Estabelecimento de protocolos in vitro e in situ para estudos de erosão dentária / Establishment of in vitro and in situ protocols for dental erosion studies

O objetivo foi comparar uma formulação de saliva artificial com e sem mucina (in vitro) com a saliva humana (in situ) na inibição da desmineralização (subprojeto I) e no reendurecimeto de lesões de erosão (subprojeto II), e a influência do tipo de dispositivo intrabucal (mandibular X palatino) no desgaste erosivo do esmalte (subprojeto III). No subprojeto I, blocos de esmalte bovino foram selecionados pela dureza de superfície e randomizados entre os grupos: GI - saliva humana (n=30), GII - saliva artificial sem mucina (n=15), GIII - saliva artificial com mucina (n=15) e GIV - água deionizada (n=15). Quinze voluntários utilizaram o dispositivo palatino por um período 2 horas (GI). Nos grupos GII, GIII e GIV, os blocos foram imersos em suas respectivas soluções por um período de 2 horas. Imediatamente após, tanto os blocos do grupo in situ quanto dos grupos in vitro foram submetidos ao desafio erosivo inicial com ácido cítrico 1% (pH 3,6) por 4 minutos. A microdureza final foi mensurada para determinar a porcentagem de perda de dureza. No subprojeto II, os blocos, após seleção, foram erodidos in vitro e randomizados entre grupos como no subprojeto I. Para a erosão, os blocos foram imersos em ácido cítrico 1% (pH 3,6) por 4 minutos. A seguir, no grupo GI, 15 voluntários utilizaram dispositivos palatinos durante 2 horas. Nos outros grupos os blocos foram imersos nas salivas artificiais com (GIII) e sem mucina (GII) e água deionizada (GIV) por 2 horas. A precipitação de minerais sobre o esmalte foi avaliada por meio da porcentagem de recuperação de dureza. No subprojeto III, após seleção dos blocos pela dureza, os mesmos foram aleatorizados em 2 grupos (n=20): GI - dispositivo palatino e GII - dispositivo mandibular. A ciclagem consistiu na imersão dos dois dispositivos em ácido clorídrico 0,01 M (pH 2,3) por 2 minutos, 4X/dia durante 5 dias. A perda do esmalte foi avaliada por perfilometria e os voluntários responderam a um questionário quanto ao conforto dos dispositivos. Nos subprojetos I e II, os dados foram submetidos aos testes ANOVA e Tukey e no subprojeto III foi aplicado o Teste T pareado (p<0,05). Nos subprojetos I e II observou-se que todas as salivas estudadas foram capazes de promover uma recuperação de dureza do esmalte e nenhuma diferença foi encontrada entre elas (p<0,05). No ensaio de desmineralização, a saliva artificial com mucina e a saliva humana (in situ) promoveram menor perda de dureza, não mostrando diferença entre elas (p<0,05). No subprojeto III os resultados mostraram que os blocos localizados no dispositivo palatino (GI) apresentaram maior desgaste erosivo quando comparados aos do dispositivo mandibular (GII). Além disso, todos voluntários relataram maior conforto no uso do dispositivo palatino. Considerando que o dispositivo palatino é mais confortável e resultou em maior perda de esmalte quando comparado ao mandibular, sugere-se o uso de dispositivos palatinos em protocolos in situ que queiram mimetizar pacientes com alto risco de erosão dentária. Para estudos in vitro, a saliva com mucina mostrou-se como uma boa substituta à saliva humana.(AU)

