SARS-CoV-2: preliminary study of infected human nasopharyngeal tissue by high resolution microscopy.

BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.

Planar cell polarity governs the alignment of the nasopharyngeal epithelium in mammals.

Planar cell polarity (PCP) signalling specifies the orientation of epithelial cells and regulates directional beating of motile cilia of multiciliated epithelial cells. Clinically, defects in cilia function are associated with nasopharyngeal symptoms. The polarity of the nasopharyngeal epithelium is poorly understood. Here, we demonstrated PCP in the nasopharyngeal epithelium. Multiciliated cells (MCCs) were uniformly aligned with their long axis parallel to the tissue axis of the nasopharynx (NP). In addition, PCP proteins exhibited an asymmetrical localisation between adjacent cells. Motile cilia were uniformly aligned in the same direction within both individual cells and neighbouring cells, which manifested as cilial polarity in MCCs. Mutation of Vangl2, a mammalian homologue of the Drosophila PCP gene, resulted in significant disruption of the orientation of epithelial cells. Finally, keratin-5-positive basal cells constantly replenished the luminal ciliated cells; the new dynamic ciliated cells were also oriented parallel to the tissue axis. These results indicate a role for the PCP pathway in the uniform orientation of dynamically replenished epithelial cells in the NP.

Morphological Characteristics of the Oropharyngeal Cavity (Tongue, Palate and Laryngeal Entrance) in the Eurasian Coot (Fulica atra, Linnaeus, 1758).

The present study represents the first definitive anatomical description of the oropharyngeal cavity of the coot Fulica atra. For this purpose, the organs of six birds were prepared to examine grossly and by SEM and stereomicroscope. The oval lingual apex had multiple overlapping branched acicular processes on its anterior and lateral border. The lingual apex and body had multiple caudally directed filiform-like papillae. By stereomicroscopy, the lingual root had a characteristic appearance and consisted of four parts. The openings of the anterior glands were present on the dorsal lingual surface of the body, while the projected papillae with wide openings of the posterior glands were present on the dorsal surface of lingual root. There was a row of caudally directed pharyngeal papillae at the caudal border of the laryngeal mound. Grossly, the pharyngeal papillae arrangement took a W-shape, while by stereomicroscopy was observed to be heart shape. The palate was divided into two regions: a small rostral non-papillary and a large caudal papillary region, but the rostral region was characterized by the presence of three longitudinal ridges. The papillary crest had two paramedian longitudinal papillary rows, which continued caudally until the beginning of the third median row. The freely distributed papillae took a caudolateral direction, while the papillae encircling the rostral part of choanal cleft took a caudomedial direction. There was a transverse papillary row between the two parts of choanal cleft. There was a transverse papillary row between the caudal border of the infundibular cleft and oesophagus.

The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens.

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.

The origin and early development of the nasal septum in human embryos.

Based on scanning electron microscopic dissections of human embryos and fetuses of the sixth to the twelfth week (Carnegie stages 16-23 and early fetus), the origin of the nasal septum was studied. The findings show that the nasal septum does not grow downwards. It is derived from the tissue between the primary choanae: as such, its anlage is present from the very beginning. Its contact and fusion with the palatal shelves is made possible by the elevation of the palatal shelves from the vertical into the horizontal position, as the tongue descends.

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.

The role of M cells of human nasopharyngeal lymphoid tissue in influenza virus sampling.

Little is known about the role of the M cells of human nasopharyngeal lymphoid tissue in the sampling of viruses that cause respiratory infections. To clarify whether M cells could function as a gateway for influenza virus into human nasopharyngeal lymphoid tissue, excised adenoid tissue was incubated in media containing influenza A virus for 30, 60, and 90 min, respectively. Transmission electron microscopic observation revealed that many influenza viruses adhered to M cell surfaces and were taken up into the cytoplasmic vesicles of M cells after 30 min incubation; the viruses had been transported into enfolded lymphoid cells after 60 min incubation. By staining M cells with Sambucus nigra lectin, which specifically recognizes the NeuAcalpha2,6 Gal linkage of sialoprotein, it was also found that abundant receptors for the human influenza virus are present on the M cell surface. Our findings indicated that M cells of human nasopharyngeal tonsils function as a major port for influenza A virus entry and that the virus could be efficiently transferred to enfolded macrophages and lymphoid cells by M cells. The transport of influenza viruses to lymphoid cells by M cells may promote antigen delivery to the immune system, and these findings may be important for systemic delivery of those influenza viruses that have the capacity to productively infect cells outside of the respiratory tract.

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.

M cells and associated lymphoid tissue of the equine nasopharyngeal tonsil.

