Structure of the Streptococcus pyogenes NAD+ Glycohydrolase Translocation Domain and Its Essential Role in Toxin Binding to Oropharyngeal Keratinocytes.

The emergence and continued dominance of a Streptococcus pyogenes (group A Streptococcus, GAS) M1T1 clonal group is temporally correlated with acquisition of genomic sequences that confer high level expression of cotoxins streptolysin O (SLO) and NAD+-glycohydrolase (NADase). Experimental infection models have provided evidence that both toxins are important contributors to GAS virulence. SLO is a cholesterol-dependent pore-forming toxin capable of lysing virtually all types of mammalian cells. NADase, which is composed of an N-terminal translocation domain and C-terminal glycohydrolase domain, acts as an intracellular toxin that depletes host cell energy stores. NADase is dependent on SLO for internalization into epithelial cells, but its mechanism of interaction with the cell surface and details of its translocation mechanism remain unclear. In this study we found that NADase can bind oropharyngeal epithelial cells independently of SLO. This interaction is mediated by both domains of the toxin. We determined by NMR the structure of the translocation domain to be a ß-sandwich with a disordered N-terminal region. The folded region of the domain has structural homology to carbohydrate binding modules. We show that excess NADase inhibits SLO-mediated hemolysis and binding to epithelial cells in vitro, suggesting NADase and SLO have shared surface receptors. This effect is abrogated by disruption of a putative carbohydrate binding site on the NADase translocation domain. Our data are consistent with a model whereby interactions of the NADase glycohydrolase domain and translocation domain with SLO and the cell surface increase avidity of NADase binding and facilitate toxin-toxin and toxin-cell surface interactions. IMPORTANCE NADase and streptolysin O (SLO) are secreted toxins important for pathogenesis of group A Streptococcus, the agent of strep throat and severe invasive infections. The two toxins interact in solution and mutually enhance cytotoxic activity. We now find that NADase is capable of binding to the surface of human cells independently of SLO. Structural analysis of the previously uncharacterized translocation domain of NADase suggests that it contains a carbohydrate binding module. The NADase translocation domain and SLO appear to recognize similar glycan structures on the cell surface, which may be one mechanism through which NADase enhances SLO pore-forming activity during infection. Our findings provide new insight into the NADase toxin and its functional interactions with SLO during streptococcal infection.

SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first detected in late December 2019 and has spread worldwide. Coronaviruses are enveloped, positive sense, single-stranded RNA viruses and employ a complicated pattern of virus genome length RNA replication as well as transcription of genome length and leader containing subgenomic RNAs. Although not fully understood, both replication and transcription are thought to take place in so-called double-membrane vesicles in the cytoplasm of infected cells. Here we show detection of SARS-CoV-2 subgenomic RNAs in diagnostic samples up to 17 days after initial detection of infection and provide evidence for their nuclease resistance and protection by cellular membranes suggesting that detection of subgenomic RNAs in such samples may not be a suitable indicator of active coronavirus replication/infection.

Comparative gross and scanning electron microscopical study of the oropharyngeal roof of young and adult domestic pigeons (Columba livia domestica).

The present study aims to compare the morphology of the oropharyngeal roof of young and adult domestic pigeon (Columba livia domestica) by gross observation, morphometric measurements, and scanning electron microscopy (SEM). The oropharyngeal roof was divided into the palate and pharyngeal roof. The palate was narrow triangular in shape and concave along its length. It could be divided into a rostral part contained three longitudinal palatine ridges and a caudal part contained the choanal slit. The choanal slit consisted of narrow rostral and wide caudal parts. The edges of the narrow part were encircled by small caudomedially directed papillae. On the contrary, the edges of the wide part of slit were free from papillae. By SEM, the palatal mucosa in young pigeon showed primordia of small papillae which increased in number and size forming a longitudinal row of papillae parallel to the edges of the rostral narrow part of slit in adult pigeon. The surface of the pharyngeal roof appeared smooth in young pigeon, while in adult pigeon, it showed dome-shaped elevations. The infundibular cleft had smooth edges. The caudal part of the pharyngeal roof formed an elevated transverse mucosal fold on which a transverse row of conical-shaped papillae was present. In conclusion, our results documented the presence of some differences between the oropharyngeal roof of young and adult pigeon, which suggest a high degree of functional adaptation in adult pigeon to their diet compared to young pigeon. Such adaptations might increase the efficiency of food prehension in adult pigeon. The present study compared the morphology of the oropharyngeal roof of young and adult domestic pigeon by gross observation, morphometry, and scanning electron microscopy. The morphometrical data showed higher values in adult pigeon compared to young pigeon. The palatal mucosa and the pharyngeal roof of adult pigeon showed papillae and elevations that were not present in young pigeon. Our results suggest a high degree of functional adaptation in adult pigeon to their diet compared to young pigeon. Such adaptations might increase the efficiency of food prehension in adult pigeon.