The aim was to compare artificial saliva formulation with and without mucin (in vitro) with human saliva (in situ) on the inhibition of erosive demineralization (subproject I) and on the rehardening of erosion lesions (subproject II), and analyze the influence of the type of intraoral appliance (mandibular X maxillary) in enamel wear caused by erosive challenges (subproject III). In the subproject I, bovine enamel blocks were selected by initial surface hardness and randomized among the groups: GI - human saliva (n=30), GII - artificial saliva without mucin (n=15), GIII - artificial saliva with mucin (n=15) and GIV - deionized water (n=15). Fifteen volunteers wore palatal appliances for 2 hours (GI). In the GII, GIII and GIV groups, the blocks were immersed to the respective solutions for 2 hours. Subsequently, both in vitro and in situ blocks were subjected to initial erosive challenge in 1% citric acid (pH 3.6) for 4 minutes. Final enamel hardness was measured to determine the protective capacity of saliva tested by percentage of hardness loss. For the subproject II, after blocks selection, they were in vitro eroded and randomly among the groups as in subproject I. For erosion, the enamel blocks were immersed on 1% citric acid (pH 3.6) for 4 minutes. Then, in the GI, 15 volunteers wore palatal appliances for 2 hours. In the other groups the blocks were immersed in artificial saliva with (GIII) and without mucin (GII) and deionized water (GIV) for 2 hours. The minerals precipitation on the enamel was evaluated by the percentage of hardness recovery. On subproject III, after enamel blocks selection by surface hardness, they were randomly divided into 2 groups (n=20): GI - palatine appliance and GII - mandibular appliance. Erosive cycling consists in immersing of both devices in 0.01 M hydrochloric acid (pH 2.3) for 2 minutes, 4X/day during 5 days. The analysis of the wear was measured by profilometry and volunteers answered a questionnaire about the comfort of the devices. In the subprojects I and II, data were analyzed by one-way ANOVA and Tukeys test, and in the subproject III It was applied paired t-test (p <0.05). In subprojects I and II it was observed that all studied saliva promoted enamel rehardening and no difference was found between them (p <0.05). In the demineralization test, the artificial saliva with mucin and human saliva (in situ) provided lower enamel hardness loss, showing no difference between them (p<0.05). In the subproject III, the results showed that the specimens allocated in palatine appliance (GI) presented significantly higher erosive wear when compared to the specimens fixed in mandibular appliance (GII). In addition, all volunteers reported greater comfort in using the palatal device. Considering the palatal device is more comfortable and resulted in higher enamel loss when compared to the mandibular device, it is suggested the use of palatine appliances in in situ protocols who want to mimic a patient at high risk of dental erosion. For in vitro studies, the saliva with mucin might be a good substitute for human saliva.(AU)

Tissue quantification of neutral and acid mucins in the mucosa of the colon with and without fecal stream in rats / Quantificação tecidual de mucinas neutras e ácidas na mucosa do cólon com e sem trânsito intestinal em ratos

PURPOSE: To quantify the intensity of the expression of neutral and acids mucins in mucosa of the colon with and without fecal stream and to correlate this with the duration of fecal transit diversion. METHODS: Thirty male Wistar rats were subjected to fecal transit deviation in the left colon by a proximal colostomy and a distal mucous fistula. The animals were divided into three experimental groups, according to whether sacrificing would be performed six, 12 or 18 weeks after surgery. The expression of neutral and acid mucins was evaluated using the histochemical techniques of Periodic Acid Schiff and Alcian Blue, respectively. The tissue mucins expression was quantified by computer-assisted image analysis software (NIS-Elements) in the segments with and without fecal stream. Student's paired t test was used to compare the quantities of mucins in colon with or without fecal stream and variance between the experimental groups by ANOVA and Newman-Keuls post-test, establishing level of signification of 5 percent (p<0.05). RESULTS: There were significant decreased quantities of acid and neutral mucins in the colon without transit, compared with the colon with fecal stream, independent of the duration of exclusion. There was increased expression of neutral mucins in the colon with fecal stream after 12 and 18 weeks of exclusion. There was no increase in the expression of acid mucins in the colon with transit as the duration of fecal transit exclusion progressed. There was increased production of acid mucins in the animals submitted to diversion of the fecal stream for 18 weeks, compared with those subjected to diversion for 6 and 12 weeks. In the colon without fecal stream, there was increased expression of neutral mucins after 12 and 18 weeks of exclusion. CONCLUSIONS: Deviation of the fecal stream decreased the expression of acid and neutral mucins in the segments without fecal transit, compared with segments with transit. Regardless of ...

