Chronic ear disease with cholesteatoma is characterized by an intrusion of keratinizing stratified squamous epithelium into the middle ear manifesting bone resorption at the interface of the perimatrix. The aim of our study was to investigate the markers of a catabolic process associated with several chronic inflammatory states. We assessed the level of catabolism of glycoconjugates in assays of cholesteatoma extracts, quantifying two lysosomal exoglycosidases: alpha-mannosidase (alpha-MAN) and beta-galactosidase (beta-GAL). Cholesteatomas (n = 15) and normal adult postauricular skin served as controls (n = 15) were collected from the patients during surgery owing to chronic otitis media. To assess exoglycosidase activity, release of p-nitrophenol from p-nitrophenol derivatives of alpha-mannose and beta-galactose was used. In 13 of 15 specimens, we observed significantly higher activity of investigated enzymes in cholesteatoma tissue compared with control tissue (postauricular skin). The mean activity of alpha-MAN from the cholesteatoma cells was 1.76 +/- 1.10 nkat/g wet tissue and 0.61 +/- 0.21 nkat/g wet tissue in the control probes. The mean activity of beta-GAL from the cholesteatoma cells was 1.77 +/- 1.07 nkat/g wet tissue and 0.87 +/- 0.20 nkat/g wet tissue in the control probes. Catabolic reactions involving glycoproteins, glycolipids, and proteoglycans may play a role in cholesteatoma-related bone resorption. The present data indicating that the lysosomal exoglycosidases alpha-MAN and beta-GAL are significantly and consistently elevated suggest the need to further correlations assessment between levels of alpha-MAN and beta-GAL and cholesteatoma behavior. Further research should also evaluate the relative importance of these particular exoglycosidases in manifesting bone resorption in considering the spectrum of identified inflammatory mediators.

Cholesteatoma is a destructive disease characterized by the progressive expansion of keratinizing squamous epithelium in the middle ear and mastoid, and chronic inflammatory reaction of the subepithelial connective tissue. N-Acetyl-beta-d-hexosaminidase (HEX) catalyzes the release of terminal non-reducing N-acetyl-d-hexosamine residues acting on glucosides and galactosides in glycoproteins, G(M2)-gangliosides and glycosaminoglycans (GAGs). In this study the activities of HEX were measured in cholesteatoma tissue and in normal skin to demonstrate a possible role of HEX in bone resorption in the area adjacent to cholesteatoma. Cholesteatomas (n = 21) and normal adult retroauricular skin (controls, n = 21), were collected from patients during surgery due to chronic otitis media. In 20 of 21 specimens a significantly higher activity of HEX was observed in cholesteatoma tissue compared with that in normal skin. Mean release of HEX from the activated cells was 68.55 +/- 30.77 nkat/g wet tissue in cholesteatoma and 31.79 +/- 10.02 nkat/g wet tissue in skin specimens. It may explain the process of bone resorption in the area adjacent to cholesteatoma, i.e. ossicles or temporal bone. This study suggests that drugs inhibiting HEX activity, such as iminocyclitols, may be useful in cholesteatoma treatment.

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No abstract

A number of different types of glycoconjugate are found associated with joint tissue and fluids, comprising glycoproteins, glycolipids and glycosaminoglycans. Oligosaccharide chains of glycoconjugates are degraded by exoglycosidases, and the dominant exoglycosidase found in human blood, synovial fluid, the synovial membrane and chondrocytes of articular cartilage is HEX (N-acetyl-β-hexosaminidase). HEX is localized mostly intracellularly in synovial cells. Serum activity of HEX may be used to monitor the course and efficiency of treatment of Lyme arthritis, and activity of HEX, above 10 μkat/kg of protein in the synovial fluid, suggests rheumatoid disease. There is a shortage of HEX inhibitors able to penetrate synoviocytes, so the development of drugs which inhibit synthesis and/or the activity of HEX will be a promising field for future investigations.