The aim of this study was to characterise the morphological and histochemical features of equine nasopharyngeal tonsillar tissue. Nasal and oropharyngeal tonsillar tissue has been described as the gatekeeper to mucosal immunity because of its strategic location at the entrance to the respiratory and alimentary tracts. A combination of light, scanning and transmission electron microscopy has revealed the presence of follicle-associated epithelium (FAE) overlying lymphoid tissue of the equine nasopharyngeal tonsil caudal to the pharyngeal opening of the guttural pouch. Membranous microvillus (M) cells were identified in the FAE on the basis of short microvilli, an intimate association with lymphocytes, cytoplasmic vimentin filaments and epitopes on the apical surface reactive with lectin GS I-B4 specific for alpha-linked galactose. CD4-positive lymphocytes were scattered throughout the lamina propria mucosae as well as forming dense aggregates in the subepithelial part. The central follicular area was heavily populated with B lymphocytes and the dome and parafollicular areas contained both CD4- and CD8-positive lymphocytes. CD8-positive lymphocytes were also present in the epithelium and, together with B lymphocytes, in small numbers in the lamina propria mucosae. These observations indicate that the nasopharyngeal tonsil is potentially an important mucosal immune induction site in the horse and an appropriate target for intranasally administered vaccines.

Development of an ex vivo model to study adherence of Mannheimia haemolytica serovar 1 to mucosal tissues of the respiratory tract of cattle.

OBJECTIVE: To develop and validate an ex vivo model for study of adherence of Mannheimia haemolytica (formerly Pasteurella haemolytica) to respiratory tract mucosa of cattle and to use this model to confirm adherence of M haemolytica serovar 1 (Mh1) to several relevant respiratory mucosal surfaces. SAMPLE POPULATION: Excised nasal, nasopharyngeal, turbinate, and tonsillar mucosal tissue from the bovine upper respiratory tract. PROCEDURE: Mh1 was radiolabeled by use of tritiated leucine. Various concentrations of labeled bacteria were incubated with bovine upper respiratory tract tissues for various times. Tissue was washed to remove nonadherent bacteria, and percentage of bacteria adhered (percentage of adherence) was estimated using radioactivity. Using an optimal inoculum concentration and incubation time, percentage of Mh1 adherence was compared on nasal, nasopharyngeal, turbinate, and tonsillar mucosal tissue, and adherence to nasopharyngeal tissue was confirmed by scanning and transmission electron microscopy. RESULTS: The optimal Mh1 inoculum concentration was 1 X 10(7) colony forming units/ml and incubation time was 3 hours. Percentage of adherence of Mh1 to nasopharyngeal tissue was greater than adherence to other tissue types. CONCLUSIONS AND CLINICAL RELEVANCE: The ex vivo model maintained the functional and structural integrity of bovine upper respiratory tract mucosa, as confirmed by light and electron microscopy. Electron microscopy revealed participation of epithelial cell cilia and surface mucus in adherence of Mh1 to nasopharyngeal tissue. Adherence of Mh1 was confirmed in repeated assays, indicating that this organism adheres to upper respiratory tract mucosa of cattle.

High frequency of chromosome 3p deletion in histologically normal nasopharyngeal epithelia from southern Chinese.

We have examined the presence of loss of heterozygosity (LOH) on chromosome 3p in histologically normal nasopharyngeal epithelia (NP), dysplastic lesions, and carcinoma of the nasopharynx from different ethnic and geographic regions. Microdissected normal NP from noncancerous individuals and nasopharyngeal carcinoma (NPC) samples from both the high-risk group (southern Chinese in Hong Kong) and two low-risk groups for NPC (central/northern Chinese in Anhui/Beijing and Caucasians in Toronto) were included. All NPC samples showed high incidence of 3p deletion (81-100%). High frequencies of LOH on 3p were also detected in normal NP (73.9%) and dysplastic lesions (75%) from the southern Chinese. Significant lower frequency of LOH on 3p was noted in normal NP from the low-risk groups (20%) than those from high-risk groups (P = 0.0003). The presence of such genetic alterations in the histologically normal NP and dysplastic lesions suggests that it is an early event in tumor development. The higher frequency of 3p LOH found in normal NP from southern Chinese compared with those from low-risk groups may be related to the distinct cancer incidence among these populations.

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.

The endolymphatic sac receives antigenetic information from the organs of the mucosa-associated lymphatic system.

