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Obstructive sleep apnea (OSA) is characterized by the collapse of the soft palate in the upper airway, resulting in chronic intermittent hypoxia during sleep. Therefore, an understanding of the molecular mechanisms underlying pathophysiological dysfunction of the soft palate in OSA is necessary for the development of new therapeutic strategies. In the present study, we observed that high mobility group protein box 1 (HMGB1) was released by a large infiltration of macrophages in the soft palate of OSA patients. The toll-like receptor 4/nuclear factor kappa B pathway was observed to be activated by the release of HMGB1, and this was accompanied by an increased expression of pro-inflammatory factors, including tumor necrosis factor-α and interleukin-6. Importantly, increased expression of toll-like receptor 4 was observed in endothelial cells, contributing to upregulation of the angiogenesis-related factors vascular endothelial-derived growth factor and matrix metalloproteinase 9. Moreover, we confirmed the effect of the HMGB1-mediated toll-like receptor 4/nuclear factor kappa B pathway on cell proliferation and angiogenesis in an in vitro cell model of human umbilical vein endothelial cells. We conclude that HMGB1 may be a potential therapeutic target for preventing angiogenesis and pathology in OSA., (© 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

We reported de novo variants in specific exons of the TBX15 and ADAMTS2 genes in a hitherto undescribed class of patients with unique craniofacial developmental defects. The nine unrelated patients represent unilateral soft palate hypoplasia, lost part of the sphenoid bone in the pterygoid process, but the uvula developed completely. Interestingly, these clinical features are contrary to the palate's anterior-posterior (A-P) developmental direction. Based on developmental characteristics, we suggested that these cases correspond to a novel craniofacial birth defect different from cleft palate, and we named it soft palate dysplasia (SPD). However, little is known about the molecular mechanism of the ADAMTS2 and TBX15 genes in the regulation of soft palate development. Phylogenetic analysis showed that the sequences around these de novo mutation sites are conserved between species. Through cellular co-transfections and chromatin immunoprecipitation assays, we demonstrate that TBX15 binds to the promoter regions of the ADAMTS2 gene and activates the promoter activity. Furthermore, we show that TBX15 and ADAMTS2 are colocalization in the posterior palatal mesenchymal cells during soft palate development in E13.5 mice embryos. Based on these data, we propose that the disruption of the TBX15-ADAMTS2 signaling pathway during embryogenesis leads to a novel SPD., (© 2022 Wiley Periodicals LLC.)

The soft palate is the only structure that reversibly separates the respiratory and gastrointestinal systems. Most species can eat and breathe at the same time. Humans cannot do this and malfunction of the soft palate may allow food to enter the lungs and cause fatal aspiration pneumonia. Speech is the most defining characteristic of humans and the soft palate, along with the larynx and tongue, plays the key roles. In addition, palatal muscles are involved in snoring and obstructive sleep apnea. Considering the significance of the soft palate, its function is insufficiently understood. The objectives of this study were to document morphometric and immunohistochemical characteristics of adult human soft palate muscles, including fiber size, the fiber type, and myosin heavy chain (MyHC) composition for better understanding muscle functions. In this study, 15 soft palates were obtained from human autopsies. The palatal muscles were separated, cryosectioned, and stained using histological and immunohistochemical techniques. The results showed that there was a fast type II predominance in the musculus uvulae and palatopharyngeus and a slow type I predominance in the levator veli palatine. Approximately equal proportions of type I and type II fibers existed in both the palatoglossus and tensor veli palatine. Soft palate muscles also contained hybrid fibers and some specialized myofibers expressing slow-tonic and embryonic MyHC isoforms. These findings would help better understand muscle functions.

BACKGROUND Chronic intermittent hypoxia (CIH) is a key feature of obstructive sleep apnea (OSA) syndrome. The pathogenesis of CIH-induced soft palate lesion is not well understood. Understanding the mechanisms of CIH-induced soft palate damage could provide new strategies for clinical treatment. MATERIAL AND METHODS Twenty male Sprague‑Dawley rats were randomized into a control group (n=10) and experimental group (n=10). The experimental group were exposed to CIH for 28 days. The control experiments were run in parallel. Morphological changes of CIH-induced soft palate were examined by hematoxylin and eosin. Peripheral nerves and vascular associated markers were analyzed by western blot and immunohistochemical staining. LC3B expression and transmission electron microscopy analysis was detected to investigate the destiny of cells in CIH-induced soft palate. RESULTS Histological studies demonstrated the thicken mucosal layer, muscular changes consistent with glands hyperplasia, and loose connective tissues of the soft palate in CIH induced rat models. CIH exposure significantly decreased the expression of annexin V but did not change argin level, suggesting that sensory nerves not motor nerves were damaged when exposed to intermittent hypoxia. Moreover, in response to CIH, the vascular vessel around the nerves and muscles became enlarged and caveolin-1 was overexpressed. Autophagy occurs in response to CIH-induced neuromuscular and vascular endothelial injury. CONCLUSIONS Sensory nerves and endothelial dysfunction contributed to the morphological damage of soft palate under intermittent hypoxia. Autophagy as a compensatory mechanism protects against CIH-induced injury. These findings have important implications for understanding mechanisms contributing to the increased soft palate lesion in patients with OSA.

A mouse single taste bud contains 10-100 taste bud cells (TBCs) in which the elongated TBCs are classified into 3 cell types (types I-III) equipped with different taste receptors. Accordingly, differences in the cell numbers and ratios of respective cell types per taste bud may affect taste-nerve responsiveness. Here, we examined the numbers of each immunoreactive cell for the type II (sweet, bitter, or umami receptor cells) and type III (sour and/or salt receptor cells) markers per taste bud in the circumvallate and foliate papillae and compared these numerical features of TBCs per taste bud to those in fungiform papilla and soft palate, which we previously reported. In circumvallate and foliate taste buds, the numbers of TBCs and immunoreactive cells per taste bud increased as a linear function of the maximal cross-sectional taste bud area. Type II cells made up approximately 25% of TBCs irrespective of the regions from which the TBCs arose. In contrast, type III cells in circumvallate and foliate taste buds made up approximately 11% of TBCs, which represented almost 2 times higher than what was observed in the fungiform and soft palate taste buds. The densities (number of immunoreactive cells per taste bud divided by the maximal cross-sectional area of the taste bud) of types II and III cells per taste bud are significantly higher in the circumvallate papillae than in the other regions. The effects of these region-dependent differences on the taste response of the taste bud are discussed., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Leishmaniasis is a parasitic infection transmitted by the bite of infected female sandflies. It principally affects the skin, and the frequency of mucosal involvement is about 5% to 20%. Considering the rarity of leishmaniasis affecting the upper aerodigestive tract mucosa, we evaluated the characteristics of mucocutaneous leishmaniasis and mucosal leishmaniasis and the diagnostic difficulty when the parasites are scarce in tissue samples. The clinical, histopathological, histochemical, immunohistochemical, and molecular features of 17 cases of mucocutaneous leishmaniasis and mucosal leishmaniasis were assessed. Mucosal disease was principally found in the soft palate, oropharynx, and nose, manifesting mainly as a solitary ulcer. In hematoxylin and eosin-stained sections, 10 cases revealed abundant amastigotes within the macrophages. Giemsa staining was not shown to be helpful to confirm the diagnosis in 6 cases with scarce or nondetectable amastigotes. Immunohistochemistry (IHC) showed high sensitivity by positive staining in 14 out of 17 cases (82.3%). Polymerase chain reaction was shown to be more sensitive than IHC with 13 out of 14 (92.8%) positive cases, including the 3 IHC negative cases; however, this technique is not available in many endemic regions. In summary, we suggest that the IHC is a simple technique with rapid results and relatively low cost, when compared with other laboratorial procedures; thus, IHC is a helpful tool that should be implemented in the routine diagnosis of leishmania.

