A Single-Cell Atlas of the Human Healthy Airways.

Rationale: The respiratory tract constitutes an elaborate line of defense that is based on a unique cellular ecosystem.Objectives: We aimed to investigate cell population distributions and transcriptional changes along the airways by using single-cell RNA profiling.Methods: We have explored the cellular heterogeneity of the human airway epithelium in 10 healthy living volunteers by single-cell RNA profiling. A total of 77,969 cells were collected at 35 distinct locations, from the nose to the 12th division of the airway tree.Measurements and Main Results: The resulting atlas is composed of a high percentage of epithelial cells (89.1%) but also immune (6.2%) and stromal (4.7%) cells with distinct cellular proportions in different regions of the airways. It reveals differential gene expression between identical cell types (suprabasal, secretory, and multiciliated cells) from the nose (MUC4, PI3, SIX3) and tracheobronchial (SCGB1A1, TFF3) airways. By contrast, cell-type-specific gene expression is stable across all tracheobronchial samples. Our atlas improves the description of ionocytes, pulmonary neuroendocrine cells, and brush cells and identifies a related population of NREP-positive cells. We also report the association of KRT13 with dividing cells that are reminiscent of previously described mouse "hillock" cells and with squamous cells expressing SCEL and SPRR1A/B.Conclusions: Robust characterization of a single-cell cohort in healthy airways establishes a valuable resource for future investigations. The precise description of the continuum existing from the nasal epithelium to successive divisions of the airways and the stable gene expression profile of these regions better defines conditions under which relevant tracheobronchial proxies of human respiratory diseases can be developed.

Tenting effect of the elevated sinus membrane over an implant with adjunctive use of a hydroxyapatite-powdered collagen membrane in rabbits.

PURPOSE: The aim of this study was to determine the de novo bone formation beneath the Schneiderian membrane supported by an implant and with the adjunctive use of a hydroxyapatite-powdered collagen (HAC) membrane without bone grafting in rabbit sinuses. MATERIAL AND METHODS: After sinus-floor elevation procedure, an experimentally devised mini-implant (4 mm in length and 3 mm in diameter) was placed in eight rabbits. For the experimental group, an HAC membrane was placed onto the elevated sinus mucosa prior to implant placement. The animals were allowed a healing period of either 4 or 8 weeks. Microcomputed tomography and histologic analyses were performed. RESULTS: All implants placed at the thin lateral bony wall were histologically osseointegrated. The topography of the newly formed bone appeared to slope gently from the medial side down to the lateral side. The volume of new bone was significantly greater in the experimental group than in the control group at 4 weeks (P < 0.05), but not at 8 weeks. However, none of the samples in the two groups was entirely covered to the apex by bone tissue. CONCLUSIONS: It can be concluded that using the HAC membrane in combination with placement of an implant resulted in substantial bone formation around the implant, which might have been influenced by the proximity of the axial bony wall.

[Morphogenesis of the stromal tissue of nasal polyps].

The objective of the present work was to study various stages of morphogenesis of the stromal tissue of nasal polyps. The materials for the investigation were harvested during endoscopic polypotomies in the nose. They were used to obtain the preparations that were stained by the histological and histochemical methods. The immunochemical studies were carried out using monoclonal antibodies against differentiation clusters, such as CD20, CD31, and vascular endothelial growth factor (VEGF). The study has demonstrated that the growth and evolution of the nasal polyps are accompanied by sequential alterations in the morphological structure of their stromal tissue. The results of the investigations were used to develop the original classification of the selected stages of morphogenesis of the stromal tissue of nasal polyps. The following stages were distinguished: edematous one, inflammatory cellular infiltration, fibrous cirrhotic alterations, and vasculogenesis or neoangiogenesis.

Bone production by human maxillary sinus mucosa cells.

