Expression of epidermal growth factor receptor in human middle ear cholesteatoma

Middle ear cholesteatoma is destructive to auditory ossicles and temporal bone, and treatment usually includes surgical removal of all epithelial content in the tympanomastoid cavity. Epidermal growth factor receptor (EGFR) is a 170 kd to 180 kd transmembrane glycoprotein and its distribution density is related to the ability of the keratinocytes to differentiate and their state of differentiation. We used the avidin-biotin complex technique and EGFR monoclonal antibody to evaluate the expression of EGFR in 29 cases of cholesteatoma and 34 samples of normal postauricular skin. Of patients with cholesteatoma, 79% (23 cases) had EGFR-positive cells in the basal layer, 66% (19 cases) in the parabasal layer, and 62% (18 cases) in the upper layer of the epithelial tissue. Among patients with normal postauricular skin, 85% (29 cases) had EGFR-positive cells in the basal layer, 79% (27 cases) in the parabasal layer, and 79% (27 cases) in the upper layer of the epithelial tissue. No statistical difference in EGFR expression between each layer of cholesteatoma and postauricular skin was noted. However, there was an intensity gradient of positive EGFR immunoreactivity from the basal to the higher layers in cholesteatoma. Our results showed that the distribution of EGFR in middle ear cholesteatoma is not deranged, but is similar to that in normal skin tissue.     

前列腺素E2在中耳胆脂瘤上皮中的表达及意义

目的探讨前列腺素E2（PGE2）在继发性胆脂瘤发生中的作用。方法采用放射免疫法检测12份胆脂瘤上皮组织匀浆液（观察组）及10份正常外耳道皮肤组织匀浆液（对照组）中PGE2水平。结果中耳胆脂瘤上皮中PGE2的表达水平明显高于正常外耳道皮肤组织（P〈0．01）。结论PGE2高度表达可促进中耳胆脂瘤的发生、发展;抑制PGE2药物可能会阻止胆脂瘤形成。     

Senescent fibroblasts induce moderate stress in lung epithelial cells in vitro

Epithelial-mesenchymal interactions contribute to functionality and integrity of the lung epithelium, which might change during ageing and associated cellular ageing. Therefore, we studied the effect of senescent versus pre-senescent lung fibroblasts (WI-38) on mitogenic and stress-protective factors in lung epithelial cells (H358). By use of conditioned medium, we found a growth promoting impact of fibroblasts compared with control medium from epithelial cells associated with activation of ERK1/2, Akt, p70S6K, and EGF receptor. Although senescent fibroblasts mediated similar growth stimulation compared with pre-senescent cells, we observed less protection against spontaneous mitochondrial dysfunction in vitro, higher production of reactive oxygen species and activation of copper/zinc superoxide dismutase. Moreover, senescent cells induced activation of caspase-3/7 in epithelial cells, which was associated with down-regulation of the caspase-inhibitory protein XIAP. In summary, senescent lung fibroblasts induce moderate stress in lung epithelial cells in vitro without affecting growth signaling.     

Induction of cell proliferation in mammalian inner-ear sensory epithelia by transforming growth factor alpha and epidermal growth factor.

Regenerative proliferation occurs in the inner-ear sensory epithelial of warm-blooded vertebrates after insult. To determine how this proliferation is controlled in the mature mammalian inner ear, several growth factors were tested for effects on progenitor-cell division in cultured mouse vestibular sensory epithelia. Cell proliferation was induced in the sensory epithelium by transforming growth factor alpha (TGF-alpha) in a dose-dependent manner. Proliferation was also induced by epidermal growth factor (EGF) when supplemented with insulin, but not EGF alone. These observations suggest that stimulation of the EGF receptors by TGF-alpha binding, or EGF (plus insulin) binding, stimulates cell proliferation in the mature mammalian vestibular sensory epithelium.     