Identification and characterization of an M cell marker in nasopharynx- and oropharynx-associated lymphoid tissue of sheep.

M cells play a pivotal role in the induction of immune responses within the mucosa-associated lymphoid tissues. M cells exist principally in the follicle-associated epithelium (FAE) of the isolated solitary lymphoid follicles as well as in the lymphoid follicles of nasopharynx-associated lymphoid tissue and gut associated lymphoid tissue (GALT). Through lymphatic cannulation it is possible to investigate local immune responses induced following nasal Ag delivery in sheep. Hence, identifying sheep M cell markers would allow the targeting of M cells to offset the problem of trans-epithelial Ag delivery associated with inducing mucosal immunity. Sheep cDNA from the tonsils of the oropharynx and nasopharynx was PCR amplified using Glycoprotein-2 (GP2)-specific primers and expressed as a poly-His-tagged recombinant sheep GP2 (56 kDa) in HEK293 cells. The recombinant GP2 protein was purified using Ni-NTA affinity chromatography and polyclonal serum against the protein was raised in rats. The antiserum recognized the recombinant sheep GP2 and purified rat IgG against GP2 stained M cells in sections of sheep tonsils from nasopharynx and oropharynx. M cells were found to be present in epithelium of the palatine tonsils (oropharynx), pharyngeal tonsils as well as tubal tonsils (nasopharynx). They were also present in the FAE of the scattered lymphoid follicles over the base of the nasopharynx. Thus, GP2 has been identified to be an important M cell marker of nasopharynx and oropharynx-associated lymphoid tissues in sheep.

Estimation of the Young's moduli of fresh human oropharyngeal soft tissues using indentation testing.

Finite element (FE)-based biomechanical simulations of the upper airway are promising computational tools to study abnormal upper airway deformations under obstructive sleep apnea (OSA) conditions and to help guide minimally invasive surgical interventions in case of upper airway collapse. To this end, passive biomechanical properties of the upper airway tissues, especially oropharyngeal soft tissues, are indispensable. This research aimed at characterizing the linear elastic mechanical properties of the oropharyngeal soft tissues including palatine tonsil, soft palate, uvula, and tongue base. For this purpose, precise indentation experiments were conducted on freshly harvested human tissue samples accompanied by FE-based inversion schemes. To minimize the impact of the probable nonlinearities of the tested tissue samples, only the first quarter of the measured force-displacement data corresponding to the linear elastic regime was utilized in the FE-based inversion scheme to improve the accuracy of the tissue samples' Young's modulus calculations. Measured Young's moduli of the oropharyngeal soft tissues obtained in this study are presented. They include first estimates for palatine tonsil tissue samples while measured Young's moduli of other upper airway tissues were obtained for the first time using fresh human tissue samples.

Comparative distribution and arrangement of Herbst corpuscles in the oropharynx of the ostrich (Struthio camelus) and emu (Dromaius novaehollandiae).

