor p53/análise , Adulto , Idoso , Área Sob a Curva , Colectomia , Colo/patologia , Colo/cirurgia , Neoplasias do Colo/etiologia , Neoplasias do Colo/patologia , Neoplasias do Colo/cirurgia , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Curva ROC , Regeneração , Adulto JovemRESUMO
A model-based inversion method was used to obtain quantitative estimates of histological parameters from multispectral images of the colon and to examine their potential for discriminating between normal and pathological tissues. Pixel-wise estimates of the mucosal blood volume fraction, density of the scattering particles and thickness were derived using a two-stage method. In the first (forward) stage reflectance spectra corresponding to given instances of the parameter values were computed using Monte Carlo simulation of photon propagation through a multi-layered tissue. In the second (inversion) stage the parameter values were obtained via optimization using an iterated conditional modes algorithm based on Discrete Markov Random Fields. The method was validated on computer generated data contaminated with noise giving a mean normalized root mean square deviation (NRMSD) of 2.04. Validation on ex vivo images demonstrated that parametric maps show gross correspondence with histological features of mucosa characteristic of cancerous, precancerous and noncancerous colon lesions. The key signs of abnormality were shown to be the increase in the blood volume fraction and decrease in the density of scattering particles.
Assuntos
Colo/química , Neoplasias do Colo/química , Processamento de Imagem Assistida por Computador/métodos , Análise Espectral/métodos , Algoritmos , Colo/patologia , Neoplasias do Colo/patologia , Hemoglobinas/análise , Hemoglobinas/química , Humanos , Cadeias de Markov , Modelos Biológicos , Método de Monte CarloRESUMO
The human gut is colonized by a complex microbiota with multiple benefits. Although the surface-attached, mucosal microbiota has a unique composition and potential to influence human health, it remains difficult to study in vivo. Therefore, we performed an in-depth microbial characterization (human intestinal tract chip (HITChip)) of a recently developed dynamic in vitro gut model, which simulates both luminal and mucosal gut microbes (mucosal-simulator of human intestinal microbial ecosystem (M-SHIME)). Inter-individual differences among human subjects were confirmed and microbial patterns unique for each individual were preserved in vitro. Furthermore, in correspondence with in vivo studies, Bacteroidetes and Proteobacteria were enriched in the luminal content while Firmicutes rather colonized the mucin layer, with Clostridium cluster XIVa accounting for almost 60% of the mucin-adhered microbiota. Of the many acetate and/or lactate-converting butyrate producers within this cluster, Roseburia intestinalis and Eubacterium rectale most specifically colonized mucins. These 16S rRNA gene-based results were confirmed at a functional level as butyryl-CoA:acetate-CoA transferase gene sequences belonged to different species in the luminal as opposed to the mucin-adhered microbiota, with Roseburia species governing the mucosal butyrate production. Correspondingly, the simulated mucosal environment induced a shift from acetate towards butyrate. As not only inter-individual differences were preserved but also because compared with conventional models, washout of relevant mucin-adhered microbes was avoided, simulating the mucosal gut microbiota represents a breakthrough in modeling and mechanistically studying the human intestinal microbiome in health and disease. Finally, as mucosal butyrate producers produce butyrate close to the epithelium, they may enhance butyrate bioavailability, which could be useful in treating diseases, such as inflammatory bowel disease.
Assuntos
Clostridium/isolamento & purificação , Clostridium/fisiologia , Colo/microbiologia , Trato Gastrointestinal/microbiologia , Modelos Biológicos , Acil Coenzima A/metabolismo , Adulto , Butiratos/metabolismo , Clostridium/classificação , Clostridium/genética , Colo/química , Ecossistema , Fezes/microbiologia , Humanos , Mucosa Intestinal/microbiologia , Masculino , Método de Monte Carlo , Mucinas/metabolismo , RNA Ribossômico 16S/genéticaRESUMO
Colorectal cancer is a major health issue worldwide. Conventional white light endoscopy (WLE) coupled to histology is considered as the gold standard today and is the most widespread technique used for colorectal cancer diagnosis. However, during the early stages, colorectal cancer is very often characterized by flat adenomas which develop just underneath the mucosal surface. The use of WLE, which is heavily based on the detection of morphological changes, becomes quite delicate due to subtle or quasi-invisible morphological changes of the colonic lining. Several techniques are currently being investigated in the scope of providing new tools that would allow such a diagnostic or assist actual techniques in so doing. We hereby present a novel technique where high spatial resolution MRI is combined with autofluorescence and reflectance spectroscopy in a bimodal endoluminal probe to extract morphological data and biochemical information, respectively. The design and conception of the endoluminal probe are detailed and the promising preliminary results obtained in vitro (home-built phantom containing eosin and rhodamine B), on an organic sample (the kiwi fruit) and in vivo on a rabbit are presented and discussed.
