Proliferation Markers, Remodeling Factors, Cytokines, Antimicrobial Peptides and Gene Proteins in Cholesteatoma

Viscera, biological liquids, and hair represent the common
samples for toxicological analyses. However, it is possible that
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which induce osteolytic process in surrounding bone [4]. If left untreated, cholesteatoma might cause severe complications as facial nerve paralysis, hearing loss, intracranial infections like meningitis and brain abscess, also the destruction of inner ear [5]. All  So, to evaluate tissue proliferation, Ki-67 is used. It presents in all phases of the cell cycle except G0. Ki-67 is located in the cell nucleus and is showed in all proliferative cells [6], therefore, is proven to be useful in cholesteatoma tissue and demonstrate hyperproliferative process in keratin cells of cholesteatoma [7].
Cholesteatoma tissue induces remodelling factors resulting as local temporal bone osteolysis [8]. These are matrix metalloproteinases (MMPs), which are a family of zinc and calcium-dependent proteinases that are responsible for tissue remodelling, cell adhesion, migration, and also proliferation. MMPs regulate the activity of nonextracellular matrix proteins, such as growth factors, cytokines, cell receptors and other MMPs [9][10][11][12]. MMP-2 is believed to be the most important remodelling factor to cause bone erosion [13]. There are still controversies whether MMPs are more active in cholesteatoma tissue than in control tissue [14]. Banerjee et al. [13] states that there is no difference in cholesteatoma from the control tissue, but Dornelles et al. [14] proved that MMP-2 is more actively expressed in cholesteatoma than in control tissue. MMPs are inhibited by tissue inhibitors of metalloproteinases (TIMPs) [15]. Even though TIMPs are proven to be the regulators of MMPs, there are almost no studies about MMPs and TIMPs correlation in cholesteatoma.
The perimatrix of cholesteatoma contains inflammatory cells -monocytes, lymphocytes, fibroblasts, endothelial cells that emit proinflammatory and immunoregulatory cytokines like interleukin (IL)-1 and IL-10. It is believed that cytokines that are present in cholesteatoma can induce cell proliferation in the basal layer of matrix, releasing resorptive enzymes and therefore inducing osteolysis [16][17][18][19]. Tissue proinflammatory cytokine IL-1 is one of the factors that is responsible for aggressiveness of cholesteatoma.
IL-10 is known to inhibit the inflammatory process [20]. It has been suggested that IL-10 possesses antiosteoclastogenic activities in cholesteatoma [18], and that is why the correlation between IL-1 and IL-10 should be evaluated. Chronic middle ear infection with cholesteatoma is mainly characterised by P.aeruginosa infection [21]. Human beta defensin (HβD)-2 is secreted as a defence factor against P.aeruginosa infection [22]. Park et al. [23] proved that HβD-2 is overexpressed in cholesteatoma matrix compared to external ear canal skin epithelium. However, nothing is known about HβD-4 role in cholesteatoma tissue. Not only in otology, but also in other fields of medicine, not much is known about HβD-4 [24].
HβD-4 is believed to be a stronger antibacterial peptide against P.aeruginosa [25] than HβD-2. Also, it might play a different role in infection than other defensins and is proven to upregulate other defensins in the inflammation process [26,27]. The main theory is that cholesteatoma arises from epithelium which migrates from external ear canal to middle ear [28]. External ear, however, develops from 1st and 2nd pharyngeal pouch [29]

Immunohistochemical Analysis
Tissues were fixed in a mixture of 2% formaldehyde and 0.2%

Statistical Analysis
The data processing was performed with SPSS software,       Slightly more positive cells were in matrix compared to perimatrix (Table 1), (Figures 3a-3d). There was no statistically significant difference in the number of IL-1 and IL-10 positive cells between the cholesteatoma group and control group (Table   1), (Figures 3e & 3f). We detected that HβD-2 containing cells in matrix varied from a few to numerous. But HβD-4 positive cells in matrix varied from no to moderate (Table 1), (Figures 4a-4c). In perimatrix, HβD-2 positive cells displayed from a few to moderate  (Tables 1 & 2), (Figures 4b & 4d).
Shh-containing cells in matrix varied from moderate to numerous, while in perimatrix mainly moderate Shh positive cells were seen (Table 1), (Figure 5a). In the control group epithelium, Shh positive cells averaged from moderate to numerous, but in connective tissue, only a few Shh positive cells were detected (Table 1), (Figure 5b).     (Tables 1 & 2). A statistically significant difference was seen between the numbers of Ki-67, TIMP-2 positive cells in matrix and control group epithelium. Also, a statistically significant difference was detected between numbers of HβD-2, HβD-4 Shh positive cells in perimatrix in comparison to control group connective tissue (Table 2). A very strong positive correlation was detected in the cholesteatoma matrix between Ki-67 and HβD-2, Shh; between MMP-2 and HβD-2, Shh; between TIMP-2 and HβD-2, HβD-4; between HβD-2 and HβD-4. And in perimatrix a very strong correlation was between TIMP-2 and IL-10; between IL-1 and IL-10; between HβD-2 and Shh. A strong correlation was detected in matrix between Ki-67 and MMP-2, HβD-4 and between TIMP-2 and IL-1 (Table 3). A very strong positive correlation was detected in control tissue epithelium between Ki-67 and TIMP-2, IL-10, Shh; between MMP-2 and TIMP-2, IL-1; between IL-10 and TIMP-2, Shh. In connective tissue, a very strong correlation was detected between Ki-67 and Shh. A strong correlation was detected in epithelium between Ki-67 and MMP-2 and between MMP-2 and IL-10 (Table 4).       Table 3: Spearman's rank correlation coefficient revealed correlations between the relative numbers of different tissue factors in cholesteatoma matrix and perimatrix.  Table 4: Spearman's rank correlation coefficient revealed correlations between the relative numbers of different tissue factors in control tissue epithelium and connective tissue. also is similar to other author data (Banerjee et al.) [13].

Ki-67 in matrix
However, more TIMP-2 positive cells were found in the control group tissue compared to the patient group, and it was statistically significant, which might suggest that TIPM-2 in cholesteatoma is suppressed and therefore an osteolytic process in the middle ear is induced by MMPs [12]. Even though there is no statistically significant difference between groups in cytokines IL-1 and IL-10, slightly more IL-1 and IL-10 positive cells are in the patient group. This data is similar to Yetiser et al. [38] and Kuczkowski et al. [20], where they show increased IL-1 activity in cholesteatoma.
We support that pro-and anti-inflammatory balance seems to be very stable in the tumour tissue that is proved by a very strong correlation between IL-1 and IL-10 in perimatrix. Our data showed that Shh gene protein was statistically significantly more expressed in cholesteatoma perimatrix than in the control group. Other researchers like Jiang et al. [39][40][41] proved that mutations in Shh gene can cause multiple embryogenetic anomalies and even cancer development [42]. The ongoing study authors suggest the Shh gene might play a major role in the development of cholesteatoma.