Expression of CAIII and Hsp70 Is Increased the Mucous Membrane of the Posterior Commissure in Laryngopharyngeal Reflux Disease

PURPOSE: We tried to evaluate the difference in the expression of carbonic anhydrase (CA) III and heat shock protein (Hsp) 70 between laryngopharyngeal reflux disease (LPRD) and non-LPRD patients. MATERIALS AND METHODS: The study involved 28 patients who underwent laryngeal microsurgery due to benign laryngeal disease from March to August 2008. Reflux symptom index (RSI) and reflux finding score (RFS) were measured for each person, and they were assigned either to the LPRD group (n=10) or non-LPRD group (n=18). Tissue samples were obtained from the mucosa of posterior commissure, and immunohistochemistry (IHC) staining of CAIII and Hsp70 was performed. The IHC scores were measured and compared with clinical features including RSI and RFS. RESULTS: Total 10 patients were assigned as LPRD group, and 18 patients were as control group. The mean IHC score of CAIII and Hsp70 was 1.70+/-1.06 and 1.90+/-0.88, respectively, in LPRD patients, whereas the mean IHC score of CAIII and Hsp70 was 0.78+/-0.73 and 0.94+/-0.87, respectively, in non-LPRD patients. The difference between two groups was statistically significant (p<0.05). CONCLUSION: CAIII and Hsp70 expressions were higher in LPRD patients that in non-LPRD patients, suggesting the possibility as one of biomomarker in LPRD diagnosis.

Glycomics expression analysis of sulfated glycosaminoglycans of human colorectal cancer tissues and non-neoplastic mucosa by electrospray ionization mass spectrometry / Análise da expressão de glicosaminoglicanos sulfatados no tecido humano neoplásico colorretal e na mucosa não neoplásica por espectrometria de massa por ionização por electrospray

ABSTRACT Objective To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression. Methods Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student’st test. Results The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03). Conclusion The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic tissues.

RESUMO Objetivo Determinar a presença de glicosaminoglicanos na matriz extracelular do tecido conjuntivo colorretal neoplásico e não neoplásico, tendo em vista seu papel central no desenvolvimento e na progressão dos tumores. Métodos Amostras de tecidos colorretais neoplásicos e não neoplásicos foram obtidas de 64 pacientes operados com carcinoma colorretal sem metástases a distância. As expressões de heparan sulfato, sulfato de condroitina e sulfato de dermatan e seus fragmentos foram analisadas por espectrometria de massa por ionização por electrospray, com técnica de extração e quantificação de glicosaminoglicanos após proteólise e eletroforese. Para análise estatística, utilizaram-se média, desvio padrão e teste t de Student. Resultados Em gel de agarose, os glicosaminoglicanos extraídos de tecido colorretal mostraram três bandas eletroforéticas. A espectrometria de massa por ionização por electrospray mostrou fragmentos de dissacarídeos característicos de glicosaminoglicanos e indicou sua característica estrutural. Alguns picos na espectrometria de massa por ionização por electrospray não foram caracterizados como fragmentos de açúcares, sugerindo a presença de fragmentos de proteínas estruturais dos proteoglicanos, formadas durante a purificação dos glicosaminoglicanos. A quantidade média de condroitina e dermatan aumentou no tecido neoplástico em relação ao tecido normal (p=0,01). Por outro lado, a quantidade média de heparan foi menor no tecido neoplásico em relação ao tecido normal (p=0,03). Conclusão O método empregado permitiu determinar o perfil estrutural dos glicosaminoglicanos nas amostras. Tecidos neoplásicos apresentaram maiores quantidades de sulfato de condroitina e sulfato de dermatan em comparação com os não neoplásicos, enquanto o sulfato de heparan foi encontrado em menores quantidades nos tecidos neoplásicos.

Efeito da mitomicina-C tópica sobre os depósitos de colágeno total na submucosa das pregas vocais íntegras de suínos / Effect of topical mitomycin-C on total collagen deposits on the submucosa of intact vocal folds in swine

OBJETIVO: Estudar o efeito da aplicação tópica de Mitomicina-C em diferentes concentrações sobre os depósitos de colágeno total na submucosa de pregas vocais íntegras de suínos. MÉTODOS: Os animais foram divididos em três grupos de acordo com o conteúdo da solução tópica aplicada sobre as pregas vocais: solução fisiológica 0,9 por cento (grupo controle); Mitomicina-C tópica 4 mg/mL (grupo 1); e Mitomicina-C 8 mg/mL (Grupo 2). Após 30 dias da aplicação, os animais foram submetidos à eutanásia, coletado amostras das pregas vocais e coradas pela técnica do picrosirius red com polarização para a quantificação computadorizada da deposição do colágeno, através do programa Image Pro plus 4.5Ò. Compararam-se as médias de área do colágeno depositado na submucosa das pregas vocais dos três grupos através do teste não paramétrico de Mann-Whitney. RESULTADOS: As médias das áreas de depósito colágeno na submucosa das pregas vocais foram de 3.110,44 micrômetros quadrados (mm²); 3.115,98 mm² e 3.105,78 mm² nos grupos controle, 1 e 2 respectivamente. Não houve diferença significativa nas áreas de depósitos de colágeno total da submucosa das pregas vocais entre os três grupos estudados (p>0,05). CONCLUSÃO: A mitomicina-C aplicada topicamente em pregas vocais suínas íntegras não alterou significativamente a deposição de colágeno na submucosa.

