Exposure to urban particulate matter alters responses of olfactory mucosal cells to SARS-CoV-2 infection.

Respiratory viruses have a significant impact on health, as highlighted by the COVID-19 pandemic. Exposure to air pollution can contribute to viral susceptibility and be associated with severe outcomes, as suggested by recent epidemiological studies. Furthermore, exposure to particulate matter (PM), an important constituent of air pollution, is linked to adverse effects on the brain, including cognitive decline and Alzheimer's disease (AD). The olfactory mucosa (OM), a tissue located at the rooftop of the nasal cavity, is directly exposed to inhaled air and in direct contact with the brain. Increasing evidence of OM dysfunction related to neuropathogenesis and viral infection demonstrates the importance of elucidating the interplay between viruses and air pollutants at the OM. This study examined the effects of subacute exposure to urban PM 0.2 and PM 10-2.5 on SARS-CoV-2 infection using primary human OM cells obtained from cognitively healthy individuals and individuals diagnosed with AD. OM cells were exposed to PM and subsequently infected with the SARS-CoV-2 virus in the presence of pollutants. SARS-CoV-2 entry receptors and replication, toxicological endpoints, cytokine release, oxidative stress markers, and amyloid beta levels were measured. Exposure to PM did not enhance the expression of viral entry receptors or cellular viral load in human OM cells. However, PM-exposed and SARS-CoV-2-infected cells showed alterations in cellular and immune responses when compared to cells infected only with the virus or pollutants. These changes are highly pronounced in AD OM cells. These results suggest that exposure of human OM cells to PM does not increase susceptibility to SARS-CoV-2 infection in vitro, but it can alter cellular immune responses to the virus, particularly in AD. Understanding the interplay of air pollutants and COVID-19 can provide important insight for the development of public health policies and interventions to reduce the negative influences of air pollution exposure.

Effect of cadmium chloride toxicity on olfactory mucosa / Efecto de la toxicidad del cloruro de cadmio en la mucosa olfativa

SUMMARY: Cadmium is a highly toxic metal and affects the respiratory mucosa. The aim of the study is to show the inflammation and degenerative effect of cadmium on the olfactory mucosa. In this study, eight-week-old Wistar rats with an average weight of 170-190 g were divided into two groups (control and experiment) with 20 animals in each group and used in the experiments. The rats in the experimental group were given 2 mg/kg/day powdered cadmium chloride dissolved in water intraperitoneally every day for two weeks. At the end of the experiment, the nasal cavity was completely removed with anesthesia. Concha nasalis superior was separated, fixed with zinc-Formalin solution and decalcified with 5 % EDTA (Ethylene-diaminetetraacetic acid). After routine histopathological procedure, APAF-1 antibody was used for expression of Hematoxylin-Eosin (HE) and immunohistochemistry. Histopathological examination revealed interruptions in the basement membrane structure due to cadmium and degenerative changes in stem cells, degeneration in sensory cells and pycnosis in nuclei, dilatation in blood vessels and increased inflammation in connective tissue. APAF-1 expression was found to increase in epithelial cells and olfactory glands (Bowman gland) cells. It has been thought that cadmium toxicity increases cell degeneration and inflammation in the olfactory mucosa and may significantly affect cell death and olfactory metabolism by inducing the pro-apoptotic process.

El cadmio es un metal altamente tóxico que afecta la mucosa respiratoria. El objetivo fue mostrar el efecto inflamatorio y degenerativo del cadmio sobre la mucosa olfativa. En este estudio, ratas Wistar de ocho semanas de edad con un peso promedio de 170-190 g se dividieron en dos grupos (control y experimental) con 20 animales en cada grupo. Las ratas del grupo experimental recibieron 2 mg/kg/día de cloruro de cadmio en polvo disuelto en agua por vía intraperitoneal todos los días durante dos semanas. En los animales se exirpó la cavidad nasal bajo anestesia. Se separó la concha nasal superior, se fijó con solución de zinc-Formalina y se descalcificó con EDTA (ácido etilendiaminotetraacético) al 5 %. Después del procedimiento histopatológico de rutina, Hematoxilina- Eosina (HE) e inmunohistoquímica, se utilizó el anticuerpo APAF-1. El examen histopatológico reveló interrupciones en la estructura de la membrana basal debido al cadmio y cambios degenerativos en las células madre, degeneración en las células sensoriales y picnosis en los núcleos, dilatación de los vasos sanguíneos y aumento de la inflamación en el tejido conjuntivo. Se encontró que la expresión de APAF-1 aumenta en las células epiteliales y en las células de las glándulas olfatorias (glándulas de Bowman). Se ha pensado que la toxicidad del cadmio aumenta la degeneración celular y la inflamación en la mucosa olfativa y puede afectar significativamente la muerte celular y el metabolismo olfativo al inducir el proceso proapoptótico.