Herbst corpuscles are widely distributed throughout the oropharynx of the ostrich and emu in contrast to the general situation in birds. Knowledge of the comparative distribution of Herbst corpuscles in the oropharynx of these two commercially important ratite species may assist in a better understanding of their feeding habits. Tissue sections representing all parts of the oropharynx of five ostrich and five emu heads collected after slaughter were prepared for light microscopy, the Herbst corpuscles counted, and the relative percentage of corpuscles calculated for defined anatomical regions. Herbst corpuscles were more widespread in the oropharynx of the emu (where they were additionally found in the tongue and laryngeal mound) than in the ostrich but were absent from the pharyngeal folds in both species. The results further indicated that Herbst corpuscles were strategically located to aid in the handling and transport of food. In this context, the high concentration of Herbst corpuscles in the prominent median palatine and ventral ridges in the ostrich denote these structures as sensory organs, namely the palatal and interramal organs. The presence of these sensory organs, coupled with the higher relative percentage of Herbst corpuscles located on the rostral oropharyngeal floor, indicate that the part of the oropharynx caudal to the mandibular and maxillary rostra forms an important sensory region in the ostrich. Additionally, species-specific concentrations of Herbst corpuscles within the oropharynx were identified which appear to assist in the accurate positioning of the tongue and laryngeal mound for cleaning the choana (internal nares) after swallowing.

The interplay between HPV and host immunity in head and neck squamous cell carcinoma.

Persistent infection with human papillomavirus (HPV) type 16 is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC), in particular oropharyngeal squamous cell carcinoma (OPSCC). The oropharyngeal epithelium differs from the mucosal epithelium at other commonly HPV16-infected sites (i.e., cervix and anogenital region) in that it is juxtaposed with the underlying lymphatic tissue, serving a key immunologic function in the surveillance of inhaled and ingested pathogens. Therefore, the natural history of infection and immune response to HPV at this site may differ from that at other anatomic locations. This review summarizes the literature concerning the adaptive immune response against HPV in the context of HNSCC, with a focus on the T-cell response. Recent studies have shown that a broad repertoire of tumor-infiltrating HPV-specific T-cells are found in nearly all patients with HPV-positive tumors. A systemic response is found in only a proportion of these. Furthermore, the local response is more frequent in OPSCC patients than in cervical cancer patients and HPV-negative OPSCC patients. Despite this, tumor persistence may be facilitated by abnormalities in antigen processing, a skewed T-helper cell response, and an increased local prevalence of T-regulatory cells. Nonetheless, the immunologic profile of HPV-positive vs. HPV-negative HNSCC is associated with a significantly better outcome, and the HPV-specific immune response is suggested to play a role in the significantly better response to therapy of HPV-positive patients. Immunoprofiling may prove a valuable prognostic tool, and immunotherapy trials targeting HPV are underway, providing hope for decreasing treatment-related toxicity.

Oral and pharyngeal epithelial keratinocyte culture.

Primary human oral epithelial cells are readily available and have been recently employed for tissue engineering. These cells are currently being widely utilized in multiple research efforts, ranging from the study of oral biology, mucosal immunity, and carcinogenesis to stem cell biology and tissue engineering. This chapter describes step-by-step protocols for the successful isolation and culture of human oral epithelial cells and fibroblasts, and techniques for their use in two-dimensional and three-dimensional culture systems. The described methods will enable to generate reconstituted tissues that resemble epithelial like structures in vitro, which can recapitulate some of the key features of the oral epithelium in vivo.

Reduction of DNA damage by curcumin and celecoxib in epithelial cell cultures of the oropharynx after incubation with tobacco smoke condensate.

BACKGROUND: Tobacco smoke, as the major risk factor for the development of squamous cell cancer of the head and neck (HNSCC), contains various xenobiotics, such as polycyclic aromatic hydrocarbons, nitrosamines, aromatic amines and phenols. Chemoprevention either by artificial agents such as celecoxib, or natural compounds such as curcumin, might offer a chance to reduce the risk of developing malignant transformation. MATERIALS AND METHODS: In order to evaluate the DNA-damaging effects of smoke condensate towards human mucosa cells of the oropharynx, mini organ cultures (MOC) of macroscopically healthy pharyngeal tissue of 40 patients with oropharyngeal SCC were used. After incubation with smoke condensate DNA damage was evaluated with the alkaline single-cell microgel electrophoresis (comet assay). The chemoprotective potential of curcumin and celecoxib was analyzed after their incubation with the condensate-treated MOCs. As DNA-damaging and chemopreventive effects might not be equally distributed over the whole DNA, fragmentation of the epithelial growth factor receptor (EGFR) gene was additionally examined by Comet fluorescence in situ hybridization (FISH). RESULTS: As expected, tobacco smoke condensate caused significant DNA fragmentation compared to the negative control. No enhanced damage was observed on the EGFR gene. DNA fragmentation was significantly reduced when MOCs were incubated with celecoxib (p ≤ 0.001) and with curcumin (p ≤ 0.001). CONCLUSION: Both celecoxib and curcumin showed considerable chemoprotective effects towards the impact of smoke condensate. No evidence was found for higher susceptibility to damage in the EGFR gene.