The Schneider membrane is the mucosa that covers the inner part of the maxillary sinus cavities. The free surface is a ciliated pseudostratified epithelium, while the deeper portion is a highly vascularized connective tissue. The stromal fraction, bordering the bony wall of the sinus, after tooth loss can exhibit increased osteoclastic activity resulting in resorption of the bone in the posterior maxilla. Goal of our study was to isolate and characterize mesenchymal progenitors in the Schneider's membrane connective net and to evaluate their self ability to differentiate toward osteoblastic lineage, in absence of osteoinductive factors and osteoconductive biomaterials of support. This should indicate that maxillary sinus membrane represents an useful an approachable source of MSCs for bone tissue engineering and cell therapy and owns the intrinsic capacity to restore maxillary bone after tooth loss without the needing of biomaterials.

Antigen-dependent rescue of nose-associated lymphoid tissue (NALT) development independent of LTbetaR and CXCR5 signaling.

Nose-associated lymphoid tissue (NALT) in the rodent upper respiratory tract develops postnatally and is considered to be independent of several factors known to be involved in the organogenesis of LN and Peyer's patches (PP). In this study we demonstrate that at least two different pathways result in NALT development. Following NALT anlage formation the intrinsic pathway relies on a signaling cascade including those mediated through the chemokine receptor CXCR5 and the lymphotoxin beta receptor (LTbetaR). This allows for the formation of high endothelial venules and thereby the recruitment of lymphocytes into NALT. Alternatively, high endothelial venule formation and lymphocyte recruitment can be induced in the NALT anlage by environmental signals, which are independent of LT-betaR and chemokine receptor CXCR5 signaling but in part rely on CD40 ligand. Thus, our study identifies a novel mechanism that facilitates the rescue of NALT development at late stages in adult life independent of the canonical LTbetaR-CXCR5 signaling axis.

Development of secondary lymphoid organs.

Secondary lymphoid organs develop during embryogenesis or in the first few weeks after birth according to a highly coordinated series of interactions between newly emerging hematopoietic cells and immature mesenchymal or stromal cells. These interactions are orchestrated by homeostatic chemokines, cytokines, and growth factors that attract hematopoietic cells to sites of future lymphoid organ development and promote their survival and differentiation. In turn, lymphotoxin-expressing hematopoietic cells trigger the differentiation of stromal and endothelial cells that make up the scaffolding of secondary lymphoid organs. Lymphotoxin signaling also maintains the expression of adhesion molecules and chemokines that govern the ultimate structure and function of secondary lymphoid organs. Here we describe the current paradigm of secondary lymphoid organ development and discuss the subtle differences in the timing, molecular interactions, and cell types involved in the development of each secondary lymphoid organ.

Histological changes in rat nasal epithelia after unilateral neonatal naris occlusion.

The authors studied the extent of the different epithelia lining the nasal fossae of the albino rat after neonatal closure of one naris. Newborn pups were anesthetized by hypothermia and the external opening of their right naris cauterized, while littermates served as controls. Animals were sacrificed at 3 months, and the occluded (OCF) and nonoccluded (NOF) fossae of experimental animals as well as both fossae of control animals (CTF) were histologically studied. In both control and experimental animals, nasal fossae were lined by five different types of epithelia: squamous stratified, transitional, metaplastic, respiratory, and olfactory epithelia. It was found that closure of one naris provokes reorganization of the epithelial lining in both the occluded and nonoccluded side. In CTF airflow, physical conditions as well as pollutants and biological agents irritate the epithelial lining, causing squamous metaplasia as well as metaplastic epithelium showing inflammation in rostral levels. In CTF caudal levels, the metaplastic epithelium appears to a lesser degree and the respiratory epithelium prevails, except for the most caudal level where the olfactory epithelium is prevalent. In OCF, the protected environment created prevents the occurrence of metaplastic epithelium, the transitional, respiratory, and olfactory epithelia developing in the corresponding area instead. In NOF, where the airflow is double, the same pattern occurs as in CTF, although metaplastic epithelium values are approximately double, suggesting a clear linear effect. An outstanding feature observed was the increased extent of the olfactory epithelium in OCF regarding NOF, although changes in its morphological structure were not found. Airflow properties, including pressure, coldness, velocity, and turbulence, as well as biological and chemical hazards present in inflow, cause histological reorganization of the nasal epithelium lining during postnatal development. Results prove the need to consider airflow changes in nasal fossae surgery and point to the protective value of naris closure in ENT clinics, supporting it as a treatment of atrophic rhinitis.