Involvement of aldehyde oxidase in the metabolism of aromatic and aliphatic aldehyde-odorants in the mouse olfactory epithelium.

Xenobiotic-metabolizing enzymes (XMEs) expressed in the olfactory epithelium (OE) are known to metabolize odorants. Aldehyde oxidase (AOX) recognizes a wide range of substrates among which are substrates with aldehyde groups. Some of these AOX substrates are odorants, such as benzaldehyde and n-octanal. One of the mouse AOX isoforms, namely AOX2 (mAOX2), was shown to be specifically expressed in mouse OE but its role to metabolize odorants in this tissue remains unexplored. In this study, we investigated the involvement of mouse AOX isoforms in the oxidative metabolism of aldehyde-odorants in the OE. Mouse OE extracts effectively metabolized aromatic and aliphatic aldehyde-odorants. Gene expression analysis revealed that not only mAOX2 but also the mAOX3 isoform is expressed in the OE. Furthermore, evaluation of inhibitory effects using the purified recombinant enzymes led us to identify specific inhibitors of each isoform, namely chlorpromazine, 17ß-estradiol, menadione, norharmane, and raloxifene. Using these specific inhibitors, we defined the contribution of mAOX2 and mAOX3 to the metabolism of aldehyde-odorants in the mouse OE. Taken together, these findings demonstrate that mAOX2 and mAOX3 are responsible for the oxidation of aromatic and aliphatic aldehyde-odorants in the mouse OE, implying their involvement in odor perception.

Benzo[a]pyrene impairs the migratory pattern of human gonadotropin-releasing-hormone-secreting neuroblasts.

Benzo[a]pyrene (BaP) is a widespread pollutant that can act as an endocrine disrupting compound (EDC) and interferes with reproductive function. The central regulatory network of the reproductive system is mediated by gonadotropin-releasing hormone (GnRH) neurons, which originate in the olfactory placode and, during ontogenesis, migrate into the hypothalamus. Given the importance of the migratory process for GnRH neuron maturation, we investigated the effect of BaP (10 µM for 24 h) on GnRH neuroblasts isolated from the human fetal olfactory epithelium (FNCB4). BaP exposure significantly reduced the mRNA level of genes implicated in FNCB4 cell migration and affected their migratory ability. Our findings demonstrate that BaP may interfere with the central neuronal network controlling human reproduction affecting GnRH neuron maturation.

Mixture interactions at mammalian olfactory receptors are dependent on the cellular environment.

Functional characterization of mammalian olfactory receptors (ORs) remains a major challenge to ultimately understanding the olfactory code. Here, we compare the responses of the mouse Olfr73 ectopically expressed in olfactory sensory neurons using AAV gene delivery in vivo and expressed in vitro in cell culture. The response dynamics and concentration-dependence of agonists for the ectopically expressed Olfr73 were similar to those reported for the endogenous Olfr73, however the antagonism previously reported between its cognate agonist and several antagonists was not replicated in vivo. Expressing the OR in vitro reproduced the antagonism reported for short odor pulses, but not for prolonged odor exposure. Our findings suggest that both the cellular environment and the stimulus dynamics shape the functionality of Olfr73 and argue that characterizing ORs in 'native' conditions, rather than in vitro, provides a more relevant understanding of ligand-OR interactions.

Magnetic Targeting of Human Olfactory Mucosa Stem Cells Following Intranasal Administration: a Novel Approach to Parkinson's Disease Treatment.