Assuntos
Colo/química , Endoscopia/métodos , Imageamento por Ressonância Magnética/métodos , Espectrometria de Fluorescência/métodos , Actinidia/química , Animais , Endoscopia/instrumentação , Amarelo de Eosina-(YS)/química , Fezes/química , Gadolínio/química , Imageamento por Ressonância Magnética/instrumentação , Óptica e Fotônica , Imagens de Fantasmas , Coelhos , Rodaminas/química , Espectrometria de Fluorescência/instrumentaçãoRESUMO
The aim of this study was to investigate the in vitro dissolution characteristics of pH-responsive polymers in a variety of simulated fluids. Prednisolone tablets were fabricated and coated with the following polymer systems: Eudragit S (organic solution), Eudragit S (aqueous dispersion), Eudragit FS (aqueous dispersion) and Eudragit P4135 (organic solution). Dissolution tests were conducted using a pH change method whereby tablets were transferred from acid to buffer. Three different buffer media were investigated: two compendial phosphate buffers (pH range 6.8-7.4) and a physiological buffer solution (Hanks buffer) with very similar ionic composition to intestinal fluid (pH 7.4). There was considerable drug release from tablets coated with Eudragit P4135 in acid, prompting discontinuation of further investigations of this polymer. Eudragit S (organic solution), Eudragit S (aqueous dispersion) and Eudragit FS on the other hand prevented drug release in acid, though subsequent drug release in the buffer media was found to be influenced by the duration of tablet exposure to acid. At pH 7.4 drug release rate from the polymer coated tablets was similar in the two compendial media, however in the physiological buffer, they were found to differ in the following order: Eudragit S (aqueous dispersion)>Eudragit FS>Eudragit S (organic solution). The results indicate that the tablets coated with the newer Eudragit FS polymer would be more appropriate for drug delivery to the ileo-colonic region in comparison to the more established Eudragit S. More importantly, however, dissolution in the physiological buffer was found to be markedly slower for all the coated tablets than in the two compendial buffers, a result akin to reported slower dissolution of enteric coated tablets in vivo. There is therefore the need to adequately simulate the ionic composition of the intestinal fluid in the dissolution media.
Assuntos
Colo/química , Íleo/química , Preparações Farmacêuticas/química , Polímeros/química , Líquidos Corporais/química , Soluções Tampão , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ácidos Polimetacrílicos/química , Prednisolona/química , Solubilidade , Comprimidos com Revestimento Entérico , Fatores de TempoRESUMO
The Epidermal Growth Factor Receptor (EGFR) plays a role in multiple tumor cell processes and is targeted by several anticancer therapies. Although EGFR mutations may determine tumor susceptibility in a small proportion of patients, knowledge of the EGFR signaling pathway status in tumors may help guide further drug development and hypothesis-driven combination studies. We aimed to validate and apply a novel computer-aided immunohistochemical (IHC) technique to characterize the status of EGFR signaling in matched colorectal tumor and normal colon tissue samples. Tissue Microarrays (TMA)were made from both cancerous and normal colorectal tissue in 18 patients and stained with antibodies against EGFR, phospho-EGFR (pEGFR), Akt, pAkt, MAPK, and pMAPK. TMA's were quantitatively scored using the Automated Cellular Imaging System (ACIS II, Chromavision, Inc). ACIS was compared against cell line Western blotting, ELISA, and visual scoring (0-3+) by a pathologist. We found that ACIS analysis was highly reproducible and results were well correlated with other techniques. A post-scan "image microdissection" technique of analyzing heterogeneous human samples showed good correlation between paired human samples [Pearson correlation for tumors, 0.922 (p < .001)]. Cancer samples had markedly higher staining of pEGFR, Akt, pAkt, MAPK, and pMAPK. We conclude that ACIS IHC of human tissue samples is quantitative, reproducible, and correlates with Western blots and ELISA in cell line pellets as well as pathologist's scores of human samples. Colorectal tumors show higher staining of pEGFR and downstream effectors compared to matched normal colorectal tissues.