The soft palate is a key component of the oropharyngeal complex that is critical for swallowing, breathing, hearing and speech. However, complete functional restoration in patients with cleft soft palate remains a challenging task. New insights into the molecular signaling network governing the development of soft palate will help to overcome these clinical challenges. In this study, we investigated whether key signaling pathways required for hard palate development are also involved in soft palate development in mice. We described the dynamic expression patterns of signaling molecules from well-known pathways, such as Wnt, Hh, and Fgf, during the development of the soft palate. We found that Wnt signaling is active throughout the development of soft palate myogenic sites, predominantly in cells of cranial neural crest (CNC) origin neighboring the myogenic cells, suggesting that Wnt signaling may play a significant role in CNC-myogenic cell-cell communication during myogenic differentiation in the soft palate. Hh signaling is abundantly active in early palatal epithelium, some myogenic cells, and the CNC-derived cells adjacent to the myogenic cells. Hh signaling gradually diminishes during the later stages of soft palate development, indicating its involvement mainly in early embryonic soft palate development. Fgf signaling is expressed most prominently in CNC-derived cells in the myogenic sites and persists until later stages of embryonic soft palate development. Collectively, our results highlight a network of Wnt, Hh, and Fgf signaling that may be involved in the development of the soft palate, particularly soft palate myogenesis. These findings provide a foundation for future studies on the functional significance of these signaling pathways individually and collectively in regulating soft palate development., Competing Interests: The authors have declared that no competing interests exist.

This study aimed to describe the morphology, expression of IgA and IgG in adult yak tonsils. The 12 clinically healthy yak tonsils [3- to 6-year old, n = 12] were examined for morphology using light, and transmission electron microscopes. Expression of IgA and IgG was measured by qRT-PCR, ELISA, and immunohistochemistry. The results showed that the palatine tonsil, the tonsil of the soft palate, and the lingual tonsil were oropharyngeal tonsils. The stratified squamous epithelia covering them had a thick underlying layer of connective tissue and their crypts were heavily infiltrated by lymphocytes. The pharyngeal tonsil and the tubal tonsil were nasopharyngeal tonsils. The epithelia of them was predominantly pseudostratified columnar ciliary epithelium, which were loosely arranged with a number of desmosomes or intermediate junctions variably connecting them. The expression levels of IgA and IgG mRNA and protein from high to low was in the pharyngeal tonsil, palatine tonsil, tonsil of the soft palate, lingual tonsil, and tubal tonsil, respectively. Interestingly, the expression of IgG was very significantly higher than that of IgA in yak tonsils (P < 0.01). Both the IgA and IgG ASCs were distributed in the subepithelial areas of the non-reticular crypt epithelium, especially areas of pseudostratified columnar ciliary epithelium, the reticular crypt epithelium, lymphoid follicles, interfollicular areas, and with some of the positive cells aggregating around the glands. The results indicated that the tonsils were not only typical secondary lymphoid organs but also lymphoepithelial structures. IgG could be a significant component of mucosal immune responses in yak tonsils. Anat Rec, 302:999-1009, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Background: The pathophysiology of obstruction and swallowing dysfunction in snores and sleep apnea patients remains unclear. Neuropathy and to some extent myopathy have been suggested as contributing causes. Recently we reported an absence and an abnormal isoform of two cytoskeletal proteins, desmin, and dystrophin, in upper airway muscles of healthy humans. These cytoskeletal proteins are considered vital for muscle function. We aimed to investigate for muscle cytoskeletal abnormalities in upper airways and its association with swallowing dysfunction and severity of sleep apnea., Methods: Cytoskeletal proteins desmin and dystrophin were morphologically evaluated in the uvula muscle of 22 patients undergoing soft palate surgery due to snoring and sleep apnea and in 10 healthy controls. The muscles were analysed with immunohistochemical methods, and swallowing function was assessed using videoradiography., Results: Desmin displayed a disorganized pattern in 21 ± 13% of the muscle fibres in patients, while these fibers were not present in controls. Muscle fibres lacking desmin were present in both patients and controls, but the proportion was higher in patients (25 ± 12% vs. 14 ± 7%, p = 0.009). The overall desmin abnormalities were significantly more frequent in patients than in controls (46 ± 18% vs. 14 ± 7%, p < 0.001). In patients, the C-terminus of the dystrophin molecule was absent in 19 ± 18% of the desmin-abnormal muscle fibres. Patients with swallowing dysfunction had 55 ± 10% desmin-abnormal muscle fibres vs. 22 ± 6% in patients without swallowing dysfunction, p = 0.002., Conclusion: Cytoskeletal abnormalities in soft palate muscles most likely contribute to pharyngeal dysfunction in snorers and sleep apnea patients. Plausible causes for the presence of these abnormalities is traumatic snoring vibrations, tissue stretch or muscle overload.

The human oropharyngeal muscles have a unique anatomy with diverse and intricate functions. To investigate if this specialization is also reflected in the cytoarchitecture of muscle fibers, intermediate filament proteins and the dystrophin-associated protein complex have been analyzed in two human palate muscles, musculus uvula (UV) and musculus palatopharyngeus (PP), with immunohistochenmical and morphological techniques. Human limb muscles were used as reference. The findings show that the soft palate muscle fibers have a cytoskeletal architecture that differs from the limb muscles. While all limb muscles showed immunoreaction for a panel of antibodies directed against different domains of cytoskeletal proteins desmin and dystrophin, a subpopulation of palate muscle fibers lacked or had a faint immunoreaction for desmin (UV 11.7% and PP 9.8%) and the C-terminal of the dystrophin molecule (UV 4.2% and PP 6.4%). The vast majority of these fibers expressed slow contractile protein myosin heavy chain I. Furthermore, an unusual staining pattern was also observed in these fibers for β-dystroglycan, caveolin-3 and neuronal nitric oxide synthase nNOS, which are all membrane-linking proteins associated with the dystrophin C-terminus. While the immunoreaction for nNOS was generally weak or absent, β-dystroglycan and caveolin-3 showed a stronger immunostaining. The absence or a low expression of cytoskeletal proteins otherwise considered ubiquitous and important for integration and contraction of muscle cells indicate a unique cytoarchitecture designed to meet the intricate demands of the upper airway muscles. It can be concluded that a subgroup of muscle fibers in the human soft palate appears to have special biomechanical properties, and their unique cytoarchitecture must be taken into account while assessing function and pathology in oropharyngeal muscles., (© 2015 Anatomical Society.)