Among the various therapeutic procedures used for improving PD, stem cell-based therapy has been shown to be a promising method. Olfactory ectomesenchymal stem cells (OE-MSCs) are a great source of stem cells for PD. Also, the intranasal administration (INA) of stem cells to the neural lesion has several advantages over the other approaches to cellular injections. However, improving the efficacy of INA to produce the highest number of cells at the lesion site has always been a controversial issue. For this purpose, this study was designed to apply the magnetically targeted cell delivery (MTCD) approach to OE-MSCs in the injured striatum area through the IN route in order to explore their outcomes in rat models of PD. Animals were randomly classified into four groups including control, PD model, treatment-NTC (treated with INA of non-target cells), and treatment-TC (treated with INA of target cells). The Alg-SPIONs-labeled OE-MSCs were stained successfully using the Prussian blue method with an intracellular iron concentration of 2.73 pg/cell. It was able to reduce signal intensity in the striatum region by increasing the number of these cells, as shown by the magnetic resonance imaging (MRI). Behavioral evaluation revealed that the administration of OE-MSCs with this novel advanced stem cell therapy alleviated Parkinson's motor dysfunction. Further, histological evaluations confirmed the functional enhancement of dopaminergic neuron cells by the expression of Nurr1, Dopamine transporter (DAT), and paired-like homeodomain transcription factor 3 (TH). Overall, this study showed that INA of OE-MSCs in the MTCD approach enhanced stem cells' therapeutic effects in PD models.

The odorant metabolizing enzyme UGT2A1: Immunolocalization and impact of the modulation of its activity on the olfactory response.

Odorant metabolizing enzymes (OMEs) are expressed in the olfactory epithelium (OE) where they play a significant role in the peripheral olfactory process by catalyzing the fast biotransformation of odorants leading either to their elimination or to the synthesis of new odorant stimuli. The large family of OMEs gathers different classes which interact with a myriad of odorants alike and complementary to olfactory receptors. Thus, it is necessary to increase our knowledge on OMEs to better understand their function in the physiological process of olfaction. This study focused on a major olfactory UDP-glucuronosyltransferase (UGT): UGT2A1. Immunohistochemistry and immunogold electronic microscopy allowed to localize its expression in the apical part of the sustentacular cells and originally at the plasma membrane of the olfactory cilia of the olfactory sensory neurons, both locations in close vicinity with olfactory receptors. Moreover, using electroolfactogram, we showed that a treatment of the OE with beta-glucuronidase, an enzyme which counterbalance the UGTs activity, increased the response to eugenol which is a strong odorant UGT substrate. Altogether, the results supported the function of the olfactory UGTs in the vertebrate olfactory perireceptor process.

Systemic corticosteroids in coronavirus disease 2019 (COVID-19)-related smell dysfunction: an international view.

The frequent association between coronavirus disease 2019 (COVID-19) and olfactory dysfunction is creating an unprecedented demand for a treatment of the olfactory loss. Systemic corticosteroids have been considered as a therapeutic option. However, based on current literature, we call for caution using these treatments in early COVID-19-related olfactory dysfunction because: (1) evidence supporting their usefulness is weak; (2) the rate of spontaneous recovery of COVID-19-related olfactory dysfunction is high; and (3) corticosteroids have well-known potential adverse effects. We encourage randomized placebo-controlled trials investigating the efficacy of systemic steroids in this indication and strongly emphasize to initially consider smell training, which is supported by a robust evidence base and has no known side effects.

Expansion of murine and human olfactory epithelium/mucosa colonies and generation of mature olfactory sensory neurons under chemically defined conditions.

Olfactory dysfunctions, including hyposmia and anosmia, affect ~100 million people around the world and the underlying causes are not fully understood. Degeneration of olfactory sensory neurons and incapacity of globose basal cells to generate olfactory sensory neurons are found in elder people and patients with smell disorders. Thus, olfactory stem cell may function as a promising tool to replace inactivated globose basal cells and to generate sensory neurons. Methods: We established clonal expansion of cells from the murine olfactory epithelium as well as colony growth from human olfactory mucosa using Matrigel-based three-dimensional system. These colonies were characterized by immunostaining against olfactory epithelium cellular markers and by calcium imaging of responses to odors. Chemical addition was optimized to promote Lgr5 expression, colony growth and sensory neuron generation, tested by quantitative PCR and immunostaining against progenitor and neuronal markers. The differential transcriptomes in multiple signaling pathways between colonies under different base media and chemical cocktails were determined by RNA-Seq. Results: In defined culture media, we found that VPA and CHIR99021 induced the highest Lgr5 expression level, while LY411575 resulted in the most abundant yield of OMP+ mature sensory neurons in murine colonies. Different base culture media with drug cocktails led to apparent morphological alteration from filled to cystic appearance, accompanied with massive transcriptional changes in multiple signaling pathways. Generation of sensory neurons in human colonies was affected through TGF-ß signaling, while Lgr5 expression and cell proliferation was regulated by VPA. Conclusion: Our findings suggest that targeting expansion of olfactory epithelium/mucosa colonies in vitro potentially results in discovery of new source to cell replacement-based therapy against smell loss.