Background. The effect of multiple infusions of infliximab (INF), a chimeric anti-tumor necrosis factor alpha antibody, on the concentration of hexosaminidase (HEX) activity in a synovial cell culture derived from human synovial inflamed fluid obtained from patients suffering from rheumatoid arthritis (RA) has been evaluated. Objectives. The aim of this study was to prove INF efficacy in RA. Material and Methods. Inflamed synovial fluid was taken from RA patients (a study group) and patients who had undergone knee trauma within 7 days (a control group). The following solutions of infliximab were used: 40, 60 and 140 µg/mL. Determination of the concentration of HEX activity in cell cultures was performed after 24, 48, 72 and 96 h of infliximab administration. To identify synoviocytes in cell culture immunohistochemical staining with vimentin and pancytokeratin was performed. Results. A predominance of fibroblast-like synovial cells has been observed in the study group. In the control group the concentration of HEX activity without adding infliximab to the cell culture was 283.00 nkat/mL. After 96 h of incubation with infliximab, the concentrations of HEX activity in cultured synoviocytes according to infliximab doses of 40, 60 and 140 µg/mL were respectively: 280.00, 271.50 and 293.50 nkat/mL. In the study group, the concentration of HEX activity without adding infliximab to the cell culture was 542.27 nkat/mL. The final concentrations of HEX activity of cultured fibroblast-like synovial cells measured after 96 h of incubation with infliximab were: 471.72, 498.27 and 556.72 nkat/mL, according to infliximab doses of 40, 60 and 140 µg/mL. In all groups (besides the infliximab concentration of 140 µg/mL after 96 h of incubation), the level of concentration of HEX activity was significantly higher in the study group compared to the control group, irrespective of infliximab concentration and time of infliximab incubation. Conclusions. Infliximab changes the concentration of HEX activity depending on the drug dose and time of administration (Adv Clin Exp Med 2015, 24, 5, 807-813).

2. Degradation of human articular cartilage Progressive destruction of articular cartilage is a common feature of OA, RA, and LA. The articular cartilage from patients with OA and RA has decreased concentrations of proteoglycans and glycosaminoglycans (GAGs), and the size of GAG molecules is also

Objectives 1) To determine the activity of N-acetylo-beta-D-hexosaminidase (HEX), alpha-mannosidase (alpha-MAN) and beta-galactosidase (beta-GAL)in acquired cholesteatoma and normal retroauricular skin specimens. 2) To assess the level of catabolism of glycoconjugates in assays of cholesteatoma extracts, quantifying HEX, alpha-MAN and beta-GAL. Methods Cholesteatomas (n=21) and normal adult post-auricular skin, served as controls(n=21), were collected during surgery in 2006–2007. Exoglycosidases activity was evaluated by determination, the amount of released p-nitrophenol from p-nitrophenol derivatives of appropriate sugars (beta-N-acetylglucosamine, alpha-mannose, and beta-galactose). Statistical analysis was conducted using the Student's t-test; p<0.05 was regarded as significant. Results In 19 of 21 specimens we observed significantly higher activity of investigated enzymes in cholesteatoma tissue compared with control tissue (p<0.05). Mean activity of HEX, alpha-MAN and beta-GAL from the cholesteatoma cells was adequate: 68.55 ±30.77 nkat/g, 1.85 ±1.14 nkat/g and 1.92 ±1.11 nkat/g wet tissue. In the control probes, adequate: HEX: 31.79 ±10.02 nkat/g, alpha-MAN: 0.69 ±0.24 nkat/g and beta-GAL: 0.86±0.22 nkat/g wet tissue. The correlation of two variables: enzyme activity in cholesteatoma and enzyme activity in skin specimens was positive. Pearson's coefficient was equal r= 0.3815 (HEX), r= 0.527 (alpha-MAN) and r= 0.489 (beta-GAL). Conclusions Catabolic reactions involving glycoproteins, glycolipids, and proteoglycans may play a role in the pathogenesis of acquired cholesteatoma. The present data indicating that lysosomal exoglycosidases HEX, alpha-MAN and beta-GAL are significantly and consistently elevated suggest the need to further assess correlations between levels of HEX, alpha-MAN and beta-GAL and cholesteatoma behavior. Further research should also evaluate the relative importance of these particular exoglycosidases in considering the spectrum of identified inflammatory mediators.