Influenza A viruses pose a major public health threat by causing seasonal epidemics and sporadic pandemics. Their epidemiological success relies on airborne transmission from person to person; however, the viral properties governing airborne transmission of influenza A viruses are complex. Influenza A virus infection is mediated via binding of the viral haemagglutinin (HA) to terminally attached α2,3 or α2,6 sialic acids on cell surface glycoproteins. Human influenza A viruses preferentially bind α2,6-linked sialic acids whereas avian influenza A viruses bind α2,3-linked sialic acids on complex glycans on airway epithelial cells. Historically, influenza A viruses with preferential association with α2,3-linked sialic acids have not been transmitted efficiently by the airborne route in ferrets. Here we observe efficient airborne transmission of a 2009 pandemic H1N1 (H1N1pdm) virus (A/California/07/2009) engineered to preferentially bind α2,3-linked sialic acids. Airborne transmission was associated with rapid selection of virus with a change at a single HA site that conferred binding to long-chain α2,6-linked sialic acids, without loss of α2,3-linked sialic acid binding. The transmissible virus emerged in experimentally infected ferrets within 24 hours after infection and was remarkably enriched in the soft palate, where long-chain α2,6-linked sialic acids predominate on the nasopharyngeal surface. Notably, presence of long-chain α2,6-linked sialic acids is conserved in ferret, pig and human soft palate. Using a loss-of-function approach with this one virus, we demonstrate that the ferret soft palate, a tissue not normally sampled in animal models of influenza, rapidly selects for transmissible influenza A viruses with human receptor (α2,6-linked sialic acids) preference.

Sox2 is proposed to regulate the differentiation of bipotential progenitor cells into taste bud cells. However, detailed expression of Sox2 remains unclear. In this report, Sox2 expression during taste bud development in the fungiform (FF), circumvallate (CV) and soft palate (SP) areas is examined together with Prox1. First, we immunohistochemically checked Prox1 expression in adults and found that almost all taste bud cells are Prox1-positive. During FF development, intense Sox2 expression was restricted to taste bud primordia expressing Prox1 at E12.5. However, at E14.5, Sox2 was intensely expressed outside the developing taste buds resolving to perigemmal Sox2 expression in adults. In the SP, at E14.5, taste bud primordia emerged as Prox1-expressing cell clusters. However, intense Sox2 expression was not restricted to taste bud primordia but was detected widely in the epithelium. During development, Sox2 expression outside developing taste buds was generally down-regulated but was retained in the perigemmal region similarly to that in the FF. In the CV, the initial stage of taste bud development remained unclear because of the lack of taste bud primordia comparable to that in the FF and SP. Here, we show that Prox1-expressing cells appear in the apical epithelium at E12.5, in the inner trench wall at E17.5 and in the outer trench wall at E18.5. Sox2 was again not restricted to developing taste bud cells expressing Prox1 during CV development. The expression patterns support that Sox2 does not serve as a cell fate selector between taste bud cells and surrounding keratinocytes but rather may contribute to them both.

It has been reported that a subset of type III taste cells express glutamate decarboxylase (GAD)67, which is a molecule that synthesizes gamma-aminobutyric acid (GABA), and that Mash1 could be a potential regulator of the development of GABAnergic neurons via Dlx transcription factors in the central nervous system. In this study, we investigated the expression of GAD67 and Dlx in the embryonic taste buds of the soft palate and circumvallate papilla using Mash1 knockout (KO)/GAD67-GFP knock-in mice. In the wild-type animal, a subset of type III taste cells contained GAD67 in the taste buds of the soft palate and the developing circumvallate papilla, whereas GAD67-expressing taste bud cells were missing from Mash1 KO mice. A subset of type III cells expressed mRNA for Dlx5 in the wild-type animals, whereas Dlx5-expressing cells were not evident in the apical part of the circumvallate papilla and taste buds in the soft palate of Mash1 KO mice. Our results suggest that Mash1 is required for the expression of GAD67 and Dlx5 in taste bud cells., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Neural responses to sweet and bitter stimuli in the rat and mouse are compared to the expression of the molecular taste receptors, Tas1r2/Tas2rs. Integrated taste responses from the greater superficial petrosal nerve (GSP) innervating the soft palate (SP) and the chorda tympani (CT) nerve innervating the fungiform papillae (FF) were recorded in C57BL mice and SD rats. The sum of the phasic and tonic response magnitudes (SRM) was calculated by summating all relative mean responses to a concentration series of QHCl (10(-6)-10(-2)M) or Suc (10(-4)-1.0M). Molecular expression was analyzed by double-colored in situ hybridization for Gα-gustducin with Tas1r2 or Tas2rs in the SP and FF. The vast majority of cells expressing Tas1r2 or Tas2rs were included in Gα-gustducin-expressing cells in the SP of both species. Unexpectedly, a comparison between species revealed that the SRM from the GSP is not positively correlated with receptor expression in the SP. In the rat SP, the percentage of Tas2rs with Gα-gustducin (Tas2rs/gust, 65%) was twice larger than that for Tas1r2/gust (33%), while the SRM to Suc in the rat GSP was 1.5 times (tonic and phasic) larger than that to QHCl. In the mouse SP, the percentage of Tas2rs/gust (46%) was less than that in the rat and similar to that of Tas1r2/gust (40%). However, the SRM to QHCl in the mouse GSP was 2.4 (phasic) and 4.7 (tonic) times larger than to Suc. On the other hand, threshold to Suc in the rat GSP was 10(-3)M, one log unit lower than in mouse, and the threshold to QHCl in the mouse GSP was 10(-6)M, one log unit lower than in rat. These results suggest that the robust GSP response to Suc in rat and to QHCl in mouse likely do not depend upon a large number of taste cells expressing the taste receptors Tas1r2 for Suc or Tas2rs for QHCl, but upon a higher density of Tas1r2/Tas2rs within the respective taste cells of the two species., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Immunohistochemistry for two nociceptive transducers, the transient receptor potential cation channel subfamily V members 1 (TRPV1) and 2 (TRPV2), was performed on the pharynx and its adjacent regions. TRPV1-immunoreactivity (IR) was detected in nerve fibers beneath and within the epithelium and/or taste bud-like structure. In the pharynx, these nerve fibers were abundant in the naso-oral part and at the border region of naso-oral and laryngeal parts. They were also numerous on the laryngeal side of the epiglottis and in the soft palate. TRPV2-IR was expressed by dendritic cells in the pharynx and epiglottis, as well as in the root of the tongue and soft palate. These cells were located in the epithelium and lamina propria. TRPV2-immunoreactive (IR) dendritic cells were numerous in the naso-oral part of the pharynx, epiglottis, and tongue. Abundance of TRPV2-IR dendritic processes usually obscured the presence of TRPV2-IR nerve fibers in these portions. However, some TRPV2-IR nerve fibers could be observed in the epithelium of the soft palate. Retrograde tracing method also revealed that sensory neurons which innervate the pharynx or soft palate were abundant in the jugular-petrosal ganglion complex and relatively rare in the nodose ganglion. In the jugular-petrosal ganglion complex, TRPV1- and TRPV2-IR were expressed by one-third of pharyngeal and soft palate neurons. TRPV2-IR was also detected in 11.5 % pharyngeal and 30.9 % soft palate neurons in the complex. Coexpression of TRPV1 and CGRP was frequent among pharyngeal and soft palate neurons. The present study suggests that TRPV1- and TRPV2-IR jugular-petrosal neurons may be associated with the regulation of the swallowing reflex.