Assuntos
Colo/química , Neoplasias Colorretais/química , Receptores ErbB/análise , Processamento de Imagem Assistida por Computador/métodos , Transdução de Sinais , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/genética , Estudos de Avaliação como Assunto , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Immunoblotting , Imuno-Histoquímica , Reprodutibilidade dos TestesRESUMO
AIMS: To investigate the monoclonal antibody M30 for the assessment of apoptosis in colorectal tissues. Although Terminal deoxyribonucleotidyl transferase mediated nick end labelling (TUNEL) and in-situ end labelling (ISEL) are the methods most often used to demonstrate and quantify apoptosis in histological tissue sections, the interpretation and specificity of these techniques have been controversial. Immunohistochemistry using the monoclonal antibody M30 that recognizes caspase-cleaved cytokeratin 18 is considered to be a promising alternative but has yet to be validated against a generally accepted standard. METHODS AND RESULTS: Paraffin sections of normal colonic mucosa (n = 30), normal mucosa obtained from resection margins from carcinomas (n = 30), colorectal adenomas (n = 84) and carcinomas (n = 40) were studied. Apoptosis of epithelial cells was assessed by M30 immunoreactivity and morphological criteria and expressed as a proportion of the total number of cells counted (apoptotic index). Mean apoptotic indices using M30 were 0.18 +/- 0.04% in normal mucosa, 0.42 +/- 0.04% in adenomas and 1.97 +/- 0.24% in carcinomas. Using morphological criteria, these indices were 0.23 +/- 0.03%, 0.62 +/- 0.06% and 1.78 +/- 0.19%, respectively. Apoptotic counts were higher in normal mucosa obtained from resection margins than in genuinely normal mucosa using the M30 antibody. Apoptotic indices obtained by M30 immunoreactivity and morphological criteria were positively correlated (r = 0.71, P < 0.01). CONCLUSION: Assessment of apoptotic cells by M30 immunoreactivity correlates well with morphological criteria. Apoptotic indices increase in the course of the adenoma-carcinoma sequence. Apoptosis in normal mucosa obtained from resection margins differs from genuinely normal mucosa necessitating caution when interpreting studies of apoptosis in normal colonic mucosa. Our findings support the use of the M30 method in the study of apoptosis in colorectal tissues.
Assuntos
Adenocarcinoma/patologia , Adenoma/patologia , Apoptose , Neoplasias Colorretais/patologia , Mucosa Intestinal/patologia , Queratinas/análise , Adenocarcinoma/química , Adenoma/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/imunologia , Contagem de Células , Colo/química , Colo/patologia , Neoplasias Colorretais/química , Feminino , Humanos , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Mucosa Intestinal/química , Queratinas/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
In order to study the clinical significance and change of interleukin (IL)-1 beta and IL-10 concentration in intestinal mucosal tissues in various stage of ulcerative colitis (UC), IL-1 beta and IL10 levels were measured by enzyme linked immunosorbent assays (ELISA). Our results showed that IL-beta level caused by spontaneous secretion in the intestinal mucous tissues in active stage of ulcerative colitis was significantly higher than that in normal controls and in remission stage of ulcerative colitis (P < 0.01, P < 0.001). IL-10 level in various stage of UC was relatively lower in controls, but there was no significantly difference between the two groups. Our study suggested that higher IL-1 beta level in active might play an important role in pathogenesis of UC, and IL-10, as an anti-inflammatory cytokine, was low in active UC, suggesting that it may be a important factor contributing to the development of higher IL-1 beta level.