The endolymphatic sac holds the entire arrangement of immunocompetent cells and functions as an immunological potent control organ for the inner ear. The evidence of secretory immunoglobulin A and other features of lymphocyte subtypes characterizes the endolymphatic sac as an organ of the mucosa-associated lymphatic system (MALT). In this system a permanent recirculation of sensitized memory lymphocytes from one organ to the other has been demonstrated experimentally as serving to dispose memory lymphocytes after renewed antigenetic stimulus. The aim of this study was to prove the possible recirculation of antigen-sensitized lymphocytes to the endolymphatic sac after antigenic stimulus of another part of the mucosa-associated lymphatic system. The results are evidence that the endolymphatic sac is provided with immunocompetent cells which derive from the lymphatic tissue of the nasopharynx. While the origin of immunocompetent cells in the endolymphatic sac still remains uncertain, this study underlines the role of lympho-epithelial tissue of the nasopharynx as a possible cell source for the endolymphatic sac. The results might explain the altered or disturbed function of the endolymphatic sac as a possible cause of certain inner ear diseases.

Intermediate epithelium.

In the nasopharynx, the larynx, the anal canal and the auditory tube, the "intermediate epithelium" occupied the transitional zone between the ciliated (or nonciliated) columnar epithelium and the stratified squamous one. The intermediate epithelium showed gradations ranging from stratified low-columnar through stratified cuboidal to stratified squamous type. It was suggested that the intermediate epithelium showed the various stages of the epithelium transforming from the columnar to the squamous epithelium, and that the basal cells of the columnar epithelium served as the germinal layer of the transformation.

Characterization of smoking-induced nasopharyngeal lymphoid hyperplasia.

The frequency of smoking-induced nasopharyngeal lymphoid hyperplasia in heavy smokers and its potential clinical implications are still unknown. Precise criteria to differentiate this entity from other types of nasopharyngeal lymphoid hyperplasia are needed. A prospective clinicopathological study of smoking-induced nasopharyngeal lymphoid hyperplasia was conducted in 17 heavy smokers. Ten nonsmoking patients, five of them with chronic sinusitis, three with adult-onset adenoid hypertrophy, and two children with adenoidal hypertrophy served as a control group. Both in smokers and in nonsmokers, lymphocytic infiltration of the mucosa was characterized immunohistochemically as T cells. In smokers, semithin (1 micron) sections revealed deformed and migrating cytotoxic lymphocytes in the nasopharyngeal mucosa. The lymphocytes were attached to epithelial, ciliated, and goblet cells, resulting in cell damage. Transmission electron microscopy of biopsies from smokers revealed emperipolesis, characterized by mucosal invasion and epithelial cell damage by an unusual population of migrating T lymphocytes that penetrate them. These findings confirm a direct effect of smoking on the nasopharyngeal lymphoid tissue, which forms part of the immune system. It is concluded that the diagnostic evaluation and therapeutic approach of heavy smokers with otological and airway symptoms should be based on thorough endoscopic examination of the nasopharynx. When the diagnosis is not clear-cut, selective tele-endoscopic biopsy and electron microscopic examination are recommended. This entity should be added to the list of known clinical manifestations of the smoking habit.

Nasopharyngeal carcinoma, with emphasis on its relationship to Epstein-Barr virus.

Nasopharyngeal carcinoma (NPC) is an epithelial tumor with a distinct geographic distribution and characteristic histologic appearance. It is rare in Europe and North America, but it is among the most common cancers in southern China. Genetic predisposition, environmental factors, and Epstein-Barr virus (EBV) all have been associated with the pathogenesis of this tumor. There is an increasing body of evidence that among all these factors, EBV appears to be the strongest and most consistently related factor. According to the current sensitive in situ hybridization methods for the detection of EBV-encoded small RNAs (EBER), almost 100% of cases of NPC, irrespective of their histologic subtypes, have demonstrable EBERs in the nuclei of the tumor cells. In this review paper, we discuss the predisposing genetic and environmental factors and the role of EBV in the pathogenesis of this tumor with particular emphasis on the role of EBV.

Damage to the nasopharyngeal mucosa induced by current levels of urban air pollution: a field study in lambs.

This study concerns the effects of urban air pollution on the nasopharyngeal epithelium, with the aim of evaluating the possible harmful activity of levels of atmospheric pollution which are not currently considered to be dangerous. Over a 3 month period, 10 lambs kept in a zone characterized by numerous vehicles were sacrificed at regular intervals, and their nasopharyngeal mucosa was examined by scanning electron microscopy and image analysis. Two lambs kept in a rural area were used as controls. The local levels of some airborne contaminants (NO(x), NO2, NO, SO2, CO and particulate matter with aerodynamic diameter < or =10 microm (PM10)) were monitored throughout the experiment. The urban air had an irritating effect, inducing hypersecretion of mucus and morphological damage to the ciliated epithelium. These alterations increased with the duration of exposure to urban air and with increasing pollution levels, although the levels remained below current legal levels. We conclude that the harmful effects of airborne contaminants are probably underestimated. Moreover, physicochemical evaluation of pollution parameters should be complemented by morphological study of upper respiratory epithelium in exposed animals, since this mucosa is a sensitive target for irritating agents.