Immunohistochemistry for transient receptor potential melastatin-8 (TRPM8), the cold and menthol receptor, was performed on the rat soft palate, epiglottis and pharynx. TRPM8-immunoreactive (IR) nerve fibers were located beneath the mucous epithelium, and occasionally penetrated the epithelium. These nerve fibers were abundant in the posterior portion of the soft palate and at the border region of naso-oral and laryngeal parts of the pharynx. The epiglottis was free from such nerve fibers. The double immunofluorescence method demonstrated that TRPM8-IR nerve fibers in the pharynx and soft palate were mostly devoid of calcitonin gene-related peptide-immunoreactivity (CGRP-IR). The retrograde tracing method also demonstrated that 30.1 and 8.7 % of sensory neurons in the jugular and petrosal ganglia innervating the pharynx contained TRPM8-IR, respectively. Among these neurons, the co-expression of TRPM8 and CGRP-IR was very rare. In the nodose ganglion, however, pharyngeal neurons were devoid of TRPM8-IR. Taste bud-like structures in the soft palate and pharynx contained 4-9 TRPM8-IR cells. In the epiglottis, the mucous epithelium on the laryngeal side had numerous TRPM8-IR cells. The present study suggests that TRPM8 can respond to cold stimulation when food and drinks pass through oral and pharyngeal cavities.

Introduction: The oral mucosa is an appropriate route for drug delivery systems, as it evades first-pass metabolism, enhances drug bioavailability and provides the means for rapid drug transport to the systematic circulation. This delivery system offers a more comfortable and convenient delivery route compared with the intravenous route. Although numerous drugs have been evaluated for oral mucosal delivery, few of them are available commercially. This is due to limitations such as the high costs associated with developing such drug delivery systems., Areas Covered: The present review covers recent developments and applications of oral transmucosal drug delivery systems. More specifically, the review focuses on the suitability of the oral soft palatal site as a new route for drug delivery systems., Expert Opinion: The novelistic oral soft palatal platform is a promising mucoadhesive site for delivering active pharmaceuticals, both systemically and locally, and it can also serve as a smart route for the targeting of drugs to the brain.

Conclusion: Genetic dysregulation may be associated with development of obstructive sleep apnea (OSA)., Objectives: Genetic expression was investigated in the soft palate of patients with OSA to evaluate the biomolecular characteristics of the tissue where airway obstruction actually occurs., Methods: Soft palate mucosa was obtained during uvulopalatal flap surgery. Three patients with apnea/hypopnea index (AHI) >30/h and three simple snoring patients with AHI < 5/h were enrolled. After total RNA was extracted and amplified, gene expression levels were determined by microarray analysis, and relative signal intensities for each gene were evaluated., Results: Of the 45,034 genes analyzed, 137 were up-regulated in OSA patients compared with simple snorers and 95 were down-regulated. Gene network analysis showed that genes related to RNA interference and transcription were most commonly involved in the functional pathophysiology of OSA. In particular, genes encoding proliferating cell nuclear antigen and proteasome 26S subunit ATPase 6 were functionally connected with other genes.

To clarify the regional differences in the expression and functional significance of Gα-gustducin in soft palate (SP) and fungiform (FF) taste buds, we examined the coexpression of Gα-gustducin with taste receptors and the impact of Gα-gustducin knockout (gKO) on neural responses to several sweet and bitter compounds. Sweet responses from both the greater superficial petrosal (GSP) and chorda tympani (CT) nerves in gKO mice were markedly depleted, reflecting overlapping expression of Gα-gustducin and Tas1r2. However, although Gα-gustducin was expressed in 87% and 88% of Tas2rs cells in the SP and FF, respectively, there were no statistically significant differences in the CT responses to quinine-HCl (QHCl) and denatonium (Den) between gKO and wild-type (WT) mice. In contrast, GSP responses to these compounds were markedly reduced in gKO mice with an apparent elevation of thresholds (>10-fold). These results suggest that 1) Gα-gustducin plays a critical role in sweet transduction in both the SP and the FF, 2) other Gα subunits coexpressed with Gα-gustducin in the FF are sufficient for responses to QHCl and Den, and 3) robust GSP responses to QHCl and Den occur in the SP by a Gα-gustducin-dependent mechanism, which is absent in the FF.

Objective: To study the mRNA expression of muscle phenotype and collagen of soft palate and pathology in obstructive sleep apnea hypopnea syndrome (OSAHS)., Method: We used the Real-time PCR to test the mRNA expression of soft palate muscle myosin heavy chain (MyHC) phenotype and collagen in 12 OSAHS patients and 8 control patients. We also distinguished the muscle isoforms I , II with ATPase staining, then counted the numbers of isoforms muscle fiber., Result: The mRNA expression of OSAHS group was more than control group in II A MyHC phenotype (P<0.01). The number of OSAHS group muscle fibre I isoform was less than control group with pH4. 3 ATPase staining (P<0.05)., Conclusion: Compare to control group, the enhancement happened in the mRNA expression of II A MyHC phenotype which can increase the velocity and power but de crease the enduring quality of muscle in OSAHS, and the reduce be in the I MyHC isoform of muscle fiber that can cause muscle velocity become slower and persistency become longer in OSAHS patients.