EGF shifts human airway basal cell fate toward a smoking-associated airway epithelial phenotype

The airway epithelium of smokers acquires pathological phenotypes, including basal cell (BC) and/or goblet cell hyperplasia, squamous metaplasia, structural and functional abnormalities of ciliated cells, decreased number of secretoglobin (SCGB1A1)-expressing secretory cells, and a disordered junctional barrier. In this study, we hypothesized that smoking alters airway epithelial structure through modification of BC function via an EGF receptor (EGFR)-mediated mechanism. Analysis of the airway epithelium revealed that EGFR is enriched in airway BCs, whereas its ligand EGF is induced by smoking in ciliated cells. Exposure of BCs to EGF shifted the BC differentiation program toward the squamous and epithelial–mesenchymal transition-like phenotypes with down-regulation of genes related to ciliogenesis, secretory differentiation, and markedly reduced junctional barrier integrity, mimicking the abnormalities present in the airways of smokers in vivo. These data suggest that activation of EGFR in airway BCs by smoking-induced EGF represents a unique mechanism whereby smoking can alter airway epithelial differentiation and barrier function.     

Alteration of fibroblast architecture and loss of Basal lamina apertures in human emphysematous lung

RATIONALE: In normal human lung, single alveolar fibroblasts link capillary endothelium to type 2 pneumocytes through apertures in the endothelial and epithelial basal laminae. These fibroblasts are hypothesized to play a role in cellular communication between the endothelium and epithelium and are positioned to provide leukocytes a surface on which they may migrate through the interstitium. OBJECTIVES: To determine whether fibroblasts link the endothelium to the epithelium in emphysematous lung and to compare basal lamina aperture frequency with previously published results. METHODS: We performed transmission electron microscopy serial section three-dimensional reconstructions of emphysematous regions of human alveolar wall and a quantitative analysis of basal lamina apertures beneath 403 type 2 pneumocytes. MEASUREMENTS AND MAIN RESULTS: Our three-dimensional reconstruction demonstrated that the fibroblasts subtending type 2 pneumocytes in emphysematous lung no longer link these epithelial cells to the capillary endothelium through basal lamina apertures. Basal lamina apertures may be absent below some type 2 pneumocytes. Our morphometric analysis showed that their frequency and area beneath type 2 pneumocytes is significantly reduced in emphysematous regions when compared with nonemphysematous regions of matched control lung. CONCLUSIONS: We conclude that the endothelial/fibroblast/epithelial linkage is disrupted in emphysematous human lungs and postulate this disruption may disturb leukocyte migration and account for their accumulation in the alveolar interstitium of emphysematous lung tissue.     

The murine forestomach: a sensitive site for graft-versus-host disease

The minor histoincompatible mouse radiation chimera provides a useful model of graft-versus-host disease (GVHD) paralleling conditions in human marrow transplant recipients. While studying the B10.D2/BALB/c model, we noted that a graft-versus-host reaction of particular severity develops in the forestomach near the squamocolumnar junction. Comparison of the dyskeratotic index of this epithelium with that of the skin of the same animal revealed the forestomach to be a more sensitive site for detection of GVHD. In normal mice, both basal forestomach squamous epithelium and cells in the lamina propria expressed class II antigens. During the course of GVHD, class II antigen expression was elevated in the squamous epithelium of the forestomach, the columnar epithelium associated with the stomach, and cells within the associated lamina propria. The demonstration here that the squamocolumnar junction (considered to be a stem cell region for the maintenance of adjacent forestomach squamous epithelium) appears to be a particularly sensitive target tissue in GVHD and earlier identification of other epithelial target tissues of GVHD (hair follicles and rete ridges of epidermis, bile ductules of liver, and crypt cells of the gut) collectively support the hypothesis that GVHD tends to target sites of epithelial proliferation overlying epithelial stem cells.     

Epidermal growth factor in the oesophagus.

