Histology, histochemistry and ultrastructure of the nasopharyngeal tonsil of the buffalo (Bubalus bubalis).

The light microscopic appearance and ultrastructure of the nasopharyngeal tonsil (tonsilla pharyngea), collected from 12 adult buffaloes of local mixed breed, were explored for the distribution of different types of epithelia, lymphoid tissue and high endothelial venules. The tonsillar mucosa was lined by pseudostratified columnar ciliated epithelium having goblet cells. The respiratory epithelium associated with the underlying lymphoid tissue formed the lymphoepithelium. The epithelium was further modified into follicle-associated epithelium (FAE) characterized by reduced epithelial height, presence of a few dome-shaped cuboidal cells equivalent of the M-cells and absence of goblet and ciliated cells. The lymphoid tissue was distributed in the form of isolated lymphoid cells, diffuse lymphoid tissue and lymphoid follicles, mainly distributed within the propria-submucosa along with the sero-mucous glandular tissue. The goblet cells of the respiratory epithelium and the acinar cells contained different mucopolysaccharides. Scanning electron microscopy of the surface mucosa demonstrated a dense mat of cilia, island-like arrangement of microvillus cells, M-cells and a few brush-like cells. The transmission electron microscopy revealed the different cell organelles of the respiratory epithelium and the FAE. Lymphocyte migration via the high endothelial venules in the propria-submucosa was also observed.

Ameloblastoma with adenoid features: A series of eight cases.

BACKGROUND: Ameloblastoma with adenoid features are characterized by the presence of duct-like structures formed from the parenchyma of the tumor. This study was conducted to report a series of eight ameloblastomas with adenoid features, highlighting their clinicopathological and immunohistochemical aspects. MATERIAL AND METHODS: Out of 71 cases of ameloblastomas, this study classified 8 cases as ameloblastomas with adenoid features. Clinicopathological data and immunohistochemistry for CK7, CK14, CK19, IMP3, p53 and Ki-67 were evaluated. RESULTS: From those cases of ameloblastoma exhibiting adenoid features, there were 4 women and 4 men, with mean age of 39 years. Most cases affected the mandible and all presented radiographically as a radiolucency. The predominant histopathological features were pseudoducts, squamous metaplasia, nuclear hyperchromatism, clear cells, whorled aspect of epithelial structures, cribriform growth pattern, proliferation of spindle cells and extracellular eosinophilic material. Immunohistochemical analysis showed high expression for CK14 (n = 6) and CK19 (n = 3) and all cases (n = 8) were negative for p53, IMP3 and CK7. In addition, all samples (n = 8) showed low expression for Ki-67. CONCLUSIONS: The similarities between the histopathological and immunohistochemical features of eight cases described in the present study and those described in previous studies support the possibility that these lesions are adenoid ameloblastomas. In addition, the immunohistochemical results of CK14, CK19, p53 and Ki-67 did not differ from those of conventional ameloblastomas.

Detection of Bacterial Biofilms in Chronic Pharyngitis Resistant to Medical Treatment.

OBJECTIVE: To evaluate the role played by adenoids as a reservoir for infection in children assigned for adenoidectomy. METHODOLOGY: The study included 35 children with adenoid hypertrophy. All patients underwent clinical examination and adenoidectomy, adenotonsillectomy, or myringotomy with insertion of aeration tube according to indications. Surgical specimens were processed for conventional bacterial culture examination and to assay for biofilm formation. The obtained adherence values using spectrophotometer at 595 nm (OD595) was used to classify isolates according to its biofilm forming capacity. RESULTS: We did adenotonsillectomy and myringotomy with insertion of aeration tube in 5 patients having adenotonsillitis with otitis media with effusion. We did adenotonsillectomy in 12 patients having adenotonsillitis and adenoidectomy in 18 patients having adenoid hypertrophy. Thirty-one surgical specimens showed bacterial growth on conventional media, while 4 specimens failed to give growth. The predominant organism was H influenzae then Staph aureus and Strept pneumoniae. Thirty-two specimens showed biofilm forming capacity (BFC) of variable extent, while others showed no BFC. CONCLUSION: Adenoids act as a bacterial reservoir secondary to bacterial biofilm formation so could induce chronicity and initiate development of complications. Determination of BFC using the proposed protocol is feasible, inexpensive, and available and spares the need for sophisticated instruments or approaches.