Objective and Design: Autopsies of the submandibular gland, the vestibular folds and the soft palate from 65-87 old humans were examined to record the immunohistochemical expression of MUC1 and the simple mucin-type antigens Tn and Sialyl-Tn., Results: (1) The serous acini in the submucosal glands from the larynx and the soft palate expressed MUC1-associated glycans that were not detectable in the serous acini from the submandibular gland. (2) Virtually all the submucosal acini at oral site of the soft palate are mucous, and in contrast to mucous acini in the vestibular folds and submandibular gland, the palatinal acini in the submucosa underneath the oral mucosa showed a well-defined cytoplasmic reaction with anti-MUC1 antibodies as wells as with anti-Tn. (3) Both the mucous acini and the ducts at the oral site of the soft palate showed reaction for Sialyl-Tn while in the vestibular folds and in the submandibular gland expression for this carbohydrate was observed only in the acini. (4) The staining obtained after incubation with the Tn antibodies showed no cross localization with the staining obtained after incubation with an anti-A blood group antibody. (5) All the autopsies showed reaction in the glands after incubation with the MUC1 antibodies while some autopsies reacted with the anti-Tn antibodies and/or with the anti-Sialyl-Tn antibodies and others did not., Conclusion: The mucin expression in the acini and ducts from the upper human aerodigestive tract strongly depended on the location of the glandular tissue., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT4), and their TrkB receptor regulate taste system development. To determine where and when gustatory neurons come in contact with these important factors, temporospatial expression patterns of Bdnf, Ntf4/5, and TrkB in the peripheral taste system were examined using RT-PCR. In the lingual epithelium, Ntf4/5 mRNA expression was higher than that of Bdnf at embryonic day 12.5 (E12.5), and the expression of both factors decreased afterwards. However, Ntf4/5 expression decreased at an earlier age than Bdnf. Bdnf and Ntf4/5 are expressed in equal amounts at E12.5 in geniculate ganglion, but Bdnf expression increased from E14.5 to birth, whereas Ntf4/5 expression decreased. These findings indicate that NT4 functions at early embryonic stages and is derived from different sources than Bdnf. TrkB expression in the geniculate ganglion is robust throughout development and is not a limiting factor for neurotrophin function in this system.

The epithelium of the mouse tongue and soft palate consists of at least three distinct epithelial cell populations: basal cells, keratinized cells organized into filiform and fungiform papillae, and taste receptor cells present in tight clusters known as taste buds in the fungiform and circumvallate papillae and soft palate. All three cell types develop from the simple epithelium of the embryonic tongue and palate, and are continually replaced in the adult by cell turnover. Previous studies using pulse-chase tritiated thymidine labeling in the adult mouse provided evidence for a high rate of cell turnover in the keratinocytes (5-7 days) and taste buds (10 days). However, little is known about the localization and phenotype of the long-term stem or progenitor cells that give rise to the mature taste bud cells and surrounding keratinocytes in these gustatory tissues. Here, we make use of a tamoxifen-inducible K14-CreER transgene and the ROSA26 LacZ reporter allele to lineage trace the mature keratinocytes and taste bud cells of the early postnatal and adult mouse tongue and soft palate. Our results support the hypothesis that both the pore keratinocytes and receptor cells of the taste bud are derived from a common K14(+)K5(+)Trp63(+)Sox2(+) population of bipotential progenitor cells located outside the taste bud. The results are also compatible with models in which the keratinocytes of the filiform and fungiform papillae are derived from basal progenitor cells localized at the base of these structures.

Although embryonic expression of Shh in the fungiform papilla placodes has a critical role in fungiform papilla patterning, it remains unclear whether its appearance indicates the differentiation of the basal cells of taste buds. To examine the embryonic development of the basal cells, the expression of Shh, Prox1, and Mash1 was determined in the anterior tongue and soft palate in mouse embryos by in situ hybridization. In the anterior tongue, Prox1 was coexpressed with Shh from the beginning of Shh expression in the fungiform papilla placodes at E12.5. Shh was expressed in the soft palate in a band-like pattern in the anteriormost region and in a punctate pattern in the posterior region at E14.5. The number (21.4 +/- 4.3, at E14.5) of locations where Shh was observed (i.e., spots) rapidly increased and reached a peak level (54.8 +/- 4.0 at E15.5). Also in the soft palate, Prox1 was coexpressed with Shh from the beginning of Shh expression. These results suggest that basal cell differentiation occurs synchronously with the patterning of Shh spots both in the anterior tongue and in the soft palate. In contrast, Mash1 expression lagged behind the expression of Shh and Prox1 and began after the number of Shh spots had reached its peak level in the soft palate. Furthermore, immunohistochemistry of PGP9.5 and Shh revealed that epithelial innervation slightly preceded Mash1 expression both in the tongue and in the soft palate. This is the first report describing the time courses of the embryonic expression of basal cell markers of taste buds., ((c) 2008 Wiley-Liss, Inc.)

A 69-year-old man with a 0.6 cm yellowish polypoid lesion in the lateral soft palate for 2 years is presented. Histological examination of the tumor revealed a small, well-differentiated, mucoepidermoid carcinoma dominated by densely packed cholesterol clefts in association with an exuberant foreign-body reaction with several giant cells. The tumor entity 'mucoepidermoid carcinoma with foreign-body giant cells' might be differentiated from 'mucoepidermoid carcinoma with an osteoclast-like giant cell stromal reaction' because differences in pathogenesis and tumor behavior are assumed.

Background and Objectives: Snoring and apnea are social and medical problems that affect as many as 2% of adult women and 4% of adult men. This study compares collagen distribution and type with the intensity of snoring and the degree of drowsiness following uvulopalatoplasty with CO2 laser and radiofrequency ablation., Study Design, Patients, and Methods: Fragments were taken from the soft palate by biopsies of 16 uvulopalatoplasty patients and 5 controls, 5 weeks after surgery, and they were stained with Sirius red and observed with polarization microscopy. Photographs were taken, and the type and changes in the amount of collagen before and after surgery were compared., Results: A histological analysis 5 weeks postoperatively in 10 patients showed an increased amount of collagen and replacement of type III collagen with type I collagen., Conclusions: The increase in the amount of collagen after uvulopalatoplasty was significantly larger in patients than in the controls, and all changes from type III to mostly type I collagen occurred in patients who had the surgery. The subjective improvement of both snoring and somnolence is associated with this increase in type I collagen.

Type III IP3 receptor (IP3R3) is one of the common critical calcium-signaling molecules for sweet, umami, and bitter signal transduction in taste cells, and the total IP3R3-expressing cell population represents all cells mediating these taste modalities in the taste buds. Although gustducin, a taste cell-specific G-protein, is also involved in sweet, umami, and bitter signal transduction, the expression of gustducin is restricted to different subsets of IP3R3-expressing cells by location in the tongue. Based on the expression patterns of gustducin and taste receptors in the tongue, the function of gustducin has been implicated primarily in bitter taste in the circumvallate (CV) papillae and in sweet taste in the fungiform (FF) papillae. However, in the soft palate (SP), the expression pattern of gustducin remains unclear and little is known about its function. In the present paper, the expression patterns of gustducin and IP3R3 in taste buds of the SP and tongue papillae in the rat were examined by double-color whole-mount immunohistochemistry. Gustducin was expressed in almost all (96.7%) IP3R3-expressing cells in taste buds of the SP, whereas gustducin-positive cells were 42.4% and 60.1% of IP3R3-expressing cells in FF and CV, respectively. Our data suggest that gustducin is involved in signal transduction of all the tastes of sweet, umami, and bitter in the SP, in contrast to its limited function in the tongue.