Epidermal growth factor (EGF) has been implicated in mitogenesis and oncogenesis in the gastrointestinal tract. To determine the role of EGF in oesophageal disease, its quantity and distribution in the oesophageal mucosa of control subjects and patients with oesophageal disease were studied. Oesophageal biopsy specimens, taken 20-40 cm from the incisors in 72 patients, were graded histologically and adjacent specimens were taken for immunohistochemical analysis of the distribution of EGF. In patients with Barrett's columnar lined oesophagus, specimens were also taken from the gastric cardia for comparison. Twenty two biopsy specimens showed oesophagitis, 20 Barrett's mucosa, and 30 were histologically normal. EGF was found in the capillary endothelium of the normal oesophageal papillae and basal mucosa. Significantly more EGF positive papillae were found in the normal mucosa (81%) than in the inflamed mucosa (42%) (p < 0.001). The 20 patients with Barrett's mucosa showed abnormal expression of EGF in 25% of the isthmus and superficial epithelial cells. This study has shown that EGF is found only in the endothelial cells of the capillaries of the normal oesophageal mucosa and that the peptide is detectable significantly less frequently than normal in the inflamed oesophageal mucosa. EGF is also abnormally present, in large quantities, in the cytoplasm of the epithelial cells of Barrett's mucosa compared with gastric mucosa.     

Progesterone regulates epidermal growth factor receptor on mucous secreting cells in the rabbit endocervix

During postnatal differentiation, epidermal growth factor (EGF) receptor is expressed by all major cell types of the cervix. Computer-assisted image analysis confirmed the highest concentration of EGF receptor is in the epithelial cells. Flow cytometric analysis of subpopulations of epithelial cells from estrous rabbits showed the mucous secreting cells had the highest concentration of EGF receptor, i.e. 1-1.5 x 10(5) receptors per cell. Because the mucous secreting cells are targets for steroid hormones it seemed likely that steroids regulate EGF receptor expression. To investigate this possibility, hormone-dependent changes in EGF receptor expression were quantified by flow cytometry. Ovariectomy and the treatment of ovariectomized animals with estradiol altered forward angle light scatter and side scatter signals which correlated with cell size and secretory granule content, respectively. However, the number of epithelial cells and the number of EGF receptors per cell were unaffected. Progesterone treatment of ovariectomized animals dramatically reduced the number of EGF receptors on the mucous secreting cells, accounting for a 43% reduction in the total EGF receptor content of the epithelium. The treatment of neonates with diethylstilbestrol did not change the number of EGF receptors in endocervical epithelial cells when examined in adulthood. However, the number of mucous secreting cells was decreased, thereby reducing the EGF receptor content of the epithelium 19-36% compared to estrous and estradiol-treated animals. These results provide the first evidence that progesterone regulates EGF receptor on mucous secreting cells in the endocervix and that diethylstilbestrol treatment alters the EGF receptor content of the epithelium by altering its cellular composition.     

Evaluation of the role of human papillomavirus in oesophageal squamous cell carcinoma in Belgium

OBJECTIVE: To evaluate the putative role of human papillomavirus (HPV) in the aetiology of oesophageal squamous cell carcinoma (OSCC) in Belgium. METHODS: The frequency of HPV infection was determined using HPV DNA PCRamplification with L1 consensus primers MY09-MY11, able to recognise about 40 different HPV types, on twenty-one formalin-fixed and paraffin-embedded oesophageal squamous cells carcinomas. Nineteen samples of histologically normal epithelium from the surgical margins of the OSCC specimens and five samples from normal oesophagus obtained at autopsy served as negative controls. RESULTS: We found only one HPV positive tumour (4.8%) out of the 21 OSCC cases. All the normal epithelium controls remained negative. CONCLUSIONS: Our data are in agreement with those previously published, suggesting that HPV infection only plays a minor role in the pathogenesis of oesophageal squamous cells carcinoma in West-European countries.     

Effect of diethylstilbestrol on cell proliferation and expression of epidermal growth factor in the developing female rat reproductive tract