Assuntos
Colite Ulcerativa/metabolismo , Interleucina-10/análise , Interleucina-1/análise , Reto/química , Adulto , Colo/química , Feminino , Humanos , Mucosa Intestinal/química , MasculinoRESUMO
AIMS: To assess whether Ki67 and p53 immunostaining may assist the diagnosis and grading of ulcerative colitis-related dysplasia. METHODS AND RESULTS: Location of Ki67 staining and location and intensity of p53 staining were assessed in ulcerative colitis (UC) cases showing the features of high-grade dysplasia (HGD, n = 14), low-grade dysplasia (LGD, n = 22), 'indefinite for dysplasia' (n = 12), or regenerative atypia (RA, n = 22). Good intra- and inter-observer reproducibilities were demonstrated in the performance of these assessments. All the dysplasia cases showed extension of Ki67 staining above the basal third of the crypt. Moderate intensity p53 staining was seen in 10/22 RA cases, but strong intensity p53 staining was seen only in cases of dysplasia. All the cases of HGD showed extension of Ki67 and p53 staining above the basal two thirds of the crypt. CONCLUSIONS: Restriction of Ki67 staining to the basal third of the crypt appears to exclude a diagnosis of dysplasia whereas strong intensity p53 staining suggests a diagnosis of dysplasia. Restriction of Ki67 or p53 staining to the basal two-thirds of the crypt appears to exclude a diagnosis of HGD.
Assuntos
Colite Ulcerativa/diagnóstico , Antígeno Ki-67/análise , Lesões Pré-Cancerosas/diagnóstico , Proteína Supressora de Tumor p53/análise , Biópsia , Colo/química , Colo/patologia , Humanos , Imuno-Histoquímica , Reprodutibilidade dos Testes , Índice de Gravidade de DoençaRESUMO
A method for the analysis of omega-hydroxy polyunsaturated fatty acids (omega-HPUFAs) in rat tissue homogenate, supplemented with NADPH and homo-gamma-linolenic acid [20:3(n-6)], arachidonic acid [20:4(n-6)], eicosapentaenoic acid [20:5(n-3)] or docosahexaenoic acid [22:6(n-3)] as a substrate was developed. By ion analysis of chromatograms obtained with reversed-phase HPLC-thermospray MS, many omega-HPUFAs corresponding to each precursor fatty acid could be characterized by the high intensity of the molecular ion (MH+) and quasimolecular ion (MNH4+, MNa+), while other common HPUFAs were characterized by the high intensity of the base ion of MH+--H2O. On a selected-ion monitoring chromatogram of rat brain homogenate, significant amounts of omega-HPUFA from each precursor fatty acid, especially from 22:6(n-3), were detected compared with the amounts found in rat large intestine homogenate. Based on these results, a highly active NADPH-dependent omega-oxidation system is suggested for rat brain homogenate resulting in extensive oxidation of 22:6(n-3).
Assuntos
Química Encefálica , Ácidos Graxos Insaturados/análise , Animais , Cromatografia Líquida de Alta Pressão , Colo/química , Masculino , Espectrometria de Massas , Oxirredução , Ratos , Ratos WistarRESUMO
Surface oximetry was used to evaluate viability of the ascending colon in 60 horses with naturally occurring colonic volvulus or displacement. Tissue surface oxygen tension (PsO2) was measured on the serosal surface of the pelvic flexure after anatomic correction of the colonic obstruction. Horses with PsO2 > 20 mm of Hg were predicted to have viable colon; whereas, horses with PsO2 < or = 20 mm of Hg were predicted to have nonviable colon. Results of surface oximetry were compared with final outcome. For surface oximetry, sensitivity (ability to accurately identify colon that was nonviable) was 53%, but specificity (ability to accurately identify bowel that was viable) was 100%. Negative predictive value (probability that a horse with PsO2 < or = 20 mm of Hg truly had nonviable bowel) was 87%, and positive predictive value (probability that a horse with PsO2 < or = 20 mm of Hg truly had nonviable bowel) was 100%. The overall accuracy was 88%. Of the 45 horses that had a colonic PsO2 > 20 mm of Hg and survived, 7 had been given, on the basis of subjective assessment of visual criteria, a good prognosis, 28 had been given a guarded prognosis, and 10 had been given a poor prognosis. Of the horses that had a colonic PsO2 > 20 mm of Hg but died after surgery because of further colonic infarction, confirmed at necropsy, 4 had been given a poor prognosis, and 3 had been given a guarded prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)