Human Nasopharyngeal Tissue Research Articles

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

BackgroundThe 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.MethodsNasopharyngeal 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.ResultsThe 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.ConclusionsThe 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.

Fluorescence Lifetimes of Normal and Carcinomatous Human Nasopharyngeal Tissues

Time-resolved fluorescence spectra of normal and carcinomatous in vitro human nasopharyngeal tissues are compared. By fitting the time-resolved emission with exponential decays, mean lifetimes were obtained. There were marked differences between the lifetimes of the carcinomatous and the normal tissues. Thus, early diagnosis of nasopharyngeal carcinoma is possible. In general, comprehensive information from human tissue autofluorescence can be acquired via both time-resolved and steady-state fluorescence spectra.

Differential expression of a subset of ribosomal protein genes in cell lines derived from human nasopharyngeal epithelium

Extraribosomal functions of human ribosomal proteins (RPs) include the regulation of cellular growth and differentiation, and are inferred from studies that linked congenital disorders and cancer to the deregulated expression of RP genes. We have previously shown the upregulation and downregulation of RP genes in tumors of colorectal and nasopharyngeal carcinomas (NPCs), respectively. Herein, we show that a subset of RP genes for the large ribosomal subunit is differentially expressed among cell lines derived from the human nasopharyngeal epithelium. Three such genes (RPL27, RPL37a and RPL41) were found to be significantly downregulated in all cell lines derived from NPC tissues compared with a nonmalignant nasopharyngeal epithelial cell line. The expression of RPL37a and RPL41 genes in human nasopharyngeal tissues has not been reported previously. Our findings support earlier suspicions on the existence of NPC-associated RP genes, and indicate their importance in human nasopharyngeal organogenesis.

Gold Nanoparticle Based Surface-Enhanced Raman Scattering Spectroscopy of Cancerous and Normal Nasopharyngeal Tissues under Near-Infrared Laser Excitation

The capabilities of using gold nanoparticle based near-infrared surface-enhanced Raman scattering (SERS) to obtain biochemical information with high spatial resolution from human nasopharyngeal tissue were presented in this paper. The gold nanoparticles used have a mean diameter of 43 nm with a standard deviation of 6 nm. The SERS bands of nasopharyngeal tissue were assigned to known molecular vibrations of nucleic acids, amino acids, proteins, and metabolites. We also observed the blinking phenomenon at the tissue level when measuring the nasopharyngeal tissue SERS spectra, most frequently in signal intensity but also occasionally in peak positions. This phenomenon is excitation light intensity dependent. This work demonstrated great potential for using SERS imaging for distinguishing cancerous and normal nasopharyngeal tissues on frozen sections without using any dye labeling or other chemical species as functionalized binding sites.

Identification of Meningococcal Genes Necessary for Colonization of Human Upper Airway Tissue

Neisseria meningitidis is an exclusively human pathogen that has evolved primarily to colonize the nasopharynx rather than to cause systemic disease. Colonization is the most frequent outcome following meningococcal infection and a prerequisite for invasive disease. The mechanism of colonization involves attachment of the organism to epithelial cells via bacterial type IV pili (Tfp), but subsequent events during colonization remain largely unknown. We analyzed 576 N. meningitidis mutants for their capacity to colonize human nasopharyngeal tissue in an organ culture model to identify bacterial genes required for colonization. Eight colonization-defective mutants were isolated. Two mutants were unable to express Tfp and were defective for adhesion to epithelial cells, which is likely to be the basis of their attenuation in nasopharyngeal tissue. Three other mutants are predicted to have lost previously uncharacterized surface molecules, while the remaining mutants have transposon insertions in genes of unknown function. We have identified novel meningococcal colonization factors, and this should provide insights into the survival of this important pathogen in its natural habitat.

