Tracheal Epithelium Research Articles

A flexible porous chiral auxetic tracheal stent with ciliated epithelium

Tracheal stent placement is a principal treatment for tracheobronchial stenosis, but complications such as mucus plugging, secondary stenosis, migration, and strong foreign body sensation remain unavoidable challenges. In this study, we designed a flexible porous chiral tracheal stent intended to reduce or overcome these complications. The stent was innovatively designed with a flexible tetrachiral and anti-tetrachiral hybrid structure as the frame and hollows filled with porous silicone sponge. Detailed finite element analysis (FEA) showed that the designed frame can maintain a Poisson's ratio that is negative or close to zero at up to 50% tensile strain. This contributes to improved airway ventilation and better resistance to migration during physiological activities such as respiration and neck movement. The preparation process combined indirect 3D printing with gas foaming and particulate leaching methods to efficiently fabricate the stent. The stent was then subjected to uniaxial tension and local radial compression tests, which indicated that it not only has the same desirable auxetic performance but also has flexibility similar to the native trachea. The porous sponge facilitated the adhesion of cells, allowed nutrient diffusion, and would prevent the ingrowth of granulation tissue. Furthermore, a ciliated tracheal epithelium similar to that of the native trachea was differentiated from normal human bronchial primary epithelial cells on the internal wall of the stent under air-liquid interface conditions. These results suggest that the designed stent has the potential for application in the treatment of tracheobronchial stenosis.

Histomorphology of the lower respiratory tract in the Indian crested porcupine (Hystrix indica).

The Indian crested porcupine (Hystrix indica) (ICP) is widely distributed in Asia; however, compared with other rodents, little is known about the structures of its respiratory system. The aim of this study was to evaluate the histomorphology of the lower respiratory portion of the ICP to provide a basis for the identification of the normal structure of this organ. The larynx, trachea and lungs of four carcasses of adult Indian crested porcupines (two males and two females) were dissected and fixed in 10% neutral-buffered formalin. The gross anatomy and histology of all specimens were evaluated. A macroscopic evaluation showed unique structures in the ICP respiratory system, including the presence of a chamber-like structure at the origin of the bronchi and a difference in epiglottis shape between males and females. Histologically, the stratified squamous epithelium covered the epiglottis and arytenoid cartilage, and the pseudostratified ciliated columnar epithelium covered the internal part of the thyroid and cricoid cartilages. Histomorphological studies showed a few goblet cells in the tracheal epithelium. In the bronchi and larger bronchioles, pseudostratified ciliated columnar epithelia were observed. Bronchi were surrounded by segments of cartilage. Distal bronchioles had a simple cuboidal/columnar epithelium with club (Clara) cells, lacked cartilaginous tissue in their walls and had a complete smooth muscle layer. These results revealed histomorphological differences between the ICP and other rodents.

Variability in tracheal mucociliary transport is not controlled by beating cilia in lambs in vivo during ventilation with humidified and nonhumidified air.

Mucociliary transport in the respiratory epithelium depends on beating of cilia to move a mucus layer containing trapped inhaled particles toward the mouth. Little is known about the relationship between cilia beat frequency (CBF) and mucus transport velocity (MTV) in vivo under normal physiological conditions and when inspired air is dry or not fully humidified. This study was designed to use video-microscopy to simultaneously measure CBF and MTV in the tracheal epithelium through an implanted optical window in mechanically ventilated lambs. The inspired air in 6 animals was heated to body temperature and fully saturated with water for 4 hours as a baseline. In another series of experiments, 5 lambs were ventilated with air at different temperatures and humidities and the mucosal surface temperature was monitored with infrared macro-imaging. In the baseline experiments, during ventilation with fully humidified air at body temperature, CBF remained constant, mean 13.9 ± 1.6 Hz but MTV varied considerably between 0.1 and 26.1 mm/min with mean 11.0 ± 3.9 mm/min, resulting in a maximum mucus displacement of 34.2 µm/cilia beat. Fully humidified air at body temperature prevented fluctuations in the surface temperature during breathing indicating a thermodynamic balance in the airways. When lambs were ventilated with dryer air, the mucosal surface temperature and MTV dropped without a significant change in CBF. When inspired air was dry, mainly latent heat (92%) was transferred to air in the trachea, reducing the surface temperature by 5 °C. Reduced humidity of the inspired air lowered the surface temperature and reduced MTV in the epithelium during ventilation.

Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF–like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.

Use of Virus-Mimicking Nanoparticles to Investigate Early Infection Events in Upper Airway 3D Models.

The current coronavirus disease-19 (COVID-19) pandemic, caused by "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), underscores the threat posed by newly emerging viruses. The understanding of the mechanisms driving early infection events, that are crucial for the exponential spread of the disease, is mandatory and can be significantly implemented generating 3D in vitro models as experimental platforms to investigate the infection substrates and how the virus invades and ravages the tissues.We here describe a protocol for the creation of a synthetic hydrogel-based 3D culture system that mimics in vitro the complex architectures and mechanical cues distinctive of the upper airway epithelia. We then expose the in vitro generated 3D nasal and tracheal epithelia to gold nanoparticles (AuNPs) that display the typical shape and size distinctive of SARS-CoV-2 and of the majority of Coronaviridae presently known.The infection platform here described provides an efficient and highly physiological in vitro model that reproduces the host-pathogen early interactions, using virus-mimicking nanoparticles, and offers a flexible tool to study virus entry into the cell. At the same time, it reduces the risk of accidental infection/spillovers for researchers, which represents a crucial aspect when dealing with a virus that is highly contagious, virulent, and even deadly.

Multi-scale alignment of respiratory cilia and its relation to mucociliary function

The tracheobronchial tree is lined by a mucociliary epithelium containing millions of multiciliated cells. Their integrated oscillatory activity continuously propels an overlying pollution-protecting mucus layer in cranial direction, leading to mucociliary clearance – the primary defence mechanism of the airways. Mucociliary transport is commonly thought to co-emerge with the collective ciliary motion pattern under appropriate geometrical and rheological conditions. Proper ciliary alignment is therefore considered essential to establish mucociliary clearance in the respiratory system. Here, we used volume electron microscopy in combination with high-speed reflection contrast microscopy in order to examine ciliary orientation and its spatial organization, as well as to measure the propagation direction of metachronal waves and the direction of mucociliary transport on bovine tracheal epithelia with reference to the tracheal long axis (TLA). Ciliary orientation is measured in terms of the basal body orientation (BBO) and the axonemal orientation (AO), which are commonly considered to coincide, both equivalently indicating the effective stroke as well as the mucociliary transport direction. Our results, however, reveal that only the AO is in line with the mucociliary transport, which was found to run along a left-handed helical trajectory, whereas the BBO was found to be aligned with the TLA. Furthermore, we show that even if ciliary orientation remains consistent between adjacent cells, ciliary orientation exhibits a gradual shift within individual cells. Together with the symplectic beating geometry, this intracellular orientational pattern could provide for the propulsion of highly viscous mucus and likely constitutes a compromise between efficiency and robustness.

Electronic Cigarette Liquid Constituents Induce Nasal and Tracheal Sensory Irritation in Mice in Regionally Dependent Fashion.

IntroductionElectronic cigarettes (e-cigs) are currently used by millions of adults and adolescents worldwide. Major respiratory symptoms, such as coughing reported by e-cig users, including patients with e-cig, or vaping, product use-associated lung injury (EVALI), indicate e-cig constituent-induced sensory irritation. However, e-cig constituent-induced nociceptive activity in nasal and tracheal respiratory epithelia (RE) and neuronal activation in the trigeminal ganglia and brainstem nuclei, which receive airway chemosensory inputs have not been examined and compared. Comparisons of physiological responses between freebase nicotine and nicotine salts are also missing.Aims and MethodsEvent-related potential (ERP) was recorded electrophysiologically to assess mouse nasal and tracheal RE chemosensory responses to various flavorings, nicotine, including freebase and nicotine salts, e-liquid mixtures, and tussigenic stimuli. Also, mice were subjected to inhalation exposure to aerosol of a vanilla-flavored e-liquid or air (control), and the activated-trigeminal nociceptive neurons and brainstem neurons were examined using immunohistochemistry.ResultsIndividual constituents and mixtures of e-liquids, capsaicin, and citric and acetic acids evoked significantly larger ERP in the nose than in the trachea with the exception of menthol. ERP responses to freebase nicotine were significantly larger than protonated nicotine. Four nicotine salts (benzoate, lactate, levulinate, and salicylate) induced similar responses. Compared with air-exposed mice, e-liquid aerosol-exposed mice showed a significant increase in numbers of activated trigeminal nociceptive neurons and brainstem neurons in the spinal trigeminal nucleus, paratrigeminal nucleus, and nucleus tractus solitarius.ConclusionsE-liquid constituents region-dependently stimulate airway nociceptive chemosensory systems, and freebase nicotine is more potent than protonated nicotine.ImplicationsNeural abnormalities have been implicated in the development of nasal and respiratory illnesses. The higher sensitivity of the nasal nociceptive chemosensory system to nicotine and flavorings may indicate a health risk for e-liquid aerosol-induced upper airway illnesses via neurogenic alteration and warrants further investigation.