Preparation of Adhesion Culture of Neural Stem/Progenitor Cells of the Olfactory Mucosa for the Treatment of Spinal Cord Injuries.

In this work, an optimal protocol was developed for obtaining adhesion culture of neural stem/progenitor cells (NSPC) of rat olfactory mucosa. During the development of the protocol, the conditions for cell culturing on adhesion substrates fibronectin and laminin in DMEM/F-12 and neurobasal media with the same culture additives were compared. Cell proliferation was maximum during culturing on both substrates in the neurobasal medium. Using the immunofluorescence method, we found that culturing on fibronectin in the neurobasal medium ensured maximum (52.22%) content of nestin-positive cells in comparison with other culturing conditions. The highest percentage of ßIII-tubulin-positive cells was detected in cultures growing on fibronectin in the neurobasal medium and in DMEM/F-12 (79.11 and 83.52%, respectively). Culturing in adhesion cultures in the neurobasal medium on fibronectin allowed obtaining cultures enriched with NSPC and neurons differentiating from them in a quantity sufficient for further transplantation. The developed protocol can be recommended for obtaining NPSC from human olfactory mucosa for the treatment of spinal cord injuries.

The effects of diazinon on the cell types and gene expression of the olfactory epithelium and whole-body hormone concentrations in the Persian sturgeon (Acipenser persicus).

The olfactory function and imprinting of odorant information of the native stream play a critical role during the homing migration in fish. Pesticides may impair olfactory imprinting by altering olfaction and hormone functions. The present study aimed to determine how diazinon impacts olfactory epithelium morphology and cell composition, as well as hormone concentrations in Persian sturgeon (Acipenser persicus) during their lifetime in freshwater and, also during diazinon-free saltwater acclimation. Fingerlings were exposed to 0, 150, 300, and 450 µg·L-1 of diazinon in freshwater for 7 days and then were transferred to diazinon-free saltwater by gradually increasing salinity up to 12 ppt. After diazinon exposure, the number of olfactory receptor cells (ORCs) and goblet cells (GCs) decreased and increased, respectively, and the expression of G-protein αolf (GPαolf) and calmodulin-dependent kinase II delta (CAMKIId) was down-regulated and up-regulated, respectively. Transferring the fish to diazinon-free saltwater (8 and 12 ppt) raised the number of ORCs, supporting cells (SCs), GCs, and GPαolf expression, and down-regulated CAMKIId without any significant differences among treatments. Exposure to diazinon increased whole-body cortisol at the high concentration, while decreased whole-body thyroxin (T4) and triiodothyronine (T3) in a dose-dependent manner. Although whole-body T4 and T3 increased in all the treatments after saltwater acclimation (8 and 12 ppt), the level of these hormones was lower in fish that had been exposed to diazinon than in the control. These results showed that diazinon can disrupt olfactory epithelium morphology and cell composition as well as hormone concentrations, which in turn may affect the olfactory imprinting in Persian sturgeon fingerlings.

Photoactivatable Odorants for Chemosensory Research.