Three-dimensional reconstruction of the pharyngeal tonsil innervation pattern in sheep.

The pharyngeal tonsil has recently been identified as a new participant in airborne contamination by the ovine scrapie agent. In the context of scrapie pathogenesis, we conducted a three-dimensional reconstruction of the innervation pattern in the lymphoid compartments of this tonsil. This model confirmed that very few nerve fibres penetrated the lymphoid follicles and suggested that the nerve fibre distribution in the interfollicular and subepithelial areas is more suitable with neuro-invasion through direct contact between these nerve fibres and prion-transporting cells prior to or after prion amplification in the germinal centre of the pharyngeal tonsil lymphoid follicles.

Examination of the reticular epithelium of the bovine pharyngeal tonsil.

The pharyngeal tonsil (adenoid), located at the posterior of the nasopharynx is ideally positioned to sample antigens passing through the nasal cavity or oral cavity. Entering antigens will first contact tonsilar epithelium. To better understand the cellular organization of this important epithelial layer, pharyngeal tonsils were collected from six, 7-month-old calves and examined by light microscopy, immunohistochemistry, and electron microscopy. Morphometric analysis showed that the epithelium overlying lymphoid follicles (reticular epithelium) contained significantly more B-cells, CD4+, and CD11c+ cells than nonreticular epithelium. In contrast, nonreticular epithelium contained significantly more, γ/δ TCR+ cells than reticular epithelium. Scanning and transmission electron microscopy of reticular epithelium identified a heterogeneous population of epithelial cells, many of which displayed morphologic characteristics of M-cells. Moreover, putative M-cells were shown to possess the capacity for microparticle uptake. Bovine pharyngeal tonsilar reticular epithelium contains key immune cells, as well as M-cells; elements essential for antigen uptake, antigen processing, and initiation of immune responses. A better understanding of the morphology and function of tonsilar lymphoepithelium will strengthen our understanding of it's role in disease pathogenesis, and potential use as an induction site for mucosal immune responses to vaccination.

Biofilm density in the pediatric nasopharynx: recurrent acute otitis media versus obstructive sleep apnea.

OBJECTIVES: We compared the biofilm surface density of adenoids removed from children with recurrent acute otitis media (RAOM) to that of adenoids removed from children with a diagnosis of obstructive sleep apnea (OSA). METHODS: We performed a comparative microanatomic study of adenoid mucosa using scanning electron microscopy in patients with diagnoses of RAOM and OSA (27 female and 41 male; age range, 3 months to 15 years). RESULTS: The adenoids removed from patients with RAOM had dense, mature biofilms covering nearly their entire mucosal surfaces. More specifically, the adenoids removed from patients with RAOM had an average of 93.53% of their mucosal surface covered, versus an average of 1.01% coverage on the adenoids removed from patients with OSA. These differences were statistically significant (p < 0.0001). CONCLUSIONS: The adenoids removed from patients with RAOM had almost their entire mucosal surface covered with biofilms, versus scant coverage for patients with OSA. Recurrent acute otitis media is notoriously resistant to antibiotic treatment, and aspirates of middle ear fluid repeatedly yield negative cultures. It is these properties that have led biofilms to become increasingly implicated in the pathogenesis of RAOM. Thus, the resistance of biofilms to antimicrobials, together with their planktonic shedding of organisms, may be an important mechanism in the development of RAOM.

Identification of adenoid biofilms with middle ear pathogens in otitis-prone children utilizing SEM and FISH.