Confocal imaging of Merkel-like basal cells in the taste buds of zebrafish.

The oropharyngeal cavity in fish supports a range of sensory modalities, including detection of chemical and mechanical stimuli. Taste buds are found throughout this tissue and may participate in both processes. We used confocal microscopy and immunohistochemistry to characterize the morphology of Merkel-like cells and their association with other cell types and nerve fibers of the taste bud in the vertebrate model, the zebrafish. In addition, we document procedures for the observation of these structures in whole-tissue preparations from larvae and adults using zebrafish-specific and monoclonal antibodies. A single microvillus Merkel-like cell was found in each taste bud regardless of age or location. Merkel-like cells were neurosecretory, as indicated by labelling with the styryl dye, FM1-43, and the synaptic vesicle marker, SV2. Merkel-like cells were associated with SV2- and calretinin-positive taste receptor cells, received innervation from discoid aggregations of nerve fibers, and retained serotonin-filled synaptic vesicles oriented within the cytoplasm toward adjacent innervation. Moreover, a ring-like formation of nerve endings was identified with the neuronal marker, zn-12 that circumscribed the taste receptor area, surrounding calretinin-immunoreactive taste cell microvilli, and appeared to associate with the nerve plexus adjacent to Merkel-like cells. We suggest that these nerve fibers are somatosensory, perhaps associated with mechanoreception or the common chemical sense.

Topical curcumin can inhibit deleterious effects of upper respiratory tract bacteria on human oropharyngeal cells in vitro: potential role for patients with cancer therapy induced mucositis?

PURPOSE: Curcumin exerts its anti-inflammatory activity via inhibition of nuclear factor κB. Oropharyngeal epithelia and residing bacteria closely interact in inflammation and infection. This in vitro model investigated the effects of curcumin on bacterial survival, adherence to, and invasion of upper respiratory tract epithelia, and studied its anti-inflammatory effect. We aimed to establish a model, which could offer insights into the host-pathogen interaction in cancer therapy induced mucositis. METHODS: Moraxella catarrhalis (Mcat) and the oropharyngeal epithelial cell line Detroit 562 were used. Time-kill curves assessed the inhibition of bacterial growth and adherence assays and gentamicin protection assays determined the effect of curcumin-preincubated cells on bacterial adherence and invasion. Curcumin-mediated inhibition of pro-inflammatory activation by Mcat was determined via interleukin-8 concentrations in the supernatants. The synergistic role of secretory IgA (sIgA) on adherence was investigated. RESULTS: Curcumin was bactericidal at concentrations >50 µM. Preincubation of Detroit cells for 60 min demonstrated that concentrations >100 µM inhibited bacterial adherence. Together with sIgA, curcumin inhibited adherence at concentrations ≥50 µM. Both 100 and 200 µM curcumin significantly inhibited Mcat cell invasion. Finally, curcumin inhibited Mcat-induced pro-inflammatory activation by strongly suppressing IL-8 release. At a concentration of 200 µM, 10 min of curcumin exposure inhibited IL-8 release significantly, and complete suppression required a pre-exposure time of ≥45 min. CONCLUSION: Curcumin, in clinically relevant concentrations for topical use, displayed strong antibacterial effect against a facultative upper respiratory tract pathogen by inhibiting bacterial growth, adherence, invasion, and pro-inflammatory activation of upper respiratory tract epithelial cells in vitro.

Immunohistochemical localization of cytokeratins in the junctional region of ectoderm and endoderm.

Although tridermic species have two junctional regions of ectoderm and endoderm between their epidermis and digestive tract, we actually know little about these particular boundaries. Cytokeratins are the major intermediate filaments of epithelial cells and show a high degree of tissue specificity. Therefore, to characterize the epithelial cells in the junctional region of ectoderm and endoderm, we immunohistochemically examined the localization of cytokeratins 5, 7/17, 14, 18, Sox17, and alpha-fetoprotein (AFP) in the oropharyngeal and anorectal regions during the mouse gastrulation process. At embryonic day (E) 9.5, cytokeratins 5, 7/17, 14, and 18 were detected in all epithelial cells of the oropharyngeal region. At E12.5, cytokeratin 5-positive cells were not observed in the middle area of the oral cavity; however, the immunoreactivity was strong in the anterior and posterior areas. The immunoreaction of cytokeratins 18 was seen only in the middle and posterior areas of the oral mucosa. Cytokeratins 7/17 and 14 were localized in all areas of the oropharyngeal region. Sox17 and AFP, which are endodermal markers, were detected in the middle and posterior areas of the oral mucosa, but not in the anterior area. Moreover, this same localization pattern of cytokeratins also existed in the anorectal region of the E12.5 embryo, suggesting that the localization of cytokeratins and endodermal markers might give an implication for the boundary between ectoderm and endoderm. These results also suggest that these cytokeratins are useful molecules for monitoring the epithelial cell differentiation in the junctional region of the germ layers.

In vivo acquisition of prophage in Streptococcus pyogenes.

Genomic analysis of 12 Streptococcus pyogenes genomes representing six different serotypes reveals that they are poly-lysogenized, with as many as seven separate phage genomes (some of which are defective). Sequence alignments of these genomes (excluding incorporated prophage) have revealed that they are approximately 90% conserved, indicating that their diversity and disease capacity might be phage related. However, because S. pyogenes are only found in humans, how are new phages acquired? In vitro and in vivo experiments show that efficient phage transfer from donor to recipient streptococci occurs in the presence of mammalian cells. This suggests that, through evolution, phage have devised a system whereby progeny phage are induced and transferred to host streptococci at a site where host organisms are more prevalent.

Candida albicans protein kinase CK2 governs virulence during oropharyngeal candidiasis.

To identify Candida albicans genes whose proteins are necessary for host cell interactions and virulence, a collection of C. albicans insertion mutants was screened for strains with reduced capacity to damage endothelial cells in vitro. This screen identified CKA2. CKA2 and its homologue CKA1 encode the catalytic subunits of the protein kinase CK2. cka2delta/cka2delta strains of C. albicans were constructed and found to have significantly reduced capacity to damage both endothelial cells and an oral epithelial cell line in vitro. Although these strains invaded endothelial cells similarly to the wild-type strain, they were defective in oral epithelial cell invasion. They were also hypersusceptible to hydrogen peroxide, but not to high salt or to cell wall damaging agents. A cka1delta/cka1delta mutant caused normal damage to both endothelial cells and oral epithelial cells, and it was not hypersusceptible to hydrogen peroxide. However, overexpression of CKA1 in a cka2delta/cka2delta strain restored wild-type phenotype. Although the cka2delta/cka2delta mutant had normal virulence in the mouse model of haematogenously disseminated candidiasis, it had significantly attenuated virulence in the mouse model of oropharyngeal candidiasis. Therefore, Cka2p governs the interactions of C. albicans with endothelial and oral epithelial cells in vitro and virulence during oropharyngeal candidiasis.

Effects of epidermal growth factor and keratinocyte growth factor on the growth of oropharyngeal keratinocytes in coculture with autologous fibroblasts in a three-dimensional matrix.

Tissue engineering of oropharyngeal mucosa is rendered complex by the fact that oropharyngeal keratinocytes are difficult to culture in the long term and do not grow well after several subcultivations. Three populations of oropharyngeal keratinocytes were isolated by a method based on different levels of beta(1)-integrin expression. In particular, keratinocytes were isolated between cell fractions that adhere rapidly on collagen-IV-coated culture dishes (RAC-IV) and populations that are less adherent (RAC-IV-D). The total fraction of both subpopulations served as a control (RAC-IV-T). The epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) were examined with regard to their effects on the growth of the three populations. Growth curves of all three cell fractions grown with or without EGF were generated, and different concentrations of EGF and KGF were tested. EGF did not change any growth characteristics of the cells, with the exception of the speed of growth. Best growth was achieved with a physiologic EGF concentration of 0.15-1.5 ng/ml and a KGF concentration of 15 ng/ml. Finally, we cocultured oropharyngeal keratinocytes and their autologous fibroblasts in a three-dimensional matrix using Matrigeltrade mark. Oropharyngeal keratinocytes grown in coculture formed larger colonies than keratinocytes grown without fibroblasts. In conclusion, we were able to optimize the supplement of EGF and KGF in standard medium for the long-term culture of primary oropharyngeal keratinocytes. The use of Matrigel as a scaffold for three-dimensional cocultures of oropharyngeal keratinocytes and fibroblasts might signify a step forward in the development of a transplantable mucosa construct.