Ultrastructure of the nasopharyngeal orifice epithelium of the eustachian tube in otitis media with effusion.

Epithelial changes in nasopharyngeal orifice of eustachian tube in 15 patients with otitis media with effusion (OME) were studied. Ultrastructural examination of the epithelium revealed distinct alterations in the ciliated cells, intermediate cells and in the columnar cells with microvilli. The ciliated cells were the predominant cell type in the epithelium and were characterized by compound cilia and apical cytoplasmic bulgings with fine granular content. The intermediate cells showed more prominent lateral cytoplasmic bulgings. Cytoplasmic bulgings of both cell types eventually pinched off and set free as cytoplasmic bodies, similar to the cytoplasmic bodies derived from lymphocytes. As a result of epithelial destruction, the lumen of nasopharyngeal orifice was occupied by epithelial cellular debris among which leucocytic cells and cytoplasmic bodies with fine granular content. This accumulation in the lumen probably developed as a result of defective mucociliary activity which is due to compound cilia formation in the ciliated cells. Moreover, ultrastructural resemblance of cytoplasmic bodies derived from ciliated cells, intermediate cells and leucocytic cells indicates the possible role of these cells in common immune defence mechanisms in chronic otitis media with effusion.

The effect of mutations in genes required for lipopolysaccharide synthesis on Haemophilus influenzae type b colonization of human nasopharyngeal tissue.

Haemophilus influenzae type b (Hib) is an upper respiratory tract commensal that can cause invasive disease, particularly in young children. Lipopolysaccharide (LPS) has been implicated as a major virulence determinant of Hib, and changes in LPS structure may influence bacterial interactions with the respiratory mucosa. We have examined the effect of variations in LPS on the interaction of Hib with human nasal turbinate tissue maintained in an organ culture model with an air-interface, by using isogenic derivatives of strains RM153 (Eagan) and RM7004 expressing truncated LPS due to mutations in genes contained within the chromosomal loci lic1 and lic2 (lic1lic2) or in the galE and galK genes (galEK). Tissue was infected with an inoculating dose of 2.3-3.3 x 10(6) colony forming units (cfu) in 2 microliters of PBS and maintained for 24 h. By scanning electron microscopy the percentage of the organ culture surface exhibiting epithelial damage increased from 5.3 +/- 1.4 in controls to 12.5 +/- 6.4-26.3 +/- 9.1 following infection, with no significant difference between parent strains and their derivatives. There was significant bacterial tropism for mucus, and to a lesser extent damaged cells, which was not influenced by the LPS phenotype. All strains caused separation of epithelial cells, adhered to non-luminal cell surfaces, and invaded the epithelium intercellularly. We conclude that Hib associated with mucus and damaged epithelium, and infrequently with normal epithelium, but changes in the LPS phenotype did not affect the interaction between Hib and the mucosal surface of human nasal turbinate tissue.

Invasion of primary nasopharyngeal epithelial cells by Neisseria meningitidis is controlled by phase variation of multiple surface antigens.

We have investigated bacterial factors required for the entry of Neisseria meningitidis serogroup B into mucosal cells using a novel in vitro infection model of primary cultures of human nasopharyngeal epithelium. An invasive meningococcal phenotype was obtained after several cycles of selection for intracellular bacteria with gentamicin. Invasive bacteria differed from those in the initial inoculum in that they lacked a capsule and pili, exhibited a nonsialylated low-molecular-weight type of lipopolysaccharide (LPS), and produced a new 28-kDa opacity outer membrane protein. LPS revertants of the selected meningococci expressed a nonsialylated L3,(7,9) type of LPS and were also invasive, while after LPS sialylation bacterial entry was inhibited. Variants lacking the 28-kDa opacity protein were poorly invasive. Coexpression of the outer membrane protein Opc and the 28-kDa opacity protein strongly inhibited microbial invasion into the primary cultured nasopharyngeal cells. Conversely, meningococcal internalization by cells of various epithelia] cell lines was correlated with the expression of Opc rather than the 28-kDa opacity protein. Our data indicate that a concurrent phase switching of multiple phase-variable bacterial surface components may be a prerequisite for meningococcal invasion into nasopharyngeal epithelium and that meningococcal class 5 proteins (Opa and Opc) may promote tissue tropism.