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.

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.

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.

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.

Highly polarized primary epithelial cells from human nasopharynx grown as spheroid-like vesicles.

The value of experimental culture models using epithelial cells often depends on the degree of polarization and other critical features observed in natural tissues, including the formation of tight junctions, desmosomes and membrane interdigitations. However, growth of normal epithelial cells as monolayers on artificial supports also leads to partial loss of the original characteristics of epithelial cells, and the quality of the monolayer is strongly influenced by the physicochemical properties of the support. In addition, not all normal epithelial cell types are able to adhere and to grow well on artificial substrata. In order to circumvent the drawbacks of two-dimensional cultures we established an in vitro model that closely resembles the in vivo situation of the intact epithelium. Human epithelial cells from nasopharynx (HNPEC) were used to prepare multicellular epithelial vesicles consisting of both non-ciliated and ciliated mucosal cells. Electron microscopy investigations showed that the morphological appearance of the epithelial cells was similar to that in situ. HNPEC vesicle cultures maintain a geometrically intact organization of individual cells that is not achieved using conventional culture conditions. HNPEC vesicles are more in vivo-like than two-dimensional cultures and therefore represent a suitable model for a variety of research purposes including studies on the pathogenesis of invasive microorganisms.

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 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.

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.

Human adenoidal organ culture: a model to study nontypable Haemophilus influenzae (NTHI) and other bacterial interactions with nasopharyngeal mucosa--implications in otitis media.

Nontypable Haemophilus influenzae (NTHI) has become the predominant cause of both acute suppurative otitis media and chronic otitis media with effusion. It has now been well-demonstrated that both outer membrane proteins and restriction fragment analysis of the bacterial genomes of concomitant nasopharyngeal and middle ear effusion isolates of NTHI are identical. It is therefore of critical importance to understand the mechanisms whereby bacteria that are present in normal healthy children in small numbers become the predominant organism in the nasopharynx in otitis media. The studies presented here suggest that nontypable Haemophilus influenzae can effectively decrease ciliary function as measured by stroboscopic illumination of ciliary beat frequency on human adenoidal organ culture. This organism also produces significant histopathologic and ultrastructural damage to the epithelial cells and cilia of adenoid organ culture, demonstrated by both light microscopy and scanning electron microscopy. The data suggest the following hypothesis: nontypable Haemophilus influenzae can destroy mucociliary function and allow increased bacterial replication in the mucus overlying the nasopharyngeal mucosa. The mucociliary system of the eustachian tube may also be involved in a similar manner, thus allowing bacteria to enter the middle ear space via the eustachian tube.

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.

Does the epithelium play a central role in the immune function of rhinopharyngeal tonsils? An immunocytochemical and ultrastructural study.

Enlarged adenoids from 10 children with chronic rhinitis and otitis media with effusion have been studied immunocytochemically and ultrastructurally, to better define the possible role of the epithelium and the dendritic accessory cells in the immune activation of lymphoid cells, and provide further insight into the pathogenesis of the disease. The presence within the columnar epithelium of lymphocytes positive for CD8 antigen, and which electron microscopically have been found frequently apposed to degenerating epithelial cells suggests that the latter cells are targets for cytotoxic activity of intraepithelial lymphocytes, rather than being engaged in antigen presentation. Furthermore, the finding of typical dendritic accessory cells, recognized by their typical immunophenotypic and ultrastructural features, in the lamina propria, indicates that antigen presentation is more likely exerted by dendritic accessory cells. This is further supported by the fact that these cells express major histocompatibility (MHC) class II molecules, which are needed for antigen presentation, whereas epithelial cells do not. A possible relationship between epithelial damage and the pathogenesis of adenoidal enlargement is discussed.

A scanning electron microscopic study of the equine upper respiratory tract.

The surface features of the upper respiratory tract of 20 clinically normal horses of various ages and types were studied with scanning electron microscopy. In the rostral part of the nasal cavity, there was a wide zone of non-ciliated epithelium whereas, caudally, the surface was well ciliated. This latter type of epithelium extended into the nasopharynx and guttural pouches although scattered areas of non-ciliated microvillous cells were also found.

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.

[Cytological diagnosis and cytohistological correlations of malignant epithelial tumors of the rhinopharynx]. / Le diagnostic cytologique et les corrélations cyto-histologiques des tumeurs épithéliales malignes du rhinopharynx

Nasopharyngeal carcinoma frequently has as its first manifestation cervical lymph node metastases before local symptoms appear. In the Instituto Nazionale per lo Studio e la Cura del Tumori of Milan, routine nasopharyngeal cytology has been carried out on these patients. Positive cytology in 55 cases of undifferentiated carcinoma (83.6%) and positive cytology of biopsies was obtained in 87.3%. The combination of cytology and histology gave a yield of 98.2% positive cases. For squamous carcinomas positive cytology was less frequently obtained, the same as for malignant lymphomas. The cytology of undifferentiated carcinoma has such marked morphological characters that it is very easy to diagnose it in a very high percentage of cases. In the differential diagnosis cytologically it is necessary to bear in mind other neoplasias, in particular some of the malignant lymphomas.

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.

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.

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.

New observations on the fine structure of the normal nasopharyngeal epithelium of the mouse.

The ciliated columnar epithelium in the mouse nasopharynx was studied by use of light and electron microscopes. The ciliated cells were divided into 3 types because of the variability in electron density of the ground cytoplasm. The dark ciliated cells were characterized by well-developed rough endoplasmic reticulum and rod-shaped mitochondria with regular arrangements of pronounced cristae. In the light ciliated cells endoplasmic reticulum was poorly developed and mitochondria were vacuolated with irregular arrangements of cristae. Furthermore, there existed the "medium ciliated cells," termed by the authors, of which the cytoplasm was medium in electron density between the dark and the light ciliated cells, and some mitochondria were rod-shaped and others were vacuolated. The medium ciliated cells are regarded to be at a stage of transformation from the dark ciliated cells to the light ones. These findings suggest that the dark ciliated cells swell to decrease their electron density with declining activity, and finally transform into the light ciliated cells. The cells, termed the "potential ciliated cells" by the authors, were scattered between the ciliated cells, reached the lumen and had no cilia. They contained many procentrioles suggesting ciliogenesis and had a cytoplasm of the same electron density as that of the dark ciliated cells. It is considered that the dark ciliated cells originate from the potential ciliated cells.

Study of the nasopharynx in man by scanning electron microscopy.

To date only few and often disagreeing studies about human nasopharynx are available. The present research has the purpose to give a contribution to the knowledge of nasopharyngeal epithelium using S.E.M. The study was carried out on biopsies taken from 20 healthy volunteers. The surface of nasopharynx is covered by ciliated cells, microvilli provided cells and goblet cells. Areas covered with squamous epithelium and presumably transitional epithelium were observed. The possibility that ciliated cells become microvilli provided cells is discussed.