Protein D of Haemophilus influenzae: A Protective Nontypeable H. influenzae Antigen and a Carrier for Pneumococcal Conjugate Vaccines

Protein D (PD) is a highly conserved 42 kDa surface lipoprotein found in all Haemophilus influenzae, including nontypeable (NT) H. influenzae. PD is involved in the pathogenesis of respiratory tract infections, in the context of which it has been shown to impair ciliary function in a human nasopharyngeal tissue culture model and to augment the capacity to cause otitis media in rats. A likely mechanism indicating that PD is a virulence factor is its glycerophosphodiesterase activity, which leads to the release of phosphorylcholine from host epithelial cells. PD has been demonstrated to be a promising vaccine candidate against experimental NT H. influenzae infection. Rats vaccinated with PD cleared NT H. influenzae better after middle ear and pulmonary bacterial challenge, and chinchillas vaccinated with PD showed significant protection against NT H. influenzae-dependent acute otitis media. In a clinical trial involving children, PD was used as an antigenically active carrier protein in an 11-valent pneumococcal conjugate investigational vaccine; significant protection was achieved against acute otitis media not only caused by pneumococci but also caused by NT H. influenzae. This may have great clinical implications, because PD is the first NT H. influenzae antigen that has induced protective responses in humans.

Analysis of bacterial glycolipids by capillary electrophoresis-electrospray mass spectrometry: Haemophilus influenzae and Neisseria meningitidis lipopolysaccharides.

AbstractA number of invasive diseases are caused by human mucosal pathogens such as those of the genera Haemophilus, Neisseria, Moraxella, Campylobacter, and Bordetella. The bacterium N. gonorrhoeae infects mucosal surfaces of the genital tract, and can enter the bloodstream, survive, and cause secondary infections if protective antibodies are not raised in time (1). Both N. gonorrhoea and H. ducreyi are highly pathogenic, causing sexually transmitted diseases such as gonorrrhea and genital ulcers, respectively (2). The pathogens H. influenzae and N. meningitidis are uniquely adapted to colonize the human respiratory tracts and can lead to disseminated infections including otitis, and bacterial meningitis in young children (3,4). Through evolution, a number of these Gram-negative bacteria have elaborated surface antigens that mimic those found in human glycosphingolipids, thereby providing a mechanism for evading the innate immune system and enhancing their survival in the challenging environmental conditions of the host mucosa. While the exact mechanisms of colonization and invasion of H. influenzae and N. meningitidis are still poorly understood, it is generally recognized that lipopolysaccharides (LPS) play an important role in the virulence and pathogenicity of these organisms (5,6) and can associate with mucus and damaged epithelium of the human nasopharyngeal tissue (7).KeywordsStainless Steel ContainerHuman Respiratory TractCore OligosaccharideInfluenzae StrainSheep Blood Agar PlateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Telomerase activity is frequently found in metaplastic and malignant human nasopharyngeal tissues.

Telomerase is a specialized ribonucleoprotein polymerase that directs the synthesis of telomere repeats at chromosome ends. Accumulating evidence has indicated that telomerase is stringently repressed in normal human somatic tissues but reactivated in cancers and immortal cells, suggesting that reactivation of telomerase plays an important role in carcinogenesis. In this study, the status of telomerase activity in diseased human nasopharyngeal lesions was determined by the telomeric repeat amplification protocol (TRAP). Fifty-four patients participated including 17 inflammation or hyperplasia, eight with squamous metaplasia, and 29 with different stages of carcinomas. Telomerase activity was detected in 1 of 17 (5.9%) inflammatory or lymphoid hyperplastic tissues, 3 of 8 (37.5%) squamous metaplastic, and 25 of 29 (86.2%) carcinoma tissues. The differences in telomerase expression in these groups is statistically significant (P< 0.001). Levels of telomerase activity correlated with tumour stage (P= 0.024). These results suggest that telomerase reactivation plays a role in the carcinogenesis of nasopharyngeal cancer. Since telomerase activity is found in the majority of nasopharyngeal cancers and a subset of metaplasia, this enzyme may be served as a reference to monitoring the status of abnormal nasopharyngeal tissues. © 2000 Cancer Research Campaign

Effects on the Ciliated Epithelium of Protein D–Producing and –Nonproducing NontypeableHaemophilus influenzaein Nasopharyngeal Tissue Cultures

A pair of isogenic, nontypeable Haemophilus influenzae strains, one expressing protein D and the other protein D-negative, was compared in their ability to cause damage in a human nasopharyngeal tissue culture model. Damage was assessed by measuring the ciliary beat frequency (CBF) of tissue specimens at 12 h intervals. Cultures inoculated with H. influenzae manifested a decrease in CBF beginning after 12 h, with a maximum decrease after 36 h. The impairment of ciliary function by the protein D-expressing strain was significantly greater than that caused by the protein D-negative mutant (P<.01). Tissue specimens examined by scanning and transmission electron microscopy after 24 h appeared normal. After 48 h of incubation, the protein D-expressing strain caused a significant loss of cilia. These findings suggest that protein D is involved in the pathogenesis of upper respiratory tract infections due to nontypeable H. influenzae, probably by enhancing functional and morphological damage to cilia.