Oral EBV and KSHV infection in HIV.

The gamma herpesviruses, Kaposi's-sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are tightly associated with the development of AIDS-associated oral disease and malignancy during immune suppression. The objective of this investigation was to characterize oral infection and pathogenesis in healthy and immune-suppressed individuals. To characterize oral EBV and KSHV infection, we examined throat washings and oral epithelial cells from HIV-positive and HIV-negative individuals. Quantitative/real-time polymerase-chain-reaction (PCR) assays, transmission electronmicroscopy, immunostaining, and sequence analysis were used to identify viral infection. Virus was isolated from throat-wash samples and was used to infect epithelial and lymphoid cell lines. We detected EBV and KSHV in the oral cavity in healthy and immune-suppressed individuals. Viral strain analysis of KSHV K1 in multiple clones from the oral cavities of healthy persons and immunosuppressed patients detected several strains previously detected in KS lesions, with minor strain variation within individuals. Immunoelectron microscopy for multiple viral antigens detected consistent expression of viral proteins and oral epithelial specimens. In oral epithelial cells infected with wild-type KSHV in vitro, the K8.1 glycoprotein associated with lytic KSHV infection was detected in both primary and telomerase immortalized oral epithelial cultures by 24 hours post-infection. Virions were detected, subsequent to infection, by scanning electron microscopy. Oral epithelial cells were also infected in vitro with wild-type EBV originating from throat washes. Analysis of these data suggests that, like EBV, KSHV infection is present in the oropharynx of healthy individuals, is transmissible in vitro, and may be transmitted by saliva.

Cytolysin-dependent evasion of lysosomal killing.

Local host defenses limit proliferation and systemic spread of pathogenic bacteria from sites of mucosal colonization. For pathogens such as streptococci that fail to grow intracellularly, internalization and killing by epithelial cells contribute to the control of bacterial growth and dissemination. Here, we show that group A Streptococcus (GAS), the agent of streptococcal sore throat and invasive soft tissue infections, evades internalization and intracellular killing by pharyngeal epithelial cells. Production of the cholesterol-binding cytotoxin streptolysin O (SLO) prevented internalization of GAS into lysosomes. In striking contrast, GAS rendered defective in production of SLO were internalized directly or rapidly transported into lysosomes, where they were killed by a pH-dependent mechanism. Because SLO is the prototype of cholesterol-dependent cytolysins produced by many Gram-positive bacteria, cytolysin-mediated evasion of lysosomal killing may be a general mechanism to protect such pathogens from clearance by host epithelial cells.

Nitric oxide formation in the oropharyngeal tract: possible influence of cigarette smoking.

Cigarette smoking reduces the level of nitric oxide (NO) in exhaled air by an unknown mechanism. The view that part of the effect of cigarette smoking on NO production should occur in the oropharyngeal tract is supported by several studies. We have therefore compared smokers and non-smokers regarding non-enzymatic formation of NO from nitrite in the oral cavity since this is a primary candidate target for cigarette smoke. We have also looked at NO synthase-dependent NO formation in the mucosa of the oropharyngeal tract as an alternative target for the inhibitory effect induced by cigarette smoke. Smokers exhaled 67% lower levels of NO than controls (p<0.01, n=15 each group). We could not detect any significant difference in salivary nitrite, nitrate or ascorbate between smokers and non-smokers. Mouthwash with the antibacterial agent chlorhexidine reduced salivary nitrite (-65%) and exhaled NO levels (-10%) similarly in the two groups. Immunohistochemical techniques revealed dense expression of inducible (but not endothelial or neuronal) NO synthase in the squamous epithelium of non-inflamed tonsillar and gingival tissue biopsies. In the same biopsies, significant Ca2+ -independent citrulline-forming activity was detected. We found no difference between smoking and non-smoking subjects regarding NO-synthase expression and in vitro activity. In another group of non-smoking subjects (n=10), spraying the oropharyngeal tract with the NO-synthase inhibitor NG-monomethyl-L-arginine (250 mg) significantly reduced exhaled NO levels for at least 30 min (-18%, p<0.01). Our data suggest that cigarette smoking does not affect non-enzymatic NO formation from nitrite in saliva. However, NO is also formed by inducible NO synthase in the squamous epithelium of the normal oropharyngeal tract. We suggest that cigarette smoking may down-regulate enzymatic NO formation in the oropharyngeal compartment as well as in the bronchial compartment.

