Margriet A. Huisman

Cholesteatoma Epithelium is Characterized by Increased Expression of Ki-67, p53 and p21, with Minimal Apoptosis

Objective To investigate differences in cell proliferation, cell cycle arrest and apoptosis between cholesteatoma and control skin. Material and Methods Immunohistochemical sections of 15 cholesteatoma and 15 paired control retro-auricular skin samples were examined for Ki-67, p53, p21 and active caspase 3, using image analysis, as well as for DNA fragmentation. Results The retro-auricular skin samples contained 5.7% ± 3.6%, Ki-67-positive cells and showed a normal expression pattern. In the cholesteatoma epithelium 11.7% ± 9.5% of the cells were Ki-67-positive and these cells were dominantly expressed in the basal and parabasal cell layers. Retro-auricular skin contained 5.8% ± 5.4% p53-positive cells and 1.0% ± 0.9%, p21-positive cells. In the cholesteatoma epithelium 17.8% ± 12.3% of the cells were p53-positive and 14.3% ± 11.6% were p21-positive The expression of Ki-67, p53 and p21 differed significantly between the two groups (p < 0.05). In the cholesteatoma epithelium a positive correlation was found between p53 and p21 expression (p = 0.016). Active caspase 3 positivity and DNA fragmentation were rarely seen in the cholesteatoma epithelium. Conclusion Our results indicate that increased cell proliferation in cholesteatoma epithelium is accompanied by an increase in p53 and p21 protein levels, whilst apoptosis is minimal.

Lipopolysaccharide concentration and bone resorption in cholesteatoma.

Hypothesis There is a relationship between the local lipopolysaccharide (LPS) concentration in cholesteatoma and local bone resorption in chronic otitis media (COM) with cholesteatoma. Background During the past decade, it has become known that the recruitment of osteoclasts is the main causative factor that induces bone destruction in COM with cholesteatoma. Cellular inflammation factors like cytokines may trigger the osteoclast. Sequel to this, LPS is able to up-regulate cytokines. This makes it of interest to study whether the local LPS concentration is related to bone resorption in cholesteatoma. Materials and Methods Twenty-four cholesteatoma samples and control tissue from COM patients without cholesteatoma were collected. During surgery, the degree of bone resorption was established and classified. Retrospectively, the authors checked whether patients had chronic purulent otorrhea. LPS concentration of the tissue samples was measured by the limulus amebocyte lysate test. The one-way analysis of variance test was used to determine the relation between LPS concentration, otorrhea, and local bone resorption. Results A significantly higher concentration of LPS was measured in samples from patients with cholesteatoma with bone resorption and otorrhea compared with cholesteatoma without bone resorption and control tissue. There were no significant differences between the LPS levels of the different groups of patients with bone resorption. Conclusion It is suggested that LPS is one of the first factors in the cascade of bone resorption in COM with cholesteatoma.

Uncomplicated differentiation of stem cells into bipolar neurons and myelinating glia.

Epidermal neural crest stem cells (EPI-NCSCs), derived from the bulge of hair follicles, appear to be promising donor stem cell candidates. In the current work, EPI-NCSCs were harvested from rodents and humans. Isolation procedures revealed high levels of nestin-positive neural stem cells and the percentage of human neural stem cells (95+/-0.6%) is even higher than the percentage found in cultures of hair follicles from rodents (90+/-0.9%). Furthermore, differentiation of EPI-NCSCs into bipolar neurons, myelinating Schwann cells and oligodendrocytes occurred by applying a simple and straightforward method. Bipolar neurons could be obtained by culturing on a collagen matrix and are of great interest for auditory neuron regeneration since auditory neurons are bipolar. We propose that this type of stem cells, would make an excellent model for autologous transplantation and offers great potential for neural regeneration in diseases, such as Parkinsons and Alzheimers disease.

Transforming Growth Factor β and Wound Healing in Human Cholesteatoma

Objective: Cholesteatoma is a nonmalignant, destructive lesion of the temporal bone that gradually expands and causes complications by the erosion of the adjacent bony structures. The consequences can be as severe as facial paralysis and intracranial complications. Until now, surgery has been the only treatment of choice. The pathogenesis of cholesteatoma remains controversial. Current concepts postulate that cholesteatoma may be a wound‐healing process, although formal proof is lacking as of yet. Several reports provide evidence for the involvement of transforming growth factor (TGF)β in both normal and abnormal wound healing.

Class III β-tubulin, a novel biomarker in the human melanocyte lineage

It is generally thought that class III β-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III β-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III β-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.

Survival signaling and terminal differentiation in cholesteatoma epithelium.

Conclusion: There is a strong indication that epithelial keratinocytes in cholesteatoma are protected against apoptosis. The late terminal differentiation program in cholesteatoma epithelium is disturbed. Objectives: Previously, minimal apoptosis has been demonstrated in cholesteatoma epithelium. The phosphoinositide 3-kinase/Akt/protein kinase B (PI3K/Akt/PKB) and the mitogen activated protein kinases (MAPK) signaling transduction pathways have been reported to protect epithelial cells against apoptosis. Both pathways have also been proven to regulate late terminal differentiation of keratinocytes. In cholesteatoma epithelium, MAPK activation has been shown to be associated with terminal differentiation. The purpose of this study was to investigate whether in human cholesteatoma epithelium protection against programmed cell death by means of PI3K/Akt survival signaling is present and associated with MAPK activation and terminal differentiation. Materials and methods: Fifteen human cholesteatoma and patient-matched retro-auricular skin samples were immunohistochemically stained for pAkt/PKB, phosphorylated extracellular regulated kinase1/2 (pERK1/2), phosphorylated JNK/SAPK, phosphorylated p38, involucrin and filaggrin. Positive cells were counted by computer-assisted digital image analysis. Results: Protein expressions of pAkt/PKB, pERK1/2, pp38, and involucrin in cholesteatoma epithelium were significantly increased when compared with retro-auricular skin (p<0.01). Filaggrin expression was significantly decreased (p=0.03). The positive correlation was confirmed between both pERK1/2 and pp38, and involucrin (p≤0.05).

Neural crest stem cells and their potential application in a therapy for deafness.

Neurosensory hearing loss is a common condition that has major social and economic implications. Recent advances in stem cell research and in cochlear implantation are offering renewed hopes to people suffering from damage to the auditory hair cells and their associated neurons. Several putative donor cell types are currently being explored, including embryonic stem cells, different types of adult stem cell and the recently described induced-pluripotent stem cells. In this review, we draw attention to the potential application of neural crest stem cells for the treatment of deafness. This population shares a similar developmental origin with the cells of the otic placode, the molecular machinery controlling their maturation and differentiation is comparable and they can produce related sensory neurons. More importantly, pockets of neural crest stem cells remain in the adult body in regions of relatively easy access, facilitating their use for autologous transplantation and therefore avoiding the need for immunosuppression and the problems of tissue rejection. Their exploration and application to hearing conditions could facilitate the development of a clinically-viable, cell-based.

Development of the stria vascularis and potassium regulation in the human fetal cochlea: Insights into hereditary sensorineural hearing loss

Sensorineural hearing loss (SNHL) is one of the most common congenital disorders in humans, afflicting one in every thousand newborns. The majority is of heritable origin and can be divided in syndromic and nonsyndromic forms. Knowledge of the expression profile of affected genes in the human fetal cochlea is limited, and as many of the gene mutations causing SNHL likely affect the stria vascularis or cochlear potassium homeostasis (both essential to hearing), a better insight into the embryological development of this organ is needed to understand SNHL etiologies. We present an investigation on the development of the stria vascularis in the human fetal cochlea between 9 and 18 weeks of gestation (W9–W18) and show the cochlear expression dynamics of key potassium‐regulating proteins. At W12, MITF+/SOX10+/KIT+ neural‐crest‐derived melanocytes migrated into the cochlea and penetrated the basement membrane of the lateral wall epithelium, developing into the intermediate cells of the stria vascularis. These melanocytes tightly integrated with Na+/K+‐ATPase‐positive marginal cells, which started to express KCNQ1 in their apical membrane at W16. At W18, KCNJ10 and gap junction proteins GJB2/CX26 and GJB6/CX30 were expressed in the cells in the outer sulcus, but not in the spiral ligament. Finally, we investigated GJA1/CX43 and GJE1/CX23 expression, and suggest that GJE1 presents a potential new SNHL associated locus. Our study helps to better understand human cochlear development, provides more insight into multiple forms of hereditary SNHL, and suggests that human hearing does not commence before the third trimester of pregnancy.

Distribution and development of peripheral glial cells in the human fetal cochlea.

The adult human cochlea contains various types of peripheral glial cells that envelop or myelinate the three different domains of the spiral ganglion neurons: the central processes in the cochlear nerve, the cell bodies in the spiral ganglia, and the peripheral processes in the osseous spiral lamina. Little is known about the distribution, lineage separation and maturation of these peripheral glial cells in the human fetal cochlea. In the current study, we observed peripheral glial cells expressing SOX10, SOX9 and S100B as early as 9 weeks of gestation (W9) in all three neuronal domains. We propose that these cells are the common precursor to both mature Schwann cells and satellite glial cells. Additionally, the peripheral glial cells located along the peripheral processes expressed NGFR, indicating a phenotype distinct from the peripheral glial cells located along the central processes. From W12, the spiral ganglion was gradually populated by satellite glial cells in a spatiotemporal gradient. In the cochlear nerve, radial sorting was accomplished by W22 and myelination started prior to myelination of the peripheral processes. The developmental dynamics of the peripheral glial cells in the human fetal cochlea is in support of a neural crest origin. Our study provides the first overview of the distribution and maturation of peripheral glial cells in the human fetal cochlea from W9 to W22.

Air-exposed tissue culture of human middle ear epithelium and meatal epidermis: a method to study the advancing front of cholesteatoma.

The suitability of an air-exposed culture model consisting of a collagen matrix was investigated for constructing an advancing front (AF) of human middle ear epithelium (MEE) and meatal epidermis (ME). Three different culture settings were used: ( i ) MEE; ( ii ) ME; and ( iii ) AF (MEE+ME). Small tissue biopsies were placed on a fibroblast-populated collagen matrix and grown at the air-liquid interface. After 3 weeks of culture, the MEE and ME outgrowth was differentiated. Light, scanning electron and transmission electron microscopy showed no visible differences compared to native MEE and ME. Cytokeratin 8 and cytokeratin 10 expressions were comparable to the expression seen in the native MEE and ME tissues. Proliferation, which was demonstrated by the expression of Ki-67, was present in the basal layers of cultured MEE and ME. A double layer of cells in which the ME covered the MEE formed the AF. In the AF, the MEE and ME showed the same morphological and immunohistochemical characteristics as in their native tissues. The results of the study show that this in vitro system is a well-defined model system offering the possibility to study the effects of external stimuli on the different epithelia of the AF involved in the pathogenesis of cholesteatoma.