OBJECTIVES: Biofilms have been implicated in the development of several chronic infections. We sought to demonstrate middle ear pathogens in adenoid biofilms using scanning electron microscopy (SEM) and fluorescent in situ hybridization (FISH) with confocal laser scanning microscopy (CLSM). METHODS: Comparative micro-anatomic investigation of adenoid mucosa using SEM and FISH with confocal scanning laser microscopic (CLSM) imaging from patients with recurrent acute otitis media (RAOM). RESULTS: All otitis-prone children demonstrated biofilm surface area presence greater than 85% by SEM. FISH accompanied by CLSM imaging also demonstrated patchy biofilms All biofilms contained middle ear pathogens and were frequent in polymicrobial distributions: 4 of 6, 4 of 6 and 3 of 6 samples contained Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, respectively. CONCLUSIONS: Dense adenoid biofilms may act as a reservoir for reinfection of the tubotympanum. Aspiration of planktonic middle ear pathogens existing in resistant adenoid biofilms during a viral upper respiratory tract infection may be an important event in the development of RAOM.

Expression of tight junction proteins in epithelium including Ck20-positive M-like cells of human adenoids in vivo and in vitro.

The human adenoid epithelium forms a continuous barrier against a wide variety of exogenous antigens. In this study, to elucidate the structures of the epithelial barrier in the human adenoid, including M-cells, we identified M-cells using an anti-cytokeratin 20 (Ck20) antibody and investigated expression of tight junction proteins in human adenoid epithelium in vivo and in vitro. In human adenoid epithelium and primary cultures, mRNAs of occludin, junctional adhesion molecule-A, ZO-1, and claudin-1, -4, -7, and -8 were detected by reverse transcription-polymerase chain reaction, whereas claudin-2 and -9 were expressed in vitro. In the epithelium in vivo, some Ck20-positive cells were randomly observed and indicated pocket-like structures, whereas Ck7 was positive in almost cells. Transmission electron microscopy revealed that Ck20-associated gold particles could be identified in M-like cells which had short microvilli and harboured the lymphocyte in the pocket-like structure. In primary cultures in vitro, Ck20-positive cells were also detected and had a function to take up fluorescent microparticles. In Ck20-positive cells in vivo and in vitro, expression of occludin, ZO-1, claudin-1 and -7 were observed at cell borders. These results indicate that the epithelial barrier of the human adenoid is stably maintained by expression of tight junction proteins in the epithelium including Ck20-positive M-like cells.

Biofilm surface area in the pediatric nasopharynx: Chronic rhinosinusitis vs obstructive sleep apnea.

OBJECTIVE: To compare the percentage of mucosal surface area of adenoids infected with biofilms removed from children with chronic rhinosinusitis (CRS) vs children with obstructive sleep apnea (OSA). DESIGN: Comparative microanatomical investigation of adenoid mucosa from patients with CRS and OSA using scanning electron microscopy. SETTING: University-affiliated hospitals and ambulatory surgery center. PATIENTS: Four girls and 12 boys ranging in age from 3 months to 10 years. MAIN OUTCOME MEASURE: Measurements of biofilm coverage of the entire adenoidal surface. RESULTS: Adenoids removed from patients with CRS had dense mature biofilms covering the mucosal surface; they had a mean of 94.9% of their mucosal surface covered with mature biofilms, compared with a mean of 1.9% coverage on the adenoids removed from patients with OSA. This difference was statistically significant at P < .001. CONCLUSIONS: Adenoids removed from patients with CRS had almost their entire mucosal surface covered with biofilms vs scant coverage for patients with OSA. Biofilms in the nasopharynx of children with CRS may act as a chronic reservoir for bacterial pathogens resistant to standard antibiotics. The mechanical debridement of the nasopharyngeal biofilms may explain the observed clinical benefit associated with adenoidectomy in this subset of pediatric patients.

Uptake of microparticles into the epithelium of human nasopharyngeal lymphoid tissue.