To evaluate mechanisms of cell proliferation in the fetal female rat reproductive tract, diethylstilbestrol (DES) effects on cell division and estrogen receptor (ER), epidermal growth factor (EGF) and EGF receptor (EGF-R) expressions were determined from gestational day (GD) 15.5 to 21.5. Reproductive tracts were evaluated within three regions along the Müllerian duct axis; these were proximal, middle and caudal, which differentiate into oviduct, uterus and upper vagina respectively. In fetuses from non-treated dams, epithelial and mesenchymal proliferation, as evaluated by 5-bromo-2'-deoxyuridine incorporation, was decreased with development in all regions of the Müllerian duct. EGF levels were determined by immunohistochemistry. Müllerian epithelial EGF immunoreactivity was intense in the proximal and middle regions on GDs 15.5 and 17.5. EGF staining remained intense only in the proximal epithelia by GD 19.5 and was weak in the caudal epithelium, but substantially reduced throughout epithelia in all regions by GD 21.5. Thus, decreased cell proliferation correlated with decreased EGF expression in the developing Müllerian duct. DES (100 microg/kg body weight) was injected from GD 15 to 19 and female fetuses were collected on GD 19.5. DES increased Müllerian duct cell proliferation in the proximal epithelium and mesenchyme but decreased it in the caudal epithelium compared with oil-treated controls. No proliferative DES effect was observed in any cell type in the middle region. Müllerian duct EGF immunoreactivity was suppressed by DES compared with oil. Competitive RT-PCR indicated DES also decreased mRNAs for EGF, ERbeta1 and ERbeta2, but not ERalpha and EGF-R. These results indicate EGF may be an important regulatory factor of Müllerian duct cell proliferation, and that DES may alter cell proliferation by disrupting normal EGF, ERbeta1 and ERbeta2 expression in the developing female rat reproductive tract.     

Congenital aural stenosis with first branchial cleft fistula,presenting with recurrent facial nerve palsy,in an elderly patient

Both duplication anomalies and external auditory canal stenosis can result in cholesteatoma due to retained epithelium. Despite their common origins, the coexistence of these anatomical abnormalities is quite unusual. This cholesteatoma may go unnoticed till adulthood and present with complications. Moreover, first branchial cleft malformations are often unrecognized or misdiagnosed for other inflammatory lesions in the periauricular region. We report a case of middle-ear cholesteatoma in a 68 year old male patient with grade II microtia with canal stenosis and first branchial cleft fistula in supra-auricular region presenting with recurrent infranuclear facial nerve palsy. The patient was managed surgically by excision of fistulous tract, canalplasty, auricular reconstruction and middle ear and mastoid exploration for cholesteatoma. This may probably be the oldest person to be managed surgically for cholesteatoma due to congenital ear anomalies and first such reported case.     

Serum-free growth of normal and tumor mouse mammary epithelial cells in primary culture.

Freshly isolated normal and tumor mouse mammary epithelial cells embedded within a collagen gel matrix undergo sustained growth when cultured for as long as 3 wk in a serum-free medium composed of a 1:1 (vol/vol) mixture of Hepesbuffered Ham's F12 and Dulbecco's modified Eagle's medium supplemented with insulin, epidermal growth factor (EGF), transferrin, bovine serum albumin fraction V, and cholera toxin. Of these additives, only insulin, EGF, and albumin are required for the growth of most normal cells. Albumin is not always an absolute requirement for growth but greatly enhances it. Lithium has been found to stimulate the growth of normal cells and can replace EGF. The collagen matrix culture system allows sustained growth of primary cultures of both normal and neoplastic mammary epithelium in serum-free conditions. This serum-free system will be useful in identifying and investigating the role of hormones, growth factors, and nutritional factors in regulating the growth of mammary epithelial cells.     

Distribution of Tn antigen recognized by an anti-Tn monoclonal antibody (MLS128) in normal and malignant tissues of the digestive tract

Alterations in the normal glycosylation process are often associated with oncogenic transformation. Using an anti-Tn monoclonal antibody, MLS128, we have investigated the immunohistochemical localization of Tn antigen in normal and malignant tissues of the digestive tract. In normal tissues, MLS128 was immunoreactive with the squamous epithelium of the esophagus and was weakly reactive with the columnar epithelia of the stomach, duodenum, colon, bile duct and pancreatic duct. In malignant, tissues, positive immunostaining was detected with high frequency (75%–100%) in carcinomas of the esophagus, stomach colon, biliary tract and pancreas, whereas 2 of 11 (18%) hepatocellular carcinomas were positive. Tn antigen was detected in the upper two-thirds of the normal squamous epithelium, and was often detected in squamous cell carcinomas with cancer pearls (keratinization). These results suggest that the expression of Tn antigen is related to the differentiation of squamous epithelium, or to keratinization. In normal columnar epithelial cells, Tn antigen was localized mainly to the Golgi area. This intracellular localization was preserved in well-differentiated papillary adenocarcinomas of the colon, but was lost in most cases of tubular adenocarcinomas.     