GPRC6A is a novel member of family C of G protein-coupled receptors with so far elusive biological function. GPRC6A has been described in human and mouse as a promiscuous l-alpha-amino acid receptor. We now report the cloning, expression analysis and, functional characterization of the rat orthologue of GPRC6A. Full-length cloning of rat GPRC6A (rGPRC6A) was accomplished using amplification of cDNA from taste tissue, and the identity of rGPRC6A confirmed at both the genomic and the protein level by similarity studies. Using selective primers, reverse transcriptase polymerase chain reaction showed that the mRNA is widely but weakly distributed, except for a high expression in the soft palate, the so-called geschmacksstreifen. On the protein level, rGPRC6A was shown to be glycosylated and most likely oligomeric, and using immunochemistry we observed that rGPRC6A is expressed at the plasma membrane of mammalian cell lines. Utilizing co-expression of rGPRC6A and the promiscuous Galpha(q)(G66D) protein in an engineered cell-based inositol phosphate turnover assay, we were able to study the ligand profile of the receptor. We found that l-ornithine is the most potent and efficacious l-amino acid agonist with an EC(50) value of 264 microM, followed by several other aliphatic, neutral, and basic amino acids. Furthermore, the divalent cation Mg(2+) was found to be a positive modulator of the l-ornithine response. The presented quantitative pharmacological data underlines the evolutionary conservation of GPRC6A to the rat and signifies the physiological importance and emerging pharmacological potential of GPRC6A.

Study Objectives: To determine whether the vibratory mechanical stimulus due to snoring induces upper-airway inflammation in an in-vivo rat model., Design: Prospective controlled animal study., Setting: University laboratory., Patients or Participants: Sixteen male Sprague-Dawley rats (250-300 g)., Interventions: The upper trachea of 8 rats was cannulated, and the upper airway was subjected to vibration (60 Hz; +/- 10 cm H2O) with a periodic pattern consisting of 1 second of vibration followed by 3 seconds of no vibration. This snoring-like vibration was applied for 3 hours. The animals breathed spontaneously through a cannula in the lower trachea. In a control group (8 rats), the animals were similarly instrumented, but no upper-airway vibration was applied., Measurements and Results: The effect of vibration was assessed by measuring the vibration-induced increase in gene expression of the pro-inflammatory cytokine tumor necrosis factor-alpha and of the neutrophil attractant chemokine macrophage inflammatory protein-2 in the soft-palate tissue. Real-time reverse-transcription polymerase chain reaction measurement of mRNA showed that vibration induced a significant overexpression of both tumor necrosis factor-alpha and macrophage inflammatory protein-2: 6.01-fold +/- 2.47-fold (p = .005) and 2.38-fold +/- 0.54 -fold (p = .021) increase when compared with control (mean +/- SEM)., Conclusions: The mechanical stimulus of vibration per se triggers an early proinflammatory process in the upper airway.

We used alpha-gustducin, a type II taste-cell-specific G protein, to investigate the onset of taste transduction and its relation to the development of the soft palate (SP) and fungiform (FF) papillae taste buds in the mouse. Paraffin wax embedded sections were prepared from the SP and anterior region of the tongue of the mouse from birth until postnatal day (PD) 63. No alpha-gustducin-immunoreactive cells were observed on the day of birth. One day later, alpha-gustducin was immunolocalised in taste buds with pores with a relatively higher frequency recorded in the SP as compared with the FF papillae. The immunoreactive cells were spindle shaped with elongated processes extending from the base to the pore of the taste buds. On PD 7, the number of taste buds containing alpha-gustducin-immunoreactive cells in the SP was three times greater than that of FF papillae. Our results indicate that taste transduction is essentially acquired from the time of birth. Moreover, the onset of taste transduction by the SP taste buds developed earlier than that achieved by taste buds in the FF papillae.

Objective: Metallothionein (MT) may play a preventive role in various carcinogenic process. 4NQO is an alkaline compound and potent mutagen that causes the formation of DNA adducts. The purpose of this study was to evaluate the immunoexpression of MT in palatal cells of mice submitted to the carcinogen 4NQO., Study Design: C57BL/6 mice received applications of 4NQO to palate for periods of 8, 16, 20 and 24 weeks (experimental group). A control group received only applications of propylene glycol for the same periods. Subsequently animals of experimental and control groups were sacrificed and the palate was histologically analysed and MT immunohistochemistry performed., Results: Although morphological atypical features were scant, the expression of MT was higher in the experimental group in comparison to controls. There was an amplified induction of MT expression in oral epithelium of mice treated by 4NQO., Conclusion: These results suggest that MT may act as an endogenous defensive factor against 4NQO in early phases of oral carcinogenesis.

Peptide 19 (PEP 19) is a 7.6-kDa polypeptide which binds to calmodulin and inhibits calcium-calmodulin signaling. In this study, PEP 19-immunoreactivity (PEP 19-IR) was examined in the rat vagal and glossopharyngeal sensory ganglia. Twenty-nine percent, 59%, and 41% of sensory neurons contained PEP 19-IR in the jugular, petrosal, and nodose ganglia, respectively. These neurons were of various sizes (jugular, mean +/- SD = 635.8 +/- 392.6 microm2, range = 105.9-1695.9 microm2; petrosal, mean +/- SD = 370.9 +/- 228.5 microm2, range = 57.7-1662.7 microm2; nodose, mean +/- SD = 380.5 +/- 157 microm2, range = 87.5-950.4 microm2) and scattered throughout these ganglia. Double immunofluorescence method revealed that PEP 19-IR neurons which had parvalbumin-IR were rare in the ganglia (jugular, 4%; petrosal, 10%; nodose, 8%). PEP 19-IR neurons which contained calbindin D-28k were abundant in the petrosal (20%) and nodose (22%) ganglia but not in the jugular ganglion (8%). Retrograde tracing method indicated that many PEP 19-IR neurons projected to the circumvallate papilla and soft palate. In the soft palate, taste buds were innervated by PEP 19-IR nerve fibers. The present study suggests that PEP 19-IR neurons include chemoreceptors in the vagal and glossopharyngeal sensory ganglia.

An ultrastructural analysis of oral balloon cell nevus of intramucosal type complemented with an immunohistochemical study was performed for the first time. The lesion was composed of large balloon cells with an admixture of small nevus cells and melanophages at the periphery. Balloon cells showed cytoplasmic accumulation of vacuoles of varying sizes and the presence of microgranular and vacuolated melanosomes were found. Residual cytoplasm contained no identifiable organelles. A spectrum of transitional forms between balloon cells and conventional nevus cells with microvacuoles was readily observed. Both cells exhibited intense immunoreactivity to multiple melanocytic markers. Ballooning phenomenon was not evident in melanophages containing a large amount of melanosome complex. It can be inferred, from the present and previous observations, that progressive vacuolization of melanosomes in nevomelanocytes may be responsible for the formation of peculiar ballooning appearance, suggesting an aberrant melanogenesis.