Rett syndrome and neuronal development.

The clinical signs of Rett syndrome, as well as neuropathology and brain imaging, suggest that the disorder disrupts neuronal circuits. Studies using receptor autoradiography demonstrate abnormalities in the density of excitatory glutamate and inhibitory gamma-aminobutyric acid (GABA) synaptic receptors in postmortem brain from young female subjects with Rett syndrome. MeCP2, the protein that is abnormal in most female individuals with Rett syndrome, is expressed predominantly in neurons and appears during development at the time of synapse formation. Studies of nasal epithelium from patients with Rett syndrome show that the maturation of olfactory receptor neurons is impeded prior to the time of synapse formation. Recent reports indicate that MeCP2 controls the expression of brain-derived neurotrophic factor and the DNA-binding homeobox protein Dlx5. Brain-derived neurotrophic factor enhances glutamate neurotransmission at excitatory synapses, whereas Dlx5 is expressed in most GABAergic neurons and stimulates the synthesis of GABA. Taken together, this information supports the hypothesis that Rett syndrome is a genetic disorder of synapse development, especially synapses that use glutamate and GABA as neurotransmitters.

Development of rhinovirus study model using organ culture of turbinate mucosa.

To better understand the pathophysiology of rhinovirus (RV) infection, a development of a study model using organ culture of turbinate mucosa was sought. Inferior turbinate mucosal tissues were cultured using air-liquid interface methods, on a support of gelfoam soaked in culture media. RV-16 was applied to the mucosal surface and washed off, and histological changes were evaluated. The success of RV infection was assayed by semi-nested RT-PCR of the mucosal surface fluid taken 48h after incubation. Intracellular RVs were visualized by in situ hybridization (ISH). Secretion of the cytokines, IL-6 and IL-8, into the culture media was quantitated by ELISA. After 7 days of culture, the turbinate mucosae did not show significant damage. A PCR product indicating successful RV infection was detected in 5 out of 10 mucosal tissues. ISH showed a very small number of positively stained cells focally located in the epithelial layer. In the beginning 24h after infection, secretion of IL-6 and IL-8 into the culture media of infected mucosae was significantly greater than into the media of control mucosae. Our results indicate that the air-liquid interface organ culture of turbinate mucosa could serve as an acceptable in vitro model for studying RV infection.

I kappa B kinase complex alpha kinase activity controls chemokine and high endothelial venule gene expression in lymph nodes and nasal-associated lymphoid tissue.

The lymphotoxin (LT) beta receptor plays a critical role in secondary lymphoid organogenesis and the classical and alternative NF-kappaB pathways have been implicated in this process. IKKalpha is a key molecule for the activation of the alternative NF-kappaB pathway. However, its precise role and target genes in secondary lymphoid organogenesis remain unknown, particularly with regard to high endothelial venules (HEV). In this study, we show that IKKalpha(AA) mutant mice, who lack inducible kinase activity, have hypocellular lymph nodes (LN) and nasal-associated lymphoid (NALT) tissue characterized by marked defects in microarchitecture and HEV. In addition, IKKalpha(AA) LNs showed reduced lymphoid chemokine CCL19, CCL21, and CXCL13 expression. IKKalpha(AA) LN- and NALT-HEV were abnormal in appearance with reduced expression of peripheral node addressin (PNAd) explained by a severe reduction in the HEV-associated proteins, glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), and high endothelial cell sulfotransferase, a PNAd-generating enzyme that is a target of LTalphabeta. In this study, analysis of LTbeta(-/-) mice identifies GlyCAM-1 as another LTbeta-dependent gene. In contrast, TNFRI(-/-) mice, which lose classical NF-kappaB pathway activity but retain alternative NF-kappaB pathway activity, showed relatively normal GlyCAM-1 and HEC-6ST expression in LN-HEV. In addition, in this communication, it is demonstrated that LTbetaR is prominently expressed on LN- and NALT-HEV. Thus, these data reveal a critical role for IKKalpha in LN and NALT development, identify GlyCAM-1 and high endothelial cell sulfotransferase as new IKKalpha-dependent target genes, and suggest that LTbetaR signaling on HEV can regulate HEV-specific gene expression.