Saccharin and cyclamate inhibit binding of epidermal growth factor.

The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.     

Apoptosis resistance in Barrett's esophagus: ex vivo bioassay of live stressed tissues

BACKGROUND AND AIMS:  Barrett's esophagus (BE) is a premalignant lesion of the distal esophagus in which squamous epithelial cells are replaced by metaplastic intestinal-like columnar epithelium that contains goblet cells. The factors that contribute to the progression from normal squamous mucosa to BE, Barrett's dysplasia, and adenocarcinoma are not well understood at the molecular level. Since reflux of bile acids is associated with BE development, we speculate that cells with an apoptosis-resistant phenotype are selected after long-term repeated exposure to pulses of bile acids. This will result in the survival of cells with unrepaired DNA damage, and a consequent increase in genomic instability leading to cancer progression. The major goal of this study is to compare sensitivity to apoptosis induced by the bile acid, deoxycholate (DOC), a known inducer of apoptosis, in normal esophageal squamous epithelium, normal colon epithelium, and BE.
METHODS :  Thirteen patients with a confirmed diagnosis of BE and four patients who had undergone clinically indicated colectomy were included in the present study. Freshly obtained biopsies were incubated with control medium or medium supplemented with 1 mM DOC for 3 h and then evaluated for apoptotic changes using transmission electron microscopy and immunohistochemical staining for two apoptotic markers, cleaved caspase 3 and cleaved cytokeratin 18.
RESULTS:  Our results indicate that BE is resistant to apoptosis induced by DOC compared to esophageal squamous epithelium and normal colon epithelium. In addition, electron micrographs revealed mitochondrial swelling in squamous epithelial cells treated ex vivo with DOC, which was absent in epithelial cells of BE. Formation of swollen mitochondria is an early marker of apoptotic cell death. Altogether, the data indicate that reduced apoptosis capability in BE tissue may contribute to progression to esophageal adenocarcinoma.     

Stimulation of intestinal epithelial cell proliferation in culture by growth factors in human and ruminant mammary secretions

Samples of human and ruminant mammary secretions stimulated the proliferation of rat intestinal epithelial (RIE-1) cells in culture. The stimulation was dose-dependent, and samples taken prepartum had greater potency than those taken after parturition. When various hormones and growth factors known to be present in milk were tested, only epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I) stimulated the proliferation of RIE-1 cells. IGF-I was effective at lower concentrations than insulin, and the maximal stimulation induced by each of these two polypeptides was greater than that induced by EGF. The maximal stimulation induced by samples of mammary secretions was similar to that induced by insulin or IGF-I.     

Epidermal growth factor receptor gene expression in avian epiphyseal growth-plate cartilage cells: effect of serum, parathyroid hormone and atrial natriuretic peptide.

Avian chondrocytes and fibroblasts, derived from epiphyseal growth-plate and skin, respectively, were cultured in vitro. In chondrocytes, epidermal growth factor (EGF) caused a dose-dependent stimulation of proliferation. EGF receptor mRNA was not detected with the v-erb B probe in chondrocytes cultured in the presence of 5% fetal calf serum (FCS). In the absence of FCS in the medium, a time-dependent increase in the level of EGF receptor mRNA was observed. Parallel changes were also observed in the level of EGF receptor, as demonstrated by immunofluorescence using antibodies directed against avian EGF receptor. In avian fibroblasts, EGF receptor mRNA and EGF receptor levels were not affected by FCS. Furthermore, FCS did not affect the level of thyroid hormone receptor mRNA (using v-erb A as a probe) in either chondrocytes or fibroblasts. Parathyroid hormone (PTH), which acts as a mitogen in avian chondrocytes attenuated--whereas atrial natriuretic peptide (ANP), a suppressor of chondrocyte proliferation, enhanced--EGF receptor mRNA. The present results show that avian growth-plate chondrocytes respond to EGF and bear EGF receptors. The levels of EGF mRNA and EGF receptor are inversely related to cell proliferation. The results also support previous suggestions that PTH and ANP play important roles in chondrocyte proliferation, possibly through their effect on the synthesis of the EGF receptor.