OBJETIVO: Quantificar a intensidade de expressão de mucinas neutras e ácidas na mucosa cólica provida e desprovida de trânsito intestinal relacionando-a ao tempo de exclusão fecal. MÉTODOS: Trinta ratos Wistar machos foram submetidos à derivação do trânsito no cólon esquerdo por colostomia proximal e fístula mucosa distal. Os animais foram divididos em três grupos experimentais segundo o sacrifício ter sido realizado seis, 12 e 18 semanas após a cirurgia. A avaliação da expressão de mucinas neutras e ácidas na mucosa cólica foi realizada com as técnicas histoquímicas do Periódico Ácido de Schiff e Azul de Alcian, respectivamente. A quantificação da expressão tecidual das mucinas foi com auxílio de programa de análise de imagem assistida por computador (NIS-Elements) nos segmentos providos e desprovidos de trânsito fecal. Utilizou-se o teste t de Student pareado na comparação da expressão de mucinas nos segmentos com e sem trânsito e a variação na expressão entre os grupos experimentais pelo teste ANOVA e pós-teste de Newmann-Keuls, estabelecendo-se nível de significância de 5 por cento (p<0,05). RESULTADOS: Houve redução na quantidade de mucinas neutras e ácidas no cólon desprovido de trânsito quando comparado ao cólon provido de trânsito, independente do tempo de exclusão. Ocorreu aumento na expressão de mucinas neutras no cólon provido de trânsito intestinal após 12 e 18 semanas de exclusão. Não houve aumento na expressão de mucinas ácidas no cólon provido de trânsito com o progredir do tempo exclusão de trânsito fecal. Ocorreu aumento na produção de mucinas ácidas nos segmentos com exclusão de trânsito por 18 semanas quando comparados aos animais submetidos à exclusão por seis e 12 semanas. No cólon desprovido de trânsito ocorreu aumento na expressão de mucinas neutras após 12 e 18 semanas de exclusão. CONCLUSÕES: A derivação do trânsito fecal diminui a expressão de mucinas ácidas e neutras nos segmentos desprovidos de trânsito fecal ...

á-N-acetylglucosamine-linked O-glycans of sialoglycoproteins (Tc-mucins) from Trypanosoma cruzi Colombiana strain

Trypanosoma cruzi sialoglycoproteins (Tc-mucins) are mucin-like molecules linked to a parasite membrane via a glycosylphosphatidylinositol anchor. We previously determined the structures of Tc-mucin O-glycan domains from several T. cruzi strains and observed significant differences among them. We now report the amino acid content and structure of Tc-mucin O-glycan chains from T. cruzi Colombiana, a strain resistant to common trypanocidal drugs. Amino acid analysis demonstrated the predominance of threonine residues (42%) and helped to identify the O-glycans as belonging to a Tc-mucin family that contain a ²-galactofuranose (²-Galf) residue attached to an á-N-acetylglucosamine (á-GlcNAc) O-4, with the most complex glycan, a pentasaccharide-GlcNAc-ol with a branched trigalactopyranose chain, on the GlcNAc O-6. The presence of ²-Galf on O-glycans from T. cruzi Colombiana mucins supports the use of glycosylation as a phylogenetic marker for the classification of Colombiana in the T. cruzi I group.

Mucinas salivales: estructura química, mecanismos de liberación y participación en la defensa no inmunológica de la cavidad bucal / Salivary mucins: chemical structure, release mechanisms and participation in the non-immunological defense of the oral cavity

En este artículo se describen: 1) las características físico-químicas de las mucinas salivales, denominadas MG1 y MG2. 2) El mecanismo de secreción por estimulación simpática y parasimpática. 3) La distinta participación de MG1 y MG2 tanto en la actividad deglutoria como en los mecanismos de defensa de la cavidad bucal, en relación con sus propiedades físico-químicas. 4) El rol de las mucinas salivales en la protección de la mucosa del tracto gastrointestinal. 5) La relación entre las mucinas saliales y las patologías de la cavidad bucal.