The chemosensory system of any animal relies on a vast array of detectors tuned to distinct chemical cues. Odorant receptors and the ion channels of the TRP family are all uniquely expressed in olfactory tissues in a species-specific manner. Great effort has been made to characterize the molecular and pharmacological properties of these proteins. Nevertheless, most of the natural ligands are highly hydrophobic molecules that are not amenable to controlled delivery. We sought to develop photoreleasable, biologically inactive odorants that could be delivered to the target receptor or ion channel and effectively activated by a short light pulse. Chemically distinct ligands eugenol, benzaldehyde, 2-phenethylamine, ethanethiol, butane-1-thiol, and 2,2-dimethylethane-1-thiol were modified by covalently attaching the photoremovable protecting group (8-cyano-7-hydroxyquinolin-2-yl)methyl (CyHQ). The CyHQ derivatives were shown to release the active odorant upon illumination with 365 and 405 nm light. We characterized their bioactivity by measuring activation of recombinant TRPV1 and TRPA1 ion channels expressed in HEK 293 cells and the electroolfactogram (EOG) response from intact mouse olfactory epithelium (OE). Illumination with 405 nm light was sufficient to robustly activate TRP channels within milliseconds of the light pulse. Photoactivation of channels was superior to activation by conventional bath application of the ligands. Photolysis of the CyHQ-protected odorants efficiently activated an EOG response in a dose-dependent manner with kinetics similar to that evoked by the vaporized odorant amyl acetate (AAc). We conclude that CyHQ-based, photoreleasable odorants can be successfully implemented in chemosensory research.

Short exposure to cadmium disrupts the olfactory system of zebrafish (Danio rerio) - Relating altered gene expression in the olfactory organ to behavioral deficits.

Fish strongly rely on olfaction as a variety of essential behaviors such as foraging and predator avoidance are mediated by the olfactory system. Cadmium (Cd) is known to impair olfaction and accumulate in the olfactory epithelium (OE) and bulb (OB) of fishes. In the present study, the acute toxicity of Cd on olfaction in zebrafish (Danio rerio) was characterized on the molecular and behavioral level. To this end, quantitative real-time PCR was performed in order to analyze the expression of selected genes in both the OE and OB. Moreover, the response of zebrafish to an alarm cue was investigated. Following 24 h of exposure to Cd, the expression of genes associated with olfactory sensory neurons was reduced in the OE. Furthermore, the antioxidant genes peroxiredoxin 1 (prdx1) and heme oxygenase 1 (hmox1), as well as the metallothionein 2 gene (mt2) were upregulated in the OE, whereas hmox1 and the stress-inducible heat shock protein 70 gene (hsp70) were upregulated in the OB upon exposure to Cd. Following stimulation with a conspecific skin extract, zebrafish displayed a considerable disruption of the antipredator behavior with increasing Cd concentration. Taken together, Cd impaired olfaction in zebrafish, thereby disrupting the antipredator response, which is crucial for the survival of individuals. Cellular stress followed by disruption of olfactory sensory neurons may have contributed to the observed behavioral deficits.

Antagonistic odor interactions in olfactory sensory neurons are widespread in freely breathing mice.

Odor landscapes contain complex blends of molecules that each activate unique, overlapping populations of olfactory sensory neurons (OSNs). Despite the presence of hundreds of OSN subtypes in many animals, the overlapping nature of odor inputs may lead to saturation of neural responses at the early stages of stimulus encoding. Information loss due to saturation could be mitigated by normalizing mechanisms such as antagonism at the level of receptor-ligand interactions, whose existence and prevalence remains uncertain. By imaging OSN axon terminals in olfactory bulb glomeruli as well as OSN cell bodies within the olfactory epithelium in freely breathing mice, we find widespread antagonistic interactions in binary odor mixtures. In complex mixtures of up to 12 odorants, antagonistic interactions are stronger and more prevalent with increasing mixture complexity. Therefore, antagonism is a common feature of odor mixture encoding in OSNs and helps in normalizing activity to reduce saturation and increase information transfer.

Smell and taste dysfunction in patients with SARS-CoV-2 infection: A review of epidemiology, pathogenesis, prognosis, and treatment options.

During the initial pandemic wave of COVID-19, apart from common presenting symptoms (cough, fever, and fatigue), many countries have reported a sudden increase in the number of smell and taste dysfunction patients. Smell dysfunction has been reported in other viral infections (parainfluenza, rhinovirus, SARS, and others), but the incidence is much lower than SARS-CoV-2 infection. The pathophysiology of post-infectious olfactory loss was hypothesized that viruses may produce an inflammatory reaction of the nasal mucosa or damage the olfactory neuroepithelium directly. However, loss of smell could be presented in COVID-19 patients without other rhinologic symptoms or significant nasal inflammation. This review aims to provide a brief overview of recent evidence for epidemiology, pathological mechanisms for the smell, and taste dysfunction in SARS-CoV-2 infected patients. Furthermore, prognosis and treatments are reviewed with scanty evidence. We also discuss the possibility of using "smell and taste loss" as a screening tool for COVID-19 and treatment options in the post-SARS-CoV-2 infectious olfactory loss.