Pathogenic Events During Infection of the Human Nasopharynx with Neisseria meningitidis and Haemophilus influenzae

The human nasopharynx is the natural habitat and reservoir for Neisseria meningitidis and Haemophilus influenzae type b (Hib). Meningococcal and Hib pathogenesis was studied in human nasopharyngeal tissue in organ culture. Inocula of greater than or equal to 10(6) meningococci or cfu of Hib were required for consistent production of infection in these cultures. By 24 hours meningococci and Hib grew to 10(8)-10(10) cfu/mL in culture supernatants, while 10(4)-10(7) cfu per organ culture were tissue associated. These studies further indicated that nasopharyngeal mucus contains components that specifically bind Hib; that both meningococci and Hib cause cytotoxicity, resulting in breakdown of tight junctions of epithelial cells, sloughing of ciliated cells, and ciliostasis; that pili are the most important components mediating initial attachment of meningococci to non-ciliated epithelial cells of the human nasopharynx; that Hib expresses both pilus and nonpilus adhesions that facilitate attachment to nonciliated cells; and that meningococci and Hib both invade the epithelial surface to reach the submucosa but do so by different routes. Meningococci and Hib have evolved successful, although divergent, mechanisms by which to infect the human nasopharynx.

The Human Nasopharynx as a Reservoir for Epstein-Barr Virus

Nineteen human nasopharyngeal tissue specimens biopsied from patients with suspected nasopharyngeal carcinoma (NPC) and 15 adenoid tissue specimens obtained by surgery were successfully grown in culture without bacterial and fungal contamination. Fibroblastic growth developed in all of the cultures. In some cases, epithelial growth was also seen among the fibroblastic growth. The cells became round and floated in the culture fluid between 19th and 57th day of culture in 6 out of 10 NPC tissue cultures, 3 out of 9 tissue cultures of apparently normal nasopharyngeal mucosa of patients referred for exclusion of NPC, and 4 out of 15 adenoid tissue cultures. The floating cells consisted of B-lymphoblastoid cells and were positive Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA). The data suggest that a small number of B-lymphocytes in the human nasopharynx serve as a reservoir for EBV.

Pathogenesis of IgA1 protease-producing and -nonproducing Haemophilus influenzae in human nasopharyngeal organ cultures.

We evaluated mucosal attachment, colonization, and invasion by Haemophilus influenzae in an experimental model of human nasopharyngeal tissue in organ culture. Nonpiliated, encapsulated, and nonencapsulated, IgA1 protease-deficient mutants of H. influenzae were compared with their isogenic IgA1 protease-producing parents. Damage to peripheral ciliary activity was first noted 6 hr after infection and was associated with sloughing of ciliated cells to which H. influenzae were not attached. Infection of organ cultures with each strain resulted in similar degrees and rates of ciliary damage. H. influenzae attached selectively to nonciliated epithelial cells or was associated with surface mucus. Later, disruption of epithelial tight junctions was observed, and clusters of H. influenzae were found between epithelial cells. Organisms were also seen within phagocytic vacuoles of mononuclear cells located above and below the basement membrane. In summary, encapsulated and nonencapsulated H. influenzae damaged the ciliary function of human nasopharyngeal organ cultures, attached to the mucosal surface, and invaded the epithelium. H. influenzae IgA1 protease, however, was not essential for the pathogenic steps observed in this human nasopharyngeal organ culture model.

Analysis of damage to human ciliated nasopharyngeal epithelium by Neisseria meningitidis.