Global pattern of interkinetic nuclear migration in tracheoesophageal epithelia of the mouse embryo: Interorgan and intraorgan regional differences.

Interkinetic nuclear migration (INM) is an apicobasal (AB) polarity-based regulatory mechanism of proliferation/differentiation in epithelial stem/progenitor cells. We previously documented INM in the endoderm-derived tracheal/esophageal epithelia at embryonic day (E) 11.5 and suggested that INM is involved in the development of both organs. We here investigated interorgan (trachea vs esophagus) and intraorgan regional (ventral vs dorsal) differences in the INM mode in the tracheal and esophageal epithelia of the mouse embryo. We also analyzed convergent extension (CE) and planar cell movement (PCM) in the epithelia based on cell distribution. The pregnant C57BL/6J mice were intraperitoneally injected with 5-ethynyl-2'-deoxyuridine at E11.5 and E12.5 and were sacrificed 1, 4, 6, 8, and 12 hours later to obtain the embryos. The distribution of labeled cell nuclei along the AB axis was chronologically analyzed in the total, ventral, and dorsal sides of the epithelia. The percentage distribution of the nuclei population was represented by histogram and the chronological change was analyzed statistically using multidimensional scaling. The interorgan comparison of the INM mode during E11.5-E12.0, but not E12.5-E13.0, showed a significant difference. During E11.5-E12.0 the trachea, but not the esophagus, showed a significant difference between ventral and dorsal sides. During E12.5-E13.0 neither organ showed regional differences. CE appeared to occur in both organs during E11.5-E12.0 while PCM was unclear in both organs. These findings suggest a difference between the trachea and esophagus, and a regional difference in the trachea, not in the esophagus, in the INM mode, which may be related with the later differential organogenesis/histogenesis of these organs.

Airway-Associated Macrophages in Homeostasis and Repair

SUMMARYThere is an increasing appreciation for the heterogeneity of myeloid lineages in the lung, but relatively little is known about populations specifically associated with the conducting airways. We use single-cell RNA sequencing, flow cytometry, and immunofluorescence to characterize myeloid cells of the mouse trachea during homeostasis and epithelial injury/repair. We identify submucosal macrophages, similar to lung interstitial macrophages, and intraepithelial macrophages. Following injury, there are early increases in neutrophils and submucosal macrophages, including M2-like macrophages. Intraepithelial macrophages are lost after injury and later restored by CCR2+ monocytes. We show that repair of the tracheal epithelium is impaired in Ccr2-deficient mice. Mast cells and group 2 innate lymphoid cells are sources of interleukin-13 (IL-13) that polarize macrophages and directly influence basal cell behaviors. Their proximity to the airway epithelium establishes these myeloid populations as potential therapeutic targets for airway disease.

Nicotine stimulates ion transport via metabotropic β4 subunit containing nicotinic ACh receptors.