Urban air particulate matter induces mitochondrial dysfunction in human olfactory mucosal cells.

BACKGROUND: The adverse effects of air pollutants including particulate matter (PM) on the central nervous system is increasingly reported by epidemiological, animal and post-mortem studies in the last decade. Oxidative stress and inflammation are key consequences of exposure to PM although little is known of the exact mechanism. The association of PM exposure with deteriorating brain health is speculated to be driven by PM entry via the olfactory system. How air pollutants affect this key entry site remains elusive. In this study, we investigated effects of urban size-segregated PM on a novel cellular model: primary human olfactory mucosal (hOM) cells. RESULTS: Metabolic activity was reduced following 24-h exposure to PM without evident signs of toxicity. Results from cytometric bead array suggested a mild inflammatory response to PM exposure. We observed increased oxidative stress and caspase-3/7 activity as well as perturbed mitochondrial membrane potential in PM-exposed cells. Mitochondrial dysfunction was further verified by a decrease in mitochondria-dependent respiration. Transient suppression of the mitochondria-targeted gene, neuronal pentraxin 1 (NPTX1), was carried out, after being identified to be up-regulated in PM2.5-1 treated cells via RNA sequencing. Suppression of NPTX1 in cells exposed to PM did not restore mitochondrial defects resulting from PM exposure. In contrast, PM-induced adverse effects were magnified in the absence of NPTX1, indicating a critical role of this protein in protection against PM effects in hOM cells. CONCLUSION: Key mitochondrial functions were perturbed by urban PM exposure in a physiologically relevant cellular model via a mechanism involving NPTX1. In addition, inflammatory response and early signs of apoptosis accompanied mitochondrial dysfunction during exposure to PM. Findings from this study contribute to increased understanding of harmful PM effects on human health and may provide information to support mitigation strategies targeted at air pollution.

The Phenomenon of Compensatory Cell Proliferation in Olfactory Epithelium in Fish Caused by Prolonged Exposure to Natural Odorants.

It was previously shown that activation of the processes of neurogenesis in the olfactory epithelium (OE) can be caused after intranasal administration of toxic or neurotrophic factors, after axon transection, or as a result of bulbectomy. Our study showed for the first time that a significant increase in olfactory cell renewal can also occur in animals due to periodic chemostimulation with natural odorants (amino acids and peptides) for 15 days. Using electron and laser confocal microscopy in fish (Paracottus knerii (Cottidae), Dybowski, 1874) from Lake Baikal, we showed that periodic stimulation of aquatic organisms with a water-soluble mixture of amino acids and peptides causes stress in OE, which leads to programmed death cells and compensatory intensification of their renewal. We estimated the level of reactive oxygen species, number of functionally active mitochondria, intensity of apoptosis processes, and mitosis activity of cells in the OE of fish in the control group and after periodic natural odorants exposure. This study showed that new stem cells are activated during enhanced odor stimulation and subsequent degenerative changes in the cells of the sensory apparatus. Those new activated stem cells are located in previously proliferatively inactive regions of OE that become involved in compensatory processes for the formation of new cells.

Increased Retinoic Acid Catabolism in Olfactory Sensory Neurons Activates Dormant Tissue-Specific Stem Cells and Accelerates Age-Related Metaplasia.