We used an in vitro model of human nasopharyngeal tissue in organ culture to evaluate the effects of Neisseria meningitidis on human cilia and ciliary function. Encapsulated, viable meningococci damaged ciliated epithelium of nasopharyngeal organ cultures, whereas Neisseria subflava, a commensal species, did not. Meningococcus-induced ciliary damage was due to loss of ciliated cells to which meningococci were not attached. Damage was seen with piliated and nonpiliated meningococci and did not appear to require the presence of other specific meningococcal surface proteins. Meningococcal viability was a requirement for both ciliary damage and interactions of meningococci with microvilli of nonciliated epithelial cells. That is, filter-sterilized supernatants from meningococcus-infected organ cultures, heat-killed meningococci at high inoculum, and purified meningococcal or gonococcal lipopolysaccharide at concentrations of 100 micrograms/ml did not damage ciliary activity of nasopharyngeal organ cultures. In contrast, meningococcal lipopolysaccharide at 10 micrograms/ml markedly damaged ciliary activity of human fallopian tube organ cultures, suggesting a selective toxicity of lipopolysaccharide for specific human ciliated cells. Damage to nasopharyngeal ciliated epithelium by N. meningitidis may be an important first step in meningococcal colonization of the human nasopharynx, but meningococcal lipopolysaccharide does not appear to be directly responsible for this toxicity.

Mechanisms of Mucosal Invasion by Pathogenic Neisseria

Neisseria gonorrhoeae are transported across the mucosa of human fallopian tubes in organ culture by mucosal cells. The steps in this process are (1) attachment of gonococci to microvilli of nonciliated cells, (2) phagocytosis of gonococci by these cells, (3) transport of phagocytic vacuoles containing gonococci to the base of the cell, and (4) exocytosis of gonococci with phagocytic vacuoles into the subepithelial tissues. In vivo gonococci in the subepithelial tissues may cause extensive local disease (e.g., salpingitis) or invade blood vessels to cause disseminated disease. Preliminary studies of human nasopharyngeal tissue in organ culture infected with Neisseria meningitidis indicate that meningococci attach to microvilli of nonciliated cells and are phagocytized by these cells. They subsequently appear in subepithelial tissues, but the route they take is not yet certain. These observations suggest that the mechanisms of attachment to and penetration of fallopian tube and nasopharyngeal mucosa by N. gonorrhoeae and N. meningitidis are similar or possibly identical.

Attachment of pathogenic Neisseria to human mucosal surfaces: role in pathogenesis.

Studies of the interaction between Neisseria gonorrhoeae and human fallopian tube mucosa in organ culture suggest that attachment of gonococci is important, not only to secure th organism in the host, but also to initiate the disease process. The steps observed in gonococcal infection of fallopian tube organ cultures are: 1) attachment of gonococci to microvilli of nonciliated cells; 2) release from gonococci of lipopolysaccharide and possibly other toxic moities to cause mucosa damage; 3) engulfment or phagocytosis of gonococci by nonciliated cells; 4) transport of phagocytic vacuoles containing gonococci to the base of the nonciliated cells; and 5) exocytosis of gonococci within phagocytic vacuoles into the subepithelial tissues. In vivo, these steps might result in extensive local disease (e.g. salpingitis) or in the invasion of blood vessels to cause disseminated disease. Preliminary studies of human nasopharyngeal tissue in organ culture infected with Neisseria meningitidis indicate that meningococci attach to microvilli of nonciliated cells and are phagocytized by these cells. Meningococci subsequently appear in subepithelial tissues, though the route they take is not yet certain. These observations suggest at least some of the ways in which attachment may play a role in disease caused by N. gonorrhoeae and N. meningitidis. Mechanisms to block this attachment may provide new approaches to the prevention of infections caused by the pathogenic Neisseria.

TRANSMISSION EXPERIMENTS WITH THE VIRUS OF POLIOMYELITIS

Since our note on the finding of the virus of anterior poliomyelitis in the tonsils of recovered monkeys,1we have been carrying on similar experimentations with human tonsillar and nasopharyngeal tissues as we have been able to obtain them from time to time. Our results in this we presented to the Boston Society of Medical Sciences late in 1911. Our conclusions were that the results of these experimental studies with the filtrates of human nasopharyngeal tissues, removed at varying periods after the acute attack, may be said to be suggestive but not conclusive for the following reasons: In three monkeys injected with three different tonsillar extracts no clinical signs of poliomyelitis followed the inoculations. In two monkeys the clinical signs were typical, but we were unable to pass the infection on to a second series of monkeys in a perfectly conclusive manner; nor were the pathologic findings absolutely typical,