OBJECTIVE: To compare the effects of topical mitomycin-C at different concentrations on submucosal collagen deposition on the vocal folds of swine. METHODS: The animals were divided into three groups according to the composition of the topical solution to be applied to the vocal folds: 0.9 percent saline solution (control group); 4 mg/ml mitomycin-C (group 1) and 8 mg/ml mitomycin-C (group 2). Thirty days after the application, all animals were sacrificed, their vocal folds were collected and stained by the picrosirius red technique, and submucosal collagen deposition areas were estimated by the Image Pro Plus 4.5® software. Mann-Whitney test was used to compare differences between parameters of each group. RESULTS: The means of the areas of submucosal collagen deposits on vocal folds were 3110.44 square micrometers (mm²), 3115.98 mm² and 3105.78 mm² for groups control, 1 and 2, respectively. There were no statistical differences across the three groups (p>0.05). CONCLUSION: Mitomycin-C topically applied to intact vocal folds of swine did not alter submucosal collagen deposition.

Expression of substance P in human laryngopharynx and gastrointestine in sudden erethistic death / 法医学杂志

OBJECTIVE@#To study the expression of substance P (SP) in human sudden erethistic death, and to seek objective morphological supports to diagnose sudden erethistic death for forensic medicine.@*METHODS@#The expression of SP was detected with immunohistochemical technique on 15 human laryngopharynx and gastrointestine of sudden erethistic death, and 20 sudden death of heart attack as control. The images of SP were analyzed by image analyzer, and the positive indexes (PI) were calculated.@*RESULTS@#SP expression in the experimental groups was significantly stronger than that in the control one (P < 0.001).@*CONCLUSION@#SP expression can offer an objective morphological reference support for forensic diagnosing sudden erethistic death.

Preparation and in vitro evaluation of liposomal/niosomal delivery systems for antifungal drug clotrimazole.

Clotrimazole, an imidazole derivative antifungal agent is widely used for the treatment of mycotic infections of the genitourinary tract. In order to develop alternative formulation for the vaginal administration of clotrimazole to provide sustained and controlled release of appropriate drug for local vaginal therapy, liposomes/niosomes were evaluated as delivery vehicles. To optimize the preparation of liposomes/niosomes with regards to size and entrapment efficiency, multilamellar liposomes/niosomes containing drug were prepared by lipid hydration method. The ability of the systems to deliver clotrimazole into and through the mucosa was evaluated in vitro using rabbit vaginal mucosa with vertical Franz diffusion cells. The in vitro permeation data showed that the liposomes/niosomes system increased the clotrimazole total penetration through the vaginal mucosa by 1.6, 1.5-fold, the accumulation of clotrimazole into the mucosa was increased by 3.1, 2.3-fold, respectively, as compared with control during 24 hr. These results suggest that the studied liposomes/niosomes systems may be appropriate vesicles for the vaginal mucosa delivery of clotrimazole for local vaginal therapy.

An investigation of the nature of bladder mucosal glycoconjugates and their role in interstitial cystitis.

The long-term objective of this study is to elucidate the role of bladder mucosal glycosaminoglycans and mucin glycoproteins in the development of interstitial cystitis and other bladder diseases. Bladder biopsies and urine samples from patients and healthy controls were analyzed for glycoconjugates by biochemical and immunochemical methods. Due to the limited availability of human bladders for research purposes, detailed analysis of rabbit bladders glycoconjugates were also carried out. Biochemical analysis of rabbit bladders indicate that while the major portion of the glycoconjugates in the urothelium is sialoglycoprotein, low levels of heparan sulfate and chondroitin sulfate are also present. The correlating immunohistochemical data show very weak staining of the rabbit bladder epithelium by antiglycosaminoglycan antibodies. In contrast, the lamina propria and muscle layers stained intensely for chondroitin sulfate and hyaluronic acid. Thus, the quantity of glycosaminoglycans associated with the bladder epithelial layer, particularly as extracellular matrix components on the luminal surface of the bladder, appears insignificant. On the other hand, several lectins and anti-epitectin (a MUC1 sialoglycoprotein) antibodies showed strong staining of the luminal surface of rabbit and normal human bladders. Further, preliminary results with anti-epitectin antibodies reveal a weaker and patchy staining of biopsy specimens from interstitial cystitis patients compared to controls. The urinary levels of glycosaminoglycans and epitectin, in interstitial cystitis patients and healthy controls were determined by chemical or immunoassays. Urinary epitectin, but not glycosaminoglycans, was decreased in interstitial cystitis patients.

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.