The M cells of nasopharyngeal lymphoid tissue (NALT) have been considered to play an important role for vaccine delivery systems in humans. A number of investigations have reported particle uptake data in NALT of rodents. However, there have been no reports indicating any involvement of the nasopharyngeal lymphoid tissue in human vaccination. In the present study, we investigated whether the epithelium of human adenoid tissues might incorporate fluorescent microparticles using electron and fluorescent microscopy. The dissected adenoid tissues were incubated with various sizes and concentrations of fluorescent microparticles for 120 min at 37 degrees C. Furthermore, the effect of surface coatings of microparticles with cations on the uptake into the epithelium of adenoid tissues was investigated. Transmission electron microscopy revealed that microparticles were taken up by the M cells of human nasopharyngeal lymphoid tissues. The NALT-M cells showed greater uptake of the smallest particles, 0.2 microm in diameter, than those of 0.5, 1.0, or 2.0 microm diameter. It was also revealed that surface coatings with poly-L: -lysin or chitosan resulted in efficient uptake into the NALT. These results indicate that nasal administration of antigenic microparticles, which were coated with cationic materials, probably leads to a useful method of transnasal vaccination against respiratory and intestinal infections in humans.

Identification of adenoid biofilms in chronic rhinosinusitis.

OBJECTIVE: To compare the percent mucosal surface area of adenoids removed from children with chronic rhinosinusitis (CRS) and those with obstructive sleep apnea (OSA) infected with biofilms. DESIGN: Comparative micro-anatomic investigation of adenoid mucosa using scanning electron microscopy from patients with CRS and OSA. SUBJECTS: 4 females and 12 males ranging from 3 months to 10 years of age. RESULTS: Adenoids removed from patients with CRS had dense mature biofilms covering the mucosal surface. More specifically, adenoids removed from patients with CRS had an average of 94.9% of their mucosal surface covered with mature biofilms vs. an average of 1.9% coverage on the adenoids removed from patients with OSA. These differences were statistically significant at the p<0.001 level. CONCLUSIONS: It is well established that adenoidectomy is useful in the treatment of CRS resistant to antibiotics. Adenoids removed from patients with CRS had almost their entire mucosal surface covered with biofilms vs. scant coverage for patients with OSA (p<0.001). Decreased metabolic activity, decreased growth rate, and transmission of resistance genes all contribute to the antibiotic resistant nature of the biofilms. These metabolically sessile communities shed planktonic microorganisms on an intermittent basis. Therefore biofilms in the nasopharynx of children with CRS may act as a chronic reservoir for bacterial pathogens resistant to standard antibiotics. Also, the mechanical debridement of the nasopharyngeal biofilms may explain the observed clinical benefit associated with adenoidectomy in this subset of pediatric patients.

Structural and immunological characteristics of chronically inflamed adenotonsillar tissue in childhood.

Recurrent or chronic adenotonsillar infections mainly affect children and frequently involve otherwise healthy subjects. Therefore, having excluded systemic immunological deficiencies, this disease may be due to a local dysfunction of the epithelial structures at either the rhino or oropharyngeal level. The aim of the present investigation was to analyze structural and immunological aspects of tonsils and adenoids in subjects who underwent adenotonsillectomy because of recurrent inflammatory episodes with fever. Histological studies and analyses of the cytokine patterns were carried out in palatine tonsils and adenoid samples from 105 patients who underwent adenoidectomy and bilateral extracapsular tonsillectomy for chronic inflammatory hypertrophy of these organs; 46 of the 105 cases examined presented hyperkeratosis of the crypt epithelium; in the remaining 59, the epithelium was hyperplastic with no signs of keratosis. Scanning electron microscopy revealed a continuous epithelial surface of polygon-shaped flattened cells with fissures towards the cryptic depressions. Titration of interleukin-1beta and tumor necrosis factor alpha in serum and tissues demonstrated higher concentrations in the adenotonsillar specimens, whereas the rise in interleukin-6 was more modest.

Light and electron microscope studies on the nasopharynx and nasopharyngeal tonsil of the horse.

Light and electron microscope studies were conducted on the nasopharynx and the nasopharyngeal tonsil of 15 young horses. The nasopharynx and nasopharyngeal tonsil was lined with pseudostratified columnar ciliated epithelium and goblet cells. The lymphoepithelium of the nasopharyngeal tonsil was folded forming crypts, the mucosa of which was modified into follicle associated epithelium characterized by stratified cuboidal epithelium, loss of cilia, absence of goblet cells and infiltration of lymphocytes. The lamina propria mucosae of the nasopharyngeal tonsil contained well-developed lymphoid tissue and clusters of seromucus acini. Scanning electron-microscopy revealed a dense mat of cilia covering the nasopharynx and nasopharyngeal tonsil. The follicle-associated epithelium consisted of different populations of microvillus cells in addition to M cells with very short microvilli and a few squamous and intermediate cells. Microvillus cells in the deeper part of the FAE had larger microvilli and their cytoplasm contained a dense population of mitochondria, smooth and rough endoplasmic reticulum, Golgi complexes and lysosomes. The flat surfaced M cell had a more electron-dense cytoplasm and contained small supranuclear vacuoles in addition to the organelles seen in microvillus cells.

The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa.

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M. avium FAP in a concentration-dependant manner. The number of bacteria adhering to fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.

Evidence of M cells as portals of entry for antigens in the nasopharyngeal lymphoid tissue of humans.

The nasopharyngeal tonsils (adenoids) are prominent components of human nasal-associated lymphoid tissues (NALT). However, the role of the nasopharyngeal tonsils in antigen uptake for initiation of the mucosal immune response is unknown. The aims of this study were to describe the ultrastructure and function of the M cells of the human nasopharyngeal tonsils and to clarify their capacity for antigen uptake. Tissues obtained from eight patients undergoing adenectomy were examined by light and electron microscopy. Lymphoepithelium covers the nasopharyngeal lymphoid tissue and consists of ciliary epithelium, non-ciliary epithelial cells, M cells, goblet cells, and many intraepithelial lymphoid cells. M cells have irregular and broad cytoplasm-containing microvilli on their surface and small vesicles in their cytoplasm. Many lymphoid cells were enfolded by M cells. The uptake of horseradish peroxidase (HRP) in the tissue in organ culture was studied using histochemical techniques. Excised adenoid tissue was incubated in RPMI 1640 culture media with HRP for 10, 30, and 60 min. HRP which had adhered to the surface was taken up in vesicles and then transported in vesicles and tubules by M cells. The M cells of nasopharyngeal lymphoid tissue were ultrastructurally and functionally similar to those in human Peyer's patches and colonic lymphoid follicles. These findings indicate that NALT bears similarities to the gut-associated lymphoid tissue, and its antigen uptake capacity may be important for initiation of immunity in the upper aerodigestive tract.

Adenoids and otitis media with effusion: a morphological study.

PURPOSE: Adenoidectomy, especially for the treatment of suppurative otitis media, has been used for a very long time. In this study, the role of adenoids in the origin of otitis media with effusion was investigated by using light microscopy, immunocytochemistry, enzyme chemistry, and electron microscopy. MATERIALS AND METHODS: A group of 28 children with otitis media with effusion (OME) was identified. Ages ranged from 3 to 12 years. A control group of 10 age-matched children without any middle ear and upper respiratory tract infection served as the basis for comparison. Specimens obtained at surgeries from both groups were divided into groups for light microscopy, immunocytochemistry, enzyme cytochemistry, and electron microscopy and then all were examined blindly. Also, quantitative analysis of antigen-presenting cells was performed blindly on 10 patients and 10 controls. RESULTS: There was an increase in the number of lymphocytes, mast cells, plasma cells, macrophages, dendritic cells, and M cells in the adenoids of patients with OME when compared with the normal cases. Stratified squamous epithelial areas, collagenous fibers, and fibrocytes were also increased in the patient group. Antigen-presenting functions of epithelial cells are shown by major histocompatibility complex (MHC) class II positivity of some ciliated-columnar epithelial cells in the patient group. CONCLUSION: Adenoid tissues of patients with OME in this study seem to be infectious foci, aggravating immune reactions, which might attack the middle ear through an ascending route.