Cutting edge: organogenesis of nasal-associated lymphoid tissue (NALT) occurs independently of lymphotoxin-alpha (LT alpha) and retinoic acid receptor-related orphan receptor-gamma, but the organization of NALT is LT alpha dependent.

Peyer's patch and nasal-associated lymphoid tissue (NALT) are mucosal lymphoid tissues that appear similar in structure and function. Surprisingly, we found that NALT, unlike Peyer's patch, was formed independently of lymphotoxin (LT)alpha. Furthermore, using mice deficient in the retinoic acid receptor-related orphan receptor-gamma, we found that NALT was formed in the absence of CD4+CD3- cells, which are thought to be the embryonic source of LTalpha. However, we also found that NALT of LTalpha-/- animals was disorganized and lymphopenic, suggesting that the organization and recruitment of lymphocytes within NALT remained dependent on LTalpha. Finally, we demonstrated that both the structure and function of NALT were restored in LTalpha-/- animals upon reconstitution with normal bone marrow. These results demonstrate that the organogenesis of NALT occurs through unique mechanisms.

[Growth of human respiratory epithelium on collagen foil]. / Wachstum humanen respiratorischen Epithels auf Kollagenfolie.

BACKGROUND: Clinical application of bioartificial tracheal prosthesis must still be regarded as an experimental concept because restoration of a functional respiratory epithelium outlining the prosthesis is still not possible. Tissue engineering as a relatively new biotechnological discipline may offer new methods in expanding differentiated respiratory epithelium in vitro. In this study we compare two different cell and tissue culture procedures for growing human nasal mucosa on commercially available collagen foil. MATERIAL AND METHODS: Harvested specimens of human nasal mucosa (n = 6, 4 x 4 cm) were placed on collagen foil and incubated as tissue cultures for 4, 6 and 8 weeks. A suspension of enzymatically dispersed nasal epithelium seeded on collagen foil (5 x 10(5) cells) served as control. Cell growth and ciliary beat were monitored through an inverted microscope with Hoffman's modulation contrast and video set-up. Histological examination was performed after 4, 6 and 8 weeks. RESULTS: In the tissue cultures, the collagen foil was initially covered with fibroblasts growing from the mucosa specimen before epithelial cells spread out. The epithelial layer showed mostly ciliated cells which developed metachronous ciliary beat after 4 weeks in vitro. Ciliary activity was observed until the end of the experiments in 8 weeks. New cells on the suspension cultures were mesenchymal and did not exhibit any ciliary activity. CONCLUSIONS: Mucosa specimens seem to be more appropriate for tissue engineering of respiratory epithelium than cell suspensions from nasal epithelium. Collagen foil as tissue scaffold initiates epithelial-mesenchymal interaction and may play an important role in epithelial differentiation of new respiratory epithelium.

Expression of olfactory receptors, G-proteins and AxCAMs during the development and maturation of olfactory sensory neurons in the mouse.