Background and PurposeMucociliary clearance is an innate immune process of the airways, essential for removal of respiratory pathogens. It depends on ciliary beat and ion and fluid homeostasis of the epithelium. We have shown that nicotinic ACh receptors (nAChRs) activate ion transport in mouse tracheal epithelium. Yet the receptor subtypes and signalling pathways involved remained unknown.Experimental ApproachTransepithelial short circuit currents (ISC) of freshly isolated mouse tracheae were recorded using the Ussing chamber technique. Changes in [Ca2+]i were studied on freshly dissociated mouse tracheal epithelial cells.Key ResultsApical application of the nAChR agonist nicotine transiently increased ISC. The nicotine effect was abolished by the nAChR antagonist mecamylamine. α‐Bungarotoxin (α7 antagonist) had no effect. The agonists epibatidine (α3β2, α4β2, α4β4 and α3β4) and A‐85380 (α4β2 and α3β4) increased ISC. The antagonists dihydro‐β‐erythroidine (α4β2, α3β2, α4β4 and α3β4), α‐conotoxin MII (α3β2) and α‐conotoxin PnIA (α3β2) reduced the nicotine effect. Nicotine‐ and epibatidine‐induced currents were unaltered in β2−/−mice, but in β4−/− mice no increase was observed. In the presence of thapsigargin (endoplasmatic reticulum Ca2+‐ATPase inhibitor) or the ryanodine receptor antagonists JTV‐519 and dantrolene there was a reduction in the nicotine‐effect, indicating involvement of Ca2+ release from intracellular stores. Additionally, the PKA inhibitor H‐89 and the TMEM16A (Ca2+‐activated chloride channel) inhibitor T16Ainh‐A01 significantly reduced the nicotine‐effect.Conclusion and Implicationsα3β4 nAChRs are responsible for the nicotine‐induced current changes via Ca2+ release from intracellular stores, PKA and ryanodine receptor activation. These nAChRs might be possible targets to stimulate chloride transport via TMEM16A.

Therapeutic Inhaled Sphingosine for Treating Lung Infection in a Mouse Model of Critical Illness.

Sphingosine, a sphingoid long chain base, is a natural lipid with antimicrobial properties. Recent animal studies have shown that preventive sphingosine inhalation can rescue susceptible mice, such as cystic fibrosis-, burn injured- or aged mice from bacterial pulmonary infection. While preventing lung infections in susceptible patients has obvious clinical merit, treatment strategies for an established infection are also direly needed, particularly in the times of rising antibiotic resistance. Here, we tested the potential of sphingosine in treating an established pulmonary infection. We used a cecal ligation and puncture (CLP) model in male CF-1 mice and a Pseudomonas aeruginosa strain that was isolated from a septic patient (P. aeruginosa 762). We determined susceptibility to intranasal infection and ascertained when the pulmonary infection was established by continuous core body temperature monitoring. We quantified sphingosine levels in the tracheal epithelium by immunohistochemistry and studied the effects on sphingosine on bacterial membrane permeabilization and intracellular acidification using fluorescent probes. We first determined that septic mice are highly susceptible to P. aeruginosa infection 2 days after indu-cing sepsis. Additionally, at this time, sphingosine levels in the tracheal epithelium are significantly reduced as compared to levels in healthy mice. Secondly, upon intranasal Pseudomonas inoculation, we ascertained that pulmonary infection was established as early as 2.5 h after inoculation as evidenced by a significant drop in core body temperature. Using these times of infection susceptibility and detection (2 days post CLP, 2.5h after inoculation) we treated with inhaled sphingosine and observed pulmonary bacterial loads reduced to levels found in infected healthy mice after inoculation and decreased infection-associated mortality. Further, our data demonstrate that sphingosine induces outer membrane permeabilization, disrupting the membrane potential and leading to intracellular acidification of the bacteria. Sphingosine shows efficacy in treating P. aeruginosa lung infections not only prophylactically, but also therapeutically.

Insulin-like growth factor 1 promotes the extension of Tracheal Epithelium in an in Vitro Tracheal organ culture model