Effect of salmeterol on Haemophilus influenzae infection of respiratory mucosa in vitro.

Haemophilus influenzae is a common bacterial pathogen causing human respiratory tract infections. We have previously shown that the beta2-agonist salmeterol reduces damage to the respiratory mucosa caused by Pseudomonas aeruginosa in vitro. We have now investigated the effect of salmeterol on H. influenzae infection of adenoid tissue in an organ culture by scanning electron microscopy. Tissue was preincubated with or without salmeterol (4x10(-7)M), prior to infection with H. influenzae and incubated for 12 or 24 h. Infected organ cultures had increased epithelial damage and decreased numbers of both ciliated and unciliated cells at 12h, which were significantly different (p < or = 0.01) from the controls at 24 h. Salmeterol (4x10(-7)M) significantly (p < or = 0.03) reduced damage and loss of ciliated cells in infected organ cultures at both 12 and 24, and significantly (p < or = 0.03) reduced loss of unciliated cells at 24 h. Salmeterol had no effect on the density of bacteria adhering to each individual mucosal feature or the total number of bacteria adhering to the organ culture. These results suggest that salmeterol protects the respiratory epithelium against Haemophilus influenzae-induced damage. The mechanism of salmeterol cytoprotection and its potential clinical relevance remain to be investigated.

Acute pharyngotonsillitis is an infection restricted to the crypt and surface secretion.

A commonly accepted hypothesis is that acute pharyngotonsillitis is caused by bacteria which first adhere to the epithelial surface and then invade the tonsillar parenchyma; however, evidence directly supporting this hypothesis is not available. In previous studies on acute pharyngotonsillitis, we found that the secretion in crypts and at the surface was infected in acute pharyngotonsillitis while no bacteria were detected in the parenchyma. Based on these results, we have proposed a new hypothesis stating that the infection is restricted to the crypt and surface secretions in acute pharyngotonsillitis. To evaluate this hypothesis further, in the present study we examined tonsillar tissue and secretion from patients with acute pharyngotonsillitis, recurrent pharyngotonsillitis and healthy tonsils. Surface secretion was studied after sampling by an imprint technique followed by routine histological preparation. Tonsillar tissue was examined by fluorescence microscopy after staining with acridine orange and by transmission electron microscopy. There were high numbers of bacteria and moderate or extensive ongoing phagocytosis in the crypt and surface secretion from patients with acute pharyngotonsillitis. Bacteria, leucocytes and phagocytosis were also present, but to less extent in the secretion from patients with recurrent pharyngotonsillitis and to even less extent in the healthy controls. In none of all the investigated tonsils were bacteria present in the parenchyma. Bacterial adherence to the epithelial surface was only very rarely observed. This study supports the hypothesis that acute pharyngotonsillitis is an infection restricted to the crypt and surface secretion and that bacterial adherence is not of significant importance in the pathogenesis of acute pharyngotonsillitis.

Effects of topical nasal steroids on human respiratory mucosa and human granulocytes in vitro.

Human respiratory mucosa and human granulocytes were exposed to topical nasal steroids in vitro. The preparations containing benzalkonium chloride and benzalkonium chloride alone destroyed the mucosa within 10 days. The same preparations also inhibited human neutrophil actin polymerization, degranulation and oxidative burst in vitro in a time and concentration dependent manner. Preparations without benzalkonium chloride, as well as the steroid compounds themselves, did not have these effects. It is concluded that benzalkonium chloride has toxic effects on human respiratory mucosa and human neutrophils in vitro.

Growth of influenza A virus in primary, differentiated epithelial cells derived from adenoids.

Epithelial cells of adenoid origin were grown in tissue culture to examine viral replication in cells that are the primary target of many human pathogens. These cells remained highly differentiated, with subpopulations of cells which retained active ciliary motility and others which demonstrated specialized secretory functions. The epithelial cells were permissive for growth of influenza A virus. Primary respiratory epithelial cells provide a model system for examining virulence, cell tropism, and receptors which replicate in the nasopharynx.