The cellular and molecular basis of metaplasia and declining neurogenesis in the aging olfactory epithelium (OE) remains unknown. The horizontal basal cell (HBC) is a dormant tissue-specific stem cell presumed to only be forced into self-renewal and differentiation by injury. Here we analyze male and female mice and show that HBCs also are activated with increasing age as well as non-cell-autonomously by increased expression of the retinoic acid-degrading enzyme CYP26B1. Activating stimuli induce HBCs throughout OE to acquire a rounded morphology and express IP3R3, which is an inositol-1,4,5-trisphosphate receptor constitutively expressed in stem cells of the adjacent respiratory epithelium. Odor/air stimulates CYP26B1 expression in olfactory sensory neurons mainly located in the dorsomedial OE, which is spatially inverse to ventrolateral constitutive expression of the retinoic acid-synthesizing enzyme (RALDH1) in supporting cells. In ventrolateral OE, HBCs express low p63 levels and preferentially differentiate instead of self-renewing when activated. When activated by chronic CYP26B1 expression, repeated injury, or old age, ventrolateral HBCs diminish in number and generate a novel type of metaplastic respiratory cell that is RALDH- and secretes a mucin-like mucus barrier protein (FcγBP). Conversely, in the dorsomedial OE, CYP26B1 inhibits injury-induced and age-related replacement of RALDH- supporting cells with RALDH1+ ciliated respiratory cells. Collectively, these results support the concept that inositol-1,4,5-trisphosphate type 3 receptor signaling in HBCs, together with altered retinoic acid metabolism within the niche, promote HBC lineage commitment toward two types of respiratory cells that will maintain epithelial barrier function once the capacity to regenerate OE cells ceases.SIGNIFICANCE STATEMENT Little is known about signals that activate dormant stem cells to self-renew and regenerate odor-detecting neurons and other olfactory cell types after loss due to injury, infection, or toxin exposure in the nose. It is also unknown why the stem cells do not prevent age-dependent decline of odor-detecting neurons. We show that (1) stem cells are kept inactive by the vitamin A derivative retinoic acid, which is synthesized and degraded locally by olfactory cells; (2) old age as well as repeated injuries activate the stem cells and exhaust their potential to produce olfactory cells; and (3) exhausted stem cells alter the local retinoic acid metabolism and maintain the epithelial tissue barrier by generating airway cells instead of olfactory cells.

Olfactory marker protein directly buffers cAMP to avoid depolarization-induced silencing of olfactory receptor neurons.

Olfactory receptor neurons (ORNs) use odour-induced intracellular cAMP surge to gate cyclic nucleotide-gated nonselective cation (CNG) channels in cilia. Prolonged exposure to cAMP causes calmodulin-dependent feedback-adaptation of CNG channels and attenuates neural responses. On the other hand, the odour-source searching behaviour requires ORNs to be sensitive to odours when approaching targets. How ORNs accommodate these conflicting aspects of cAMP responses remains unknown. Here, we discover that olfactory marker protein (OMP) is a major cAMP buffer that maintains the sensitivity of ORNs. Upon the application of sensory stimuli, OMP directly captured and swiftly reduced freely available cAMP, which transiently uncoupled downstream CNG channel activity and prevented persistent depolarization. Under repetitive stimulation, OMP-/- ORNs were immediately silenced after burst firing due to sustained depolarization and inactivated firing machinery. Consequently, OMP-/- mice showed serious impairment in odour-source searching tasks. Therefore, cAMP buffering by OMP maintains the resilient firing of ORNs.

A new nanovesicular system for nasal drug administration.

The goal of this work was to study the characteristics of a new phospholipid nanovesicular carrier for nasal administration of drugs. Multilamellar vesicles were visualized by electron microscopy, and their mean distribution size of 200 nm was evaluated by DLS. Measured pH and viscosity values were found adequate for a nasal delivery carrier. CLS micrographs of the nasal mucosa of rats following administration of the carrier incorporating probes with various properties show delivery into the nasal mucosa layers. Tramadol containing systems were characterized and tested for their analgesic effect in two pain animal models. In mice, a significantly higher antinociceptive effect and a rapid onset of action were obtained as compared to other nasal delivery carriers and to oral treatment. This enhanced analgesic effect was further confirmed in rat pain model and sustained by drug plasma and brain levels. To test the systems behavior in a larger animal, a pharmacokinetic crossover study was carried out in sheep after administrating Tramadol nasally in the nanocarrier and IV. The plasma and CSF absolute bioavailability values were 1.09 and 0.87, respectively. HPLC and LC-MS/MS methods for quantification of Tramadol in plasma, brain and CSF were developed and are presented here. It is noteworthy that no pathological alterations or inflammation signs were observed in rat nasal mucosa following sub-chronic treatment. The results obtained in this work encourage further investigation of using the new carrier for nasal delivery of drugs in humans.