Inflammatory cell infiltration and afferent neuropathy have been shown in the upper airway (UA) mucosa of subjects with obstructive sleep apnea (OSA). We hypothesized that inflammatory and denervation changes also involve the muscular layer of the pharynx in OSA. Morphometric analysis was performed on UA tissue from nonsnoring control subjects (n = 7) and patients with OSA (n = 11) following palatal surgery. As compared with control subjects, inflammatory cells were increased in the muscular layer of patients with OSA, with CD4+ and activated CD25+ T cells (both increased approximately threefold) predominating. Inflammation was also present in UA mucosa, but with a different pattern consisting of CD8+ (2.8-fold increase) and activated CD25+ (3.2-fold increase) T cell predominance. As ascertained by immunoreactivity for the panneuronal marker PGP9.5, there was a dramatic (5.7-fold) increase in intramuscular nerve fibers in OSA patients compared with control subjects, as well as direct evidence of denervation based on positive immunostaining of the muscle fiber sarcolemmal membrane for the neural cell adhesion molecule in patients with OSA. These data suggest that inflammatory cell infiltration and denervation changes affect not only the mucosa, but also the UA muscle of patients with OSA. This may have important implications for the ability to generate adequate muscular dilating forces during sleep.

Head and neck squamous cell carcinomas (HNSCC) are characterized by a marked propensity for local invasion and spread to cervical lymph nodes, with distant metastases developing in 30-40% of cases. HPV-16 is an important risk factor for HNSCC. How HPV enhances susceptibility to HNSCC is not fully understood, but seems to involve cofactors. In this study, we examined the effect of the cooperation between HPV-16 and the tyrosine kinase receptor ErbB-2 on E-cadherin/catenin complex patterns and neoplastic transformation of human normal oral epithelial (NOE) cells. We report that overexpression of ErbB-2 or E6/E7 alone does not affect E-cadherin/catenin complex patterns nor does it induce cell transformation of NOE cells. In contrast, coexpression of E6/E7 and ErbB-2 downregulates E-cadherin and catenin expression. This is accompanied by cytoplasmic localization of E-cadherin, as well as nuclear translocation of alpha, beta, and gamma-catenins. Furthermore, we demonstrate that E6/E7 cooperate with overexpressed ErbB-2 to induce tumor formation in nude mice and to upregulate cyclin D1 and c-myc expression. Our data suggest that E6/E7 cooperate with ErbB-2 in head and neck carcinogenesis, at least in part, via the conversion of beta-catenin from a cell adhesion to a nuclear function, that is, to act as a potential transcriptional regulator. This conversion leads to the upregulation of cyclin D1, c-myc and other oncoproteins necessary for alteration of the E-cadherin/catenin complex and cell transformation of NOE cells.

Study Objectives: We utilized novel three-dimensional volumetric analysis techniques with magnetic resonance imaging (MRI) to study the upper airway and surrounding soft-tissue structures. These MRI techniques allowed us to objectively quantify the volume of the tongue, soft palate, parapharyngeal fat pads, and lateral pharyngeal walls., Design: We first validated our volumetric imaging techniques on a phantom and then demonstrated that our results were reliable and reproducible in normal subjects who did not lose weight. Finally, we studied 12 obese, nonapneic women during wakefulness before and after weight loss. We hypothesized that our novel magnetic-resonance computer-reconstruction techniques would allow us to detect small reductions in the volume of the tongue, soft palate, lateral pharyngeal walls, and parapharyngeal fat pads and increases in the volume of the upper airway with weight loss., Setting: University medical center., Participants: Normal controls and 12 obese nonapneic women., Interventions: Weight loss., Measurements and Results: Following a mean 17.1+/-8.62 kg (17.3%) reduction in weight, upper airway volume increased (p = 0.06) in both the retropalatal and retroglossal regions. This increase in upper airway volume was mediated by significant reductions in the volume of the lateral pharyngeal wall (p = 0.0001) and parapharyngeal fat pads (p = 0.001). However, the volume of the tongue (p = 0.35) and soft palate (p = 0.39) were not reduced significantly with weight loss., Conclusions: These data indicate that volumetric MRI is a powerful tool to study anatomic changes in the upper airway and surrounding soft-tissue structures and is sensitive enough to detect changes in these structures.

Palatal muscle biopsies from the cleft margin of children were subjected to cytophotometrical and immunohistochemical analysis. Muscle fiber types were classified according to the enzyme activity of myofibrillic adenosine triphosphatase, glycerol-3-phosphate-dehydrogenase and succinate dehydrogenase assessed cytophotometrically. Fiber type-related immunoreactivity of nitric oxide synthase (NOS) isoforms I, II, III, as a physiological modulator of skeletal muscle function, was related to the oxidative and glycolytic activity of the muscle fibers. Fast oxidative glycolytic fibers with high oxidative activity showed strong NOS I immunoreactivity, whereas fast glycolytic fibers with high glycolytic activity were stronger immunolabelled for NOS III. NOS II expression was similar in all fiber types. No differences in NOS immunoreactivity were found between the two investigated forms of deformity. Additionally to the usual skeletal muscle fiber types, a slow tonic fiber type was for the first time identified in cleft palate muscles. Comparison of two forms of cleft palate, isolated cleft palate and combined cleft lip and palate has shown decreased enzyme activities in muscle fibers of palatal muscles from combined cleft lip and palate. Fast oxidative glycolytic fibers were mainly effected. Cytophotometrical and immunohistochemical analysis indicated a depressed performance of the cleft palatal muscles from combined cleft lip and palate as a stronger deformity compared with isolated cleft palate.

The taste buds of the fungiform papillae, circumvallate papilla, foliate papillae, soft palate and epiglottis of the rat oral cavity were examined by lectin histochemistry to elucidate the relationships between expression of glycoconjugates and innervation. Seven out of 21 lectins showed moderate to intense staining in at least more than one taste bud. They were succinylated wheat germ agglutinin (s-WGA). Dolichos biflorus agglutinin (DBA), Bandeiraea simplicifolia lectin-I (BSL-I), Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA), Ulex europaeus agglutinin-I (UEA-I) and Phaseolus vulgaris agglutinin-L (PHA-L). UEA-I and BSL-I showed moderate to intense staining in all of the taste buds examined. They strongly stained the taste buds of the epiglottis, which are innervated by the cranial nerve X. UEA-I intensely stained the taste buds of the fungiform papillae and soft palate, both of which are innervated by the cranial nerve VII. The taste buds of circumvallate papilla and foliate papillae were innervated by the cranial nerve IX and strongly stained by BSL-I. Thus, UEA-I and BSL-I binding glycoconjugates, probably alpha-linked fucose and alpha-D-galactose, respectively, might be specific for taste buds. Although the expression of these glycoconjugates would be related to the innervation of the cranial nerve X, the differential expression of alpha-linked fucose and alpha-D-galactose might be related to the innervation of the cranial nerve VII and IX, respectively.

We studied modulation of release of nitric oxide (NO) into nasal passages by physical characteristics (airflow, temperature, humidity) or gases (oxygen, carbon dioxide) in nasal air of humans. Each characteristic or gas in nasal air was changed during voluntary soft palate elevation (to isolate nasal passages). Increasing airflow through the nose caused incremental increases in NO release from 211+/-23 nl/(min x m(-2)) at 1 L/min to 312+/-40 nl/(min x m(-2)) at 22 L/min (P<0.001, n = 6). Decreased humidity (dry airflow, 1-22 L/min) reduced NO release only at the highest airflow rate. Changing temperature (range 46 to 0 degrees C) had no effect on NO release. Hypoxia (below 4% O2) rapidly and reversibly decreased NO release (200+/-40 nl/(min x m(-2)) at 21% O2 versus 99+/-17 nl/(min x m(-2)) at 0% O2 for 3 min, (P<0.001, n = 5). Carbon dioxide (5%) reduced NO release slightly. We conclude that airflow, reduced humidity, carbon dioxide concentration, and oxygen concentration modulate NO release into nasal passages.

Helospectin (HS) and pituitary adenylate cyclase activating peptide (PACAP) are newly discovered peptides isolated from the salivary gland venom of the lizard Heloderma horridum and the ovine hypothalamus, respectively. They show chemical similarities to vasoactive intestinal polypeptide (VIP), appear to have similar functions and are present in gut, brain, lung, male and female genitourinary tract. In the present study, the distribution of the helospectin and PACAP-27 in the human upper respiratory system was investigated using indirect immunofluorescence and electron-microscopical ABC-pre-embedding methods. Immunohistochemistry revealed helospectin-like (HS-LI) and PACAP-like (PACAP-LI) immunoreactivity in nerve fibers in human nasal, the larynx (vocal cord, ventricular fold, epiglottis), the tongue and the soft palate mucosa. Helospectin-LI and PACAP-LI containing nerve fibers were mainly found in close association to blood vessels and glandular structures. Colocalization studies carried out by application of double immunofluorescence showed that HS and/(or) PACAP-LI coexist with VIP in apparently the same nerve fibers in the upper respiratory system, although single nerve fibers seem to exclusively express helospectin. The localization patterns of helospectin and PACAP-LI in the human upper respiratory system suggests their possible involvement in the regulation of secretory activities and local blood flow.

Electron microscopic data on methylene blue staining of dendritic cells in the epithelia of the soft palate and skin of the mouse after supravital dye injection are presented. The ultra-structural details were compared with corresponding light microscopic findings. Methylene blue stained tissue was fixed by immersion in a paraformaldehyde-glutaraldehyde solution containing phosphomolybdic acid. The ensuing dye precipitate was stabilized by ammonium heptamolybdate. The light microscopic investigation revealed that selective staining of dendritic cells depended on the presence of ambient oxygen. In addition, delicate morphological characteristics, like spinous structures of the dendrites, were visible. Some cells also showed terminal enlargements of the dendrites close to the surface of the epithelium. In general, visualization of morphological detail was superior to that obtained by conventional histological and immunohistochemical procedures. Nerve fibers were also stained within the epithelium as well as the subepithelial connective tissue. At the electron microscopic level, the dye was clearly identified as an electron dense precipitate that accumulated primarily within the cytoplasm near the plasma membrane. Furthermore, it was bound to the chromatin of the nuclei. No significant staining of mitochondria or other organelles was seen. Within the cytoplasm, the oxygen-dependent binding sites may be associated with heme proteins that attract both the dye in its reduced lipophilic leuco form and oxygen, followed by generation of oxygen radicals and a reoxidation of the leuco form to the cationic blue dye. Because of its selectivity for intraepithelial dendritic cells, the method described here supplements immunocytochemical procedures at both the light and electron microscopic levels.

The synthesis and hydration of glycosaminoglycans (GAG) has been implicated in the generation of a palatal shelf-elevating force in mammals. This study quantifies the GAG composition in the palatal shelves of Wistar rat fetuses at various stages of palatogenesis. Hyaluronan, heparin sulphate and chondroitin-4-sulphate were detected but not dermatan sulphate or chondroitin-6-sulphate. The distribution of the GAG differed in the anterior and posterior regions (i.e. the presumptive hard and soft palates) and with the stage of development. At the time of palatal-shelf reorientation, there were no significant differences for either the total amount of GAG or for the percentages of specific GAG types between anterior and posterior regions. Indeed, the most marked differences were detected at the stages of histogenesis after shelf elevation. Nevertheless, the results support the view that hyaluronan is involved in palatal-shelf reorientation, its percentage being initially high and decreasing after shelf elevation. No changes were detected for the other (sulphated) GAG during the time of elevation. The findings point to the need to correlate the events during histogenesis with changes in the ground substance of palatal-shelf mesenchyme and indicate that there are different developmental mechanisms within the presumptive hard and soft palates.

We have compared, at the ultrastructural level, the carbohydrate structure of glycoconjugates of the different types of secretory cells of the human tracheal mucosa (HTM) and the frog palate mucosa (FPM), proposed as a model for studying bacterial adherence to mucus-coated respiratory epithelium. In addition to reactivity with Concanavalin A and Lens cullinaris agglutinin, reactivity of Epon-embedded HTM and FPM secretory granules was studied by transmission electron microscopy using neuraminidase-gold complex and colloidal gold-adsorbed lectins with affinity for sugar residues of human mucins, namely the following: Helix pomatia, Lotus tetragonolobus, Ricinus communis II, Wheat germ and Limax flavus agglutinins. The affinity of HTM and FPM mucous and serous cells for the different colloidal-gold complexes was very similar, however Limax flavus agglutinin labelled only HTM and not FPM secretory granules. The FPM mucous and serous secretory granules were nevertheless intensely labelled by the neuraminidase-gold complex, demonstrating the presence of sialic acid residues. The close ultrastructural and histochemical similarities between HTM and FPM suggest that the FPM may be a valuable model for studying the specific interaction between microbial lectins and mucus glycoproteins in the bacterial adherence phenomenon.

The effect of diphenylhydantioin (DPH) upon the degradation of in vivo [35S]-sulphate-labelled proteoglycans was studied in cat palatal mucosa during organ culture. 8-week-old cats were injected intraperitoneally with [35S]-sulphate and 24 hours later the palatal mucosa was taken to organ culture. The release of radioactivity into the culture medium was taken as a measure of degradation of sulphated macromolecules, presumably proteoglycans, and the release of hydroxyproline as an indicator for collagen degradation. A parallel decrease in the release of radioactivity and in the release of hydroxyproline was observed when the culture was done in the presence of DPH (20 mg/l). Chromatography of the culture medium upon Sephadex G-25 revealed that the reduced release of radioactivity was due to a reduction of macromolecular degradation products leaving the amounts of free sulphate in the medium unchanged. The results were interpreted using a two compartment theory for proteoglycan degradation, extracellular breakdown of the protein core resulting in the production of macromolecular degradation products and intracellular lysosomal degradation resulting in free sulphate as the identifiable product. The results indicate that DPH inhibited the extracellular enzymatic degradation of proteoglycans without influencing their intracellular degradation.

Responses of the epithelial and mesenchymal cells of the developing hamster secondary palate to hadacidin, 5-fluorouracil, and hydrocortisone were analysed. Correlation of data on cellular response with morphological and biochemical observations suggested that several aspects of differentiation of palatal cells needs to be analysed before pathogenesis of teratogen-induced cleft palate can be defined.