Expresion diferencial de los carbohidratos de mucina en endometrios humanos / Differential expression of mucin carbohydrates in human endometriuns

La aplicación de Lectinas para la identificación de oligosacáridos constituyentes de las Mucinas presentes en la superficie celular, es una herramienta técnica muy útil, debido a que, probablemente estas sustancias estén involucrados en procesos tales como invasión y metástasis. En este trabajo, nosotros estudiamos tejido endometrial normal com entidades benignas y malignas, para investigar la presencia de Galactosa beta 1-3 N Acetilgalactosamina (Galbeta 1-3GalNacalpha) y de Galactosa beta 1-3 N Acetilgalactosamina(Galbeta 1-3GalNa alpha y beta) empleando dos Lectinas: Agaricus bisporus (ABL) y Arachis hipogea (PNA) respectivamente. Lo controles fueron realizados com baños de galactosa para PNA y com mucina porcina de estómago de cerdo para ABL. El uso de estas dos Lectinas, permitió encontrar diferencias en los patrones de unión, ya que si bien ambas se unen a los mismos oligosacáridos, ABL realiza la unión en presencia de Acido Siálico mientras que PNA no. Se observaron significativas diferencias en los patrones de unión de ambas lectinas en tejidos, com entidades benignas, malignas y tejido normal. En este último, la marcación fue simpre continua com ambas Lectinas, mientras que fue irregular en el carcinoma.

Study of oesophageal mucin in cases of reflux oesophagitis

The aim of this work was to study the possible qualitative changes in the oesophageal mucins in the presence of reflux oesophagitis. Twenty patients suffering from reflux oesophagitis as well as ten control subjects were included in this study. All patients and controls were subjected to: full history taking, thorough clinical examination, blood picture, barium swallow and upper gastro-intestinal endoscopy with multiple biopsies to emphasize a greater diagnostic yield. These specimens were fixed in 10% formalin solution and paraffin sections were made. Staining was then done by 3 techniques i.e. Haematoxylin and Eosin, Alcian blue and combined Alcian blue and Periodic Acid Schieff technique. Heart burn was the prevelant complaint in patients with GERD [gastroesophageal reflux disease]. This work revealed a statistically significant correlation between hiatus hernia and reflux oesophagitis. [P<0.05]. Alteration of the mucin pattern of the lower oesophagus has been definitely proved in this study. In the present work where was a significant correlation between the predominance of sulfomucin variety and the presence of reflux oesophagitis [P<0.05]. The predominance of sulfomucin variety was also statistically significant over the neutral variety in reflux oesophagitis patients

Histochemical study of epithelial mucin in endometrial hyperplasia and adenocarcinomas

The pattern and type of mucin were studied on 37 curettage specimens. These cases were formed of 25 endometria showing normal proliferative and secretory phase and 12 endometria showing hyperplastic changes. Fifteen hysterectomy specimens from cases of endometrial adenocarcinoma were also examined in this work. All cases were stained by Hematoxylin and Eosin, periodic acid-schiff [P.A.S.] and alcian blue. The cases of endometrial hyperplasia showed the same distribution of both types of mucin secreted by the normal endometrial glands, although increased in quantity. Most of endometrial adenocarcinomas contained different quantities of cellular mucin, which was found to be more abundant and widely distributed in the well differentiated than in the moderately or poorly differentiated ones. It was observed that the histochemical properties, site and pattern of mucin secretion in these cases strongly resembled that seen in the normal endometrium. This may play a role in differentiating adenocarcinoma of endometrial origin from other carcinomas in the female genital tract like that of endocervical origin

Sialic acid variants in benign versus malignant colorectal lesions

The histochemical mucin structure of colorectal lesions has been subject to several studies. Some reports relate changes in mucin pattern to the grade of epithelial dysplasia. Alterations in sialomucin with both its O-and N-residues have been described in colonic malignancy. This work was, thus, carried out on 50 benign and malignant colorectal lesions to study the histochemical structure of sialic acid, by applying the PAT/KPH/PAS technique [introducing a counterstain to the original method described] and the mPAS technique for demonstrating the O-and N-acetyl residues respectively. The PAT/KOH/PAS technique was more informative. It gave a spectrum of colours ranging from red through purple to blue, reflecting normal, partial loss and absence of the O-acetyl residue respectively. In contrast, however, were the results of the mPAS technique. Positive cases have shown only scattered foci of mild positivity in the form of pale, red granular deposits. Among all non-neoplastic lesions examined [n = 9], the PAT/KOH/PAS technique gave a red reaction reflecting normal pattern. In adenomatous polyps without atypia [n = 10] 70% gave a normal, red, reaction while in 30% the reaction was purple. Polyps with atypia [n = 12] revealed more loss of O-residue in cases showing atypia. There was also a notable loss of O-residue with higher grades of malignancy. None of the malignant cases examined revealed a normal red reaction [n = 19], while 25% of well, 75% of moderately and 100% of poorly differentiated adenocarcinomas gave a blue mucin reaction. Results confirm higher loss of O-acetyl residue with severe dysplasia and less differentiated malignant tumors of the colon. The PAT/KOH/PAS technique can, thus, be helpful in predicting the outcome of colonic adenoma by assuming that a red colour reaction reflects its benign nature, while a purple to blue reaction implies the danger of malignant transformation. It can also be used in the interpretation of equivocal malignancies in small colonoscopic biopsies

Peptide mapping reveals differences in the non-glycosylated domains of cystic fibrosis and normal tracheobronchial mucins.

Tracheobronchial mucins from lung mucus secretions of healthy individuals and from patients with cystic fibrosis (CF) were purified according to a protocol established in our laboratory. Following digestion of the purified, reduced-alkylated mucin (free of 118 kDa and 70 kDa components) with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2 and TR-3) were observed upon chromatography on a Superose 6 column using FPLC. TR-1 (glycosylated fraction) contained all of the carbohydrate, while TR-2 and TR-3 fractions had no detectable sugars. Comparison of the amino acid composition of TR-1 fractions from normal and CF individuals revealed no significant differences, while the TR-2 fractions from these mucins showed noticeable differences. Peptide mapping of TR-2 fractions from normal and CF mucins was performed on a C18 reverse phase column using FPLC. The peptide maps of normal mucins were markedly different from CF mucins. A greater number of peptides were seen in the TR-2 fractions of normal mucins when compared to CF mucin TR-2 fractions. In addition, normal TR-2 fractions appeared to be comprised of more hydrophobic peptides when compared to CF TR-2 fractions. These data provide evidence of possible structural differences in the non-glycosylated regions of CF and non-CF mucins, since the TR-2 fractions are essentially derived from the T-domains in the "naked" stretches of the mucin polypeptide backbone.

Aspectos histoquímicos do muco do epitélio de revestimento do oviduto de coelha / Cytochemical observations on the nature of the mucin of the surface epithelium of the rabbit oviduct

Procurou-se detectar, por intermédio de métodos histoquímicos a natureza carboidrática do muco elaborado pelas células secretoras do epitélio que reveste as porçöes do infundíbulo, ampola e istmo do oviduto de coelha em anestro. Com base nos resultados obtidos, foi possível concluir que o oviduto de coelha é revestido por uma camada de muco. Este muco está representado por uma fraçäo neutra e outra ácida. Com relaçäo ao componente ácido, encontramos que as três porçöes do oviduto estäo revestidas por carboximucina, sendo que no infundíbulo encontramos também uma sialomucina e na ampola e no istmo uma sulfomucina. Os autores procuram relacionar estes dados com aspectos fisiológicos