ObjectiveRapid epithelialization is crucial to maintain tracheal patency and prevent potential graft failure in tracheal reconstruction after tracheal resection for cancer with tracheal infiltration or tracheal stenosis. Insulin-like growth factor 1 is a liver-secreted endocrine molecule that controls cell proliferation, differentiation, and apoptosis and has been reported to promote epithelialization in several organs. Here, we utilized mouse tracheal organ cultures to examine the effect of insulin-like growth factor 1 on tracheal epithelialization. MethodsThe trachea was resected from thirteen-week-old female ICR mice, and cut into small plate-shaped tracheal sections. First, the expression of insulin-like growth factor 1 receptor was assessed by immunohistochemistry. Secondly, the tracheal sections were cultured for seven days in the culture medium, and the morphological change during the seven-day culture was assessed by immunohistochemistry, hematoxylin and eosin staining, and scanning electron microscopy. Moreover, the tracheal sections were cultured for 48 h with different concentration of insulin-like growth factor 1 (0, 0.1, 1 and 10 µg/mL) in the culture medium, and the extension length of the tracheal epithelium during culture was measured in order to assess the effect of topical IGF1 on tracheal epithelialization. ResultsImmunohistochemistry showed that insulin-like growth factor 1 receptor was expressed in tracheal epithelium. Immunohistochemistry, hematoxylin and eosin staining, and scanning electron microscopy showed that the tracheal organ cultures were stable for at least seven days without apparent morphological damage. The effect of insulin-like growth factor 1 on tracheal epithelialization was examined in plate-shaped tracheal sections cultured in medium supplemented with or without insulin-like growth factor 1 for 48 h. We also found that the epithelial edge of plate-shaped tracheal sections extended further along the surface of the tracheal section in culture medium containing insulin-like growth factor 1 compared with that in culture medium without insulin-like growth factor 1. ConclusionThe current study using an in vitro mouse tracheal organ culture model demonstrated that topical insulin-like growth factor 1 treatment promoted the extension of tracheal epithelium, suggesting the potential utility of insulin-like growth factor 1 in aiding rapid tracheal epithelialization in patients requiring tracheal reconstruction using tissue-engineered tracheas.

Comparative Study of the Pathogenesis of Rhinopneumonitis Induced by Intranasal Inoculation of Hamsters with Equine Herpesvirus-9, Equine Herpesvirus-1 strain Ab4p and Zebra-borne Equine Herpesvirus-1

Equine herpesvirus-9 (EHV-9), equine herpesvirus-1 (EHV-1) and zebra-borne EHV-1 are members of the family Herpesviridae and cause encephalitis and rhinopneumonitis in a range of animal species. The aim of this study was to characterize and compare the rhinopneumonitis induced by experimental intranasal inoculation of groups of hamsters with EHV-9, EHV-1 strain Ab4p or zebra-borne EHV-1 viruses. Animals inoculated with EHV-9 had earlier and more severe neurological and respiratory signs than those inoculated with EHV-1 strain Ab4p or zebra-borne EHV-1. At 4-5 days post inoculation (dpi), hamsters inoculated with EHV-9 had significantly increased expression of open reading fame (ORF) 30, the viral gene encoding the DNA polymerase, in lung tissue. ORF 30 expression at these time points was higher in the hamsters infected with EHV-9 than in those inoculated with the other two viruses. Severe, mild or very mild rhinitis was seen in animals inoculated with EHV-1 strain Ab4p, EHV-9 and zebra-borne EHV-1, respectively. Viral antigen was detected in olfactory receptor neurons, inflammatory cells and desquamated epithelial cells in animals in all groups until 5 dpi. Tracheitis was also seen in all three virus-infected groups with viral antigen detected in tracheal epithelium. Inoculated hamsters developed interstitial pneumonia of increasing severity over the course of the experiment. Bronchopneumonia and vasculitis were also seen in all three infected groups. These results confirm that, in addition to their neurotropism, EHV-9 and zebra-borne EHV-1 are pneumotropic viruses. EHV-1 strain Ab4p caused more severe upper respiratory tract disease, but no significant differences were detected in the severity of pneumonia induced by each virus.

MiR-196-5p involvement in selenium deficiency-induced immune damage via targeting of NFκBIA in the chicken trachea.