Expression of proteolytic enzymes in head and neck cancer-associated fibroblasts.

OBJECTIVE: To elucidate tumor-stromal interactions during tumor invasion by assessing the expression of proteolytic enzymes by carcinoma-associated fibroblasts (CAFs) in vivo using complementary DNA (cDNA) array analysis. METHODS: Tumor-associated stroma was isolated from tumor and adjacent mucosal specimens of the same patient by laser capture microdissection, and the messenger RNA (mRNA) was assessed by cDNA microarray specific for proteolytic enzymes and their inhibitors. Protein overexpression was then analyzed by immunoblotting of primary fibroblast isolates derived from skin, mucosa, and tumor specimens. RESULTS: Array analysis of 4 tumor and 4 adjacent mucosal samples demonstrated significant (2.6-fold) overexpression of membrane type 1 matrix metalloproteinase (MT1-MMP) but not of serine proteases or other matrix metalloproteinases. Analysis of normal dermal fibroblasts, normal mucosal fibroblasts, and CAFs similarly demonstrated up-regulation of MT1-MMP. CONCLUSIONS: These results suggest that MT1-MMP mRNA is specifically up-regulated in CAFs in vivo whereas MT1-MMP protein is specifically up-regulated in CAFs in vitro. Known to induce tumor cell invasion when expressed in tumor cells, CAF expression of MT1-MMP may be important in the stromal response to tumor cells that characterizes the desmoplastic reaction.

Efficiency of adenovirus-mediated gene transfer to oropharyngeal epithelial cells correlates with cellular differentiation and human coxsackie and adenovirus receptor expression.

Adenovirus-mediated gene transfer is a novel treatment strategy for head and neck squamous cell carcinoma (HNSCC) that may improve the unacceptable morbidity and mortality associated with conventional treatment. Efficient adenoviral (AdV) infection largely depends on cellular expression of the human coxsackie and adenovirus receptor (hCAR); however, the relatively recent identification of this receptor precludes a comprehensive description of its tissue distribution. We have created tissue culture model systems that approximate the differentiation and three-dimensional structure of stratified squamous epithelium characteristic of head and neck mucosa. Using these systems, we have found that expression of hCAR in native and modeled normal oropharyngeal epithelium decreased as cells differentiated with the most superficial and differentiated cells expressing no detectable hCAR. In contrast, modeled stratified HNSCC cells, which did not differentiate morphologically and did not express cytokeratin markers of differentiation, had equivalent expression of hCAR in superficial and basal layers. The expression of hCAR in our models correlated not only with the undifferentiated state, but also with efficiency of AdV infection. Despite expression of hCAR in underlying basal and suprabasal cells, topical application of AdV to normal modeled epithelium resulted in inefficient transduction of the most superficial cell layer without any infection of underlying cells. These data suggest that in normal epithelium the overlying squamous cells act as a barrier preventing infection of underlying cells that would otherwise be easily infected. In modeled stratified HNSCC, transduction was much more efficient and occurred up to four cell layers deep, suggesting that unlike normal superficial epithelial cells, the superficial cells of stratified HNSCC do not act as an effective barrier to adenoviral infection. The distribution of hCAR in native tissue and the enhanced susceptibility of undifferentiated oropharyngeal epithelial cells, including undifferentiated cancer cells, to AdV infection has important implications for the development of AdV-based targeting strategies for the treatment of head and neck cancer or premalignancies.