Three mouse olfactory receptors have been cloned and sequenced and were found to be expressed in different zones of the olfactory epithelium. In situ hybridisation (ISH) results showed that each olfactory receptor was expressed at an early stage in development (E12), was not dependent on the maturation of the receptor neurons, and was present long before the onset of odour detection. Cells positive for these same olfactory receptors and the G-protein (Gbeta) were also found in non-neural regions of the nasal epithelium in the earlier stages of development (E12-16). Ncam, and Big-2 expression were, however, restricted to the region of developing olfactory neurons. Ncam expression appeared in advance of the olfactory receptor expression, while Big-2 appeared after olfactory receptor expression and neither were expressed in cells outside the olfactory epithelium. Both showed the highest number of positive cells in the early post-partum period when olfactory detection is functional. Ncam is known to be involved in guidance of the developing olfactory axons and was expressed earlier than any of the olfactory receptors, while Big-2 appears somewhat later (E14) at a time when developing axons reach the olfactory bulb. Moreover the highest periods of expression occur at post-natal day 7 when a proliferation of bulbar glomeruli are observed, suggesting the role of Big-2 to be primarily concerned with synaptogenesis.

Regeneration of olfactory mucosa in mice after inhalation exposure to perchloroethylene.

An experimental group of 20 male pure-bred mice was exposed to perchloroethylene gas at 300 ppm for 6 h daily for 5 days. Histopathological study of the nasal mucosa, particularly the olfactory mucosa, was performed sequentially 2 weeks to 3 months after exposure, to clarify the process of regeneration. The tissue damage due to perchloroethylene gas was more persistent in the nasal mucosa of the olfactory region than in the respiratory region. Two weeks after exposure, ciliated epithelial cells, as well as normal pseudostratified nonciliated columnar epithelium, began to appear in the area previously covered by olfactory epithelium and remained for up to 3 months after exposure. A basement membrane was present under the ciliated epithelium, suggesting a possible persistence of basal cells. The olfactory epithelium may thus be replaced by ciliated epithelium. The lamina propria of the olfactory mucosa, however, lost its normal structure with atrophy of the olfactory nerves and Bowman's glands.

Transplantation of postnatal vomeronasal organ in the CNS of newborn rats.

The present study was conducted to examine the survival and development of intracerebral transplanted neonatal rat vomeronasal organs (VNs). Complete neonatal (P5-P10) VNs were transplanted into the parietal cortex region of littermates and examined at 10-100 days by light microscopy. The VN survived and was organized into a series of vesicles lined by respiratory and/or sensory epithelia. Sensory neurons grew long axons that fasciculated and invaded the surrounding brain parenchyma. The newly developed axons did not prefer a specific brain region. The axons developed a complex fiber plexus either at the interface between transplant and host tissue or deep within the host brain parenchyma. Vomeronasal axons consistently formed glomerular-like structures within the fiber plexus. Our results suggest that glomerular formation is not dependent on specific target of length of axon development, but rather on a set of complementary axons that display mutual recognition.

Differential expression of alpha, mu, and pi classes of glutathione S-transferases in chemosensory mucosae of rats during development.

The expression of three classes of glutathione S-transferases (GSTs), Alpha, Mu, and Pi was investigated in the nasal mucosae of rats during development using immunohistochemical methods. GST Alpha and Mu were first detected in the supranuclear region of sustentacular cells on embryonic days 16. The Bowman's glands expressed differential patterns of immunoreactivity during development, beginning at postnatal day (P) 2 and P6 for Alpha and Mu classes, respectively and being greatest at P11 for both. The acinar cells of vomeronasal glands in the vomeronasal organ expressed Alpha and Mu classes of GSTs from P11 onwards. In the septal organ of Masera, the supranuclear region of sustentacular cells expressed GSTs from P11 with little or no variation during development. In the respiratory mucosa, Alpha and Mu classes of GSTs were detected at the brush borders of ciliated cells and in the acinar cells of posterior septal glands, but not in anterior septal or respiratory glands located on the turbinates. Compared to olfactory mucosa, the changes in immunoreactivity for GSTs were less pronounced in the respiratory mucosa during development. Specific GST Pi immunoreactivity was not detected in the nasal mucosae at any stage of development studied. The occurrence of GSTs in the nasal mucosa, including olfactory, vomeronasal, septal, and respiratory epithelia, suggests that the GSTs are actively involved in the biotransformation of xenobiotics including odorants and pheromones, and may also participate in perireceptor processes such as odorant clearance. In addition, we have developed a working model describing the cellular localization of certain phase I (e.g., cytochrome P-450s) and phase II (e.g., GSTs, gamma-glutamyl transpeptidase) biotransformation enzymes in the olfactory mucosa and their proposed roles in xenobiotic metabolism.

Neurogenesis in the olfactory epithelium.

The initial step in the recognition of odors occurs when individual odorant molecules enter the nasal cavity and interact with the sensory endings of neurons located in the olfactory epithelium. These neurons are unique in several respects. First, they are directly accessible to the external environment. Second, perhaps because of their exposure to toxic substances in the environment, they degenerate and are replenished continuously from a population of stem cells at the base of the epithelium. Third, the sensory neurons born in the olfactory epithelium of adults retain the ability to differentiate and establish synaptic contact with target cells in the mature olfactory bulb. These unique features, which are conserved phylogenetically (for example, see Blaustein et al), provide substantial rationale for studying neuronal genesis and differentiation in the olfactory epithelium. We will summarize much of what is currently known about the development of the olfactory epithelium, the birth and differentiation of olfactory receptor neurons, and the molecular correlates of these events.

Reticulum cells in the ontogeny of nasal-associated lymphoid tissue (NALT) in the rat.

This study concerns the ontogeny of reticulum cells (RC) in the nasal-associated lymphoid tissue (NALT) of Wistar and Brown-Norway rats. A panel of monoclonal antibodies (mAb) directed against RC in peripheral lymphoid organs (antibodies ED10-ED15) was used, together with a recently developed antibody ED17, which recognizes macrophages and Langerhans cells. Early in embryogenesis, staining with common connective tissue markers, ED14 and ED15, was found. ED17-positive cells were present before cells positive to ED1, a pan-macrophage marker, or Ia glycoproteins were observed. The first differentiation of reticulum was seen at the day of birth, when ED10 recognized a distinct area in the nasal mucosa. The first T-lymphocytes were found at the same time. Two days after birth, B-cells and ED11-positive cells were present in the NALT area. Fourteen days after birth, T- and B-cell compartments were recognizable. ED10 was found predominantly in the T-cell area and ED11 was mainly confined to the B-cell compartment. We conclude that the development of the NALT is closely accompanied by the phenotypic specialization of the reticulum. This suggests that the reticulum plays an important role in the compartmentalization of NALT tissue and in the retention of lymphocyte subsets within these compartments.

The development and structure of mouse nasal-associated lymphoid tissue: an immuno- and enzyme-histochemical study.

This study deals with the structure and development of Nasal-Associated Lymphoid Tissue (NALT) in the mouse. NALT is present in the nasal cavity on both sides at the entrance of the pharyngeal duct. The lymphocytes are organized in B- and T-cell areas covered by an epithelium in which M-cells are present. Immuno- and enzyme-histochemistry showed that NALT is already present at birth. Before birth, accumulations of Ia-positive dendritic cells and a few B lymphocytes were the first signs of the formation of NALT. These cell accumulations occurred just under the epithelium in the nasal floor. At birth, NALT could be distinguished as an accumulation of mainly B lymphocytes and Ia positive dendritic cells in a network of reticulum cells. The covering simple epithelium showed crypt-like invaginations. B- and T-cell areas appeared at seven days after birth. High endothelial venules (HEV) were observed from day 7 onward; from day 14 they could be stained with the HEV-specific monoclonal antibody MECA-325. Ia-positive dendritic cells increased in number during ontogeny. They occurred both in NALT and between the covering epithelial cells. Also, some epithelial cells expressed the Ia-antigen. The number of acid phosphatase positive macrophages increased steadily during ontogeny. The cells were mainly observed in the connective tissue surrounding NALT and just under the epithelium. They displayed antigenic determinants characteristic of macrophages (Moma-1, Moma-2). The structure and development of NALT are compared with those of other parts of the mucosa-associated lymphoid tissue.