Dietary selenium (Se) deficiency can induce multifarious immune injury in tissues, accompanied by inflammation and a decreased expression of selenoproteins. The results of previous studies indicated that these issues are associated with Se-mediated microRNAs involved in immune regulation, although the specific mechanisms associated with these interactions have not been reported in the trachea of chickens. To explore the effects of Se deficiency in the trachea of chickens and the role of miR-196-5p, we established correlational models of tracheal injury in chickens. One hundred broilers were divided into four groups, including a control group (C group), a Se deficient group (L group), a lipopolysaccharide (LPS)-induced control group (C + LPS group) and a LPS-induced Se deficient group (L + LPS group). Light microscopy observations indicated that the infiltration of inflammatory cells was the major histopathological change caused by Se deficiency. Furthermore, ultrastructural observation of the tracheal epithelium and ciliary showed typical inflammatory signs owing to Se deficiency. We determined the targeting relationship between miR-196-5p and NFκBIA by bioinformatics analysis. In the case of Se deficiency, the changes were detected as follows: 19 selenoproteins showed different degrees of decrease (p < 0.05). Significant inhibition of both antimicrobial peptides and immunoglobulin production were observed (p < 0.05). IκB-α (NFκBIA) expression degraded with the increasing miR-196-5p (p < 0.05), and the NF-κB pathway was activated. Thereafter, we can see a significant increase in the mRNA levels of inflammatory cytokines-related genes (tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E (PTGE), interleukin (IL)-1β, IL-6) and protein expression of NF-κB/iNOS pathway-related genes (NF-κB, iNOS, TNF-α, COX-2) (p < 0.05). The release of IL-2, interferon (IFN)-γ inhibited (p < 0.05) and the secretion of IL-4, IL-6 increased, suggesting the imbalance of Th1/Th2 (Th, helper T cell) cytokines. Compared to the control, the mRNA and protein expression levels of the anti-inflammatory system components with antioxidant activity (PPAR-γ/HO-1) were in an inhibitory state (p < 0.05). Antioxidases (SOD, CAT, GSH-Px) activities were suppressed. The activities of the peroxide markers (MDA, H2O2) were enhanced (p < 0.05). In addition, Se deficiency had a positive effect on the pathological changes of inflammation and the exceptional immunity in LPS-treated groups (p < 0.05). The results confirmed the relationship between miR-196-5p and NFκBIA in chickens, revealing that Se deficiency causes respiratory mucosal immune dysfunction via the miR-196-5p-NFκBIA axis, oxidative stress and inflammation. Moreover, Se deficiency exacerbates the inflammatory damage stimulated by LPS. Our work provides a theoretical basis for the prevention of tracheal injury owing to Se deficiency and can be used as a reference for comparative medicine. Furthermore, the targeted regulation of miR-196-5p and NFκBIA may contribute to the protection of the tracheal mucosa in chickens.

Anosmia: an evolution of our understanding of its importance in COVID-19 and what questions remain to be answered

BackgroundFrom the start of the pandemic, many European otolaryngologists observed an unprecendented number of anosmic patients. Early reports proposed that anosmia could be the first or even the only symptom of COVID-19 infection, prompting calls for self-isolation in affected patients.MethodsIn the present article, we review the COVID-19 anosmia literature and try to answer the following two questions: first, why is COVID-19 infection responsible for such a high incidence of anosmia? Second, in patients with more severe forms is anosmia really less prevalent and why?ResultsIn terms of the etiology of olfactory dysfunction, several hypotheses were proposed at the outset of the pandemic; that olfactory cleft inflammation and obstruction caused a localized conductive loss, that there was injury to the sustentacular supporting cells in the olfactory epithelium or, given the known neurotropic potential of coronavirus, that the virus could invade and damage the olfactory bulb. Olfactory cleft obstruction may contribute to the olfactory dysfunction in some patients, perhaps most likely in those that show very early resolution, it cannot account for the loss in all patients.Moreover, disordered regrowth and a predominance of immature neurons have been shown to be associated with parosmia, which is a common finding amongst patients with Covid-related anosmia. A central mechanism therefore certainly seems to be consistent with the group of patients with more prolonged olfactory deficits. Sustentacular cells showing ACE-2 immunohistochemical expression 200 to 700 times greater than nasal or tracheal epithelia seem to be the main SARS-CoV-2 gateway.As the pathophysiology of COVID-19 anosmia seems to be better understood, the question of why patients with a moderate to severe form of COVID-19 infection have less olfactory involvement remains unresolved. Different potential explanations are discussed in this review.Conclusions The last 5 months have benefited from great international collaborative research, first highlighting and then proving the value of loss of smell and taste as a symptom of COVID-19. Adoption of loss of smell into the case definition by international public health bodies will facilitate control of disease transmission.

Effects of transport stress on pathological injury and main heat shock protein expression in the respiratory system of goats.

The aim of the present study was to investigate the pathological injury and the expression of heat shock proteins in the caprine lung, trachea and bronchus under transport stress. 12 healthy male goats were selected and randomly divided into three groups. The control group (non-transported group), 2hr transport-treated group and 6hr transport-treated group. Morphological changes as well as the expression of heat shock proteins (HSPs, mainly HSP27, HSP70 and HSP90) in three parts of the respiratory tract were examined. Our results showed swollen mucosa and congestive blood vessels in mucous layer and submucosa, inflammatorycellinfiltration as well as degeneration and necrosis of mucosal epithelial cells in trachea and bronchus of the transport-treated groups. The epithelial cells were degenerated, and the exfoliated cells and debris could be seen in the alveolar cavity. The results of immunohistochemistry showed that HSP27 and HSP70 were strongly expressed in tracheal and bronchial epithelium, glandular epithelium, vascular endothelium and bronchiole epithelium. And the amount of positive inflammatory cells was increased in transport-treated groups. Western blot results indicated that the expression of all three proteins had no obvious difference among the three groups in bronchi (p>.05). In trachea, there was no significant difference in the expression of heat shock proteins among the three groups except that the expression of HSP70 which was obviously higher in the two transported groups than the control group (p<.05). The expression level of HSP70 in the 2hr transport-treated group was significantly higher than the 6hr group (p<.05) and control groups (p<.05). However, there was no significant difference in the expression level of HSP27 and HSP90 in three groups (p>.05). In conclusion, our data showed that transport stress could damage the caprine respiratory system.

Infectious Laryngotracheitis in Layer Birds from Tamil Nadu, India

Infectious laryngotracheitis (ILT), caused by Gallid herpesvirus1, is one among the respiratory diseases of poultry gradually spreading worldwide, including Indian subcontinent. Present study was carried out to identify the pathogen from the suspected cases of the disease. The tracheal tissue samples (pooled) were collected from the birds suspected to have died of ILT from 26 commercial poultry farms located in and around Namakkal district of Tamil Nadu state of India. On post-mortem examination, haemorrhagic caseous exudate and fibrinous pseudo-membrane adhered to tracheal mucosa were noticed. A total of 22 farms were found positive through PCRs targeted against ICP4 gene and the thymidine kinase (TK) gene followed by confirmation through sequencing. Histopathology examination revealed decilliation, hyperplasia, degeneration and/or loss of tracheal epithelium with severe submucosal edema. There was infiltration with numerous lymphocytes, macrophages and plasma cells in milder infections, whereas presence of fibrinous exudates admixed with numerous erythrocytes and inflammatory cells like heterophils and lymphocytes were seen indicating the severe acute form of the disease. A fibrinous pseudomembrane was seen firmly attached to the inflamed and necrotic mucosa in subacute cases. Further, virus was isolated from randomly selected 5 PCR positive tracheal tissue samples in embryonated chicken eggs through chorioallantoic membrane (CAM) route. The typical pock lesions were observed on CAM along with engorged blood vessels and thickening of the membrane. Present study has reported the disease ILT among poultry flocks in the Namakkal district of Tamil Nadu and raises the concern for thorough investigation of the nature of prevailing pathogen in the region.

Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of an ongoing pandemic, with increasing deaths worldwide. To date, documentation of the histopathological features in fatal cases of the disease caused by SARS-CoV-2 (COVID-19) has been scarce due to sparse autopsy performance and incomplete organ sampling. We aimed to provide a clinicopathological report of severe COVID-19 cases by documenting histopathological changes and evidence of SARS-CoV-2 tissue tropism.MethodsIn this case series, patients with a positive antemortem or post-mortem SARS-CoV-2 result were considered eligible for enrolment. Post-mortem examinations were done on 14 people who died with COVID-19 at the King County Medical Examiner's Office (Seattle, WA, USA) and Snohomish County Medical Examiner's Office (Everett, WA, USA) in negative-pressure isolation suites during February and March, 2020. Clinical and laboratory data were reviewed. Tissue examination was done by light microscopy, immunohistochemistry, electron microscopy, and quantitative RT-PCR.FindingsThe median age of our cohort was 73·5 years (range 42–84; IQR 67·5–77·25). All patients had clinically significant comorbidities, the most common being hypertension, chronic kidney disease, obstructive sleep apnoea, and metabolic disease including diabetes and obesity. The major pulmonary finding was diffuse alveolar damage in the acute or organising phases, with five patients showing focal pulmonary microthrombi. Coronavirus-like particles were detected in the respiratory system, kidney, and gastrointestinal tract. Lymphocytic myocarditis was observed in one patient with viral RNA detected in the tissue.InterpretationThe primary pathology observed in our cohort was diffuse alveolar damage, with virus located in the pneumocytes and tracheal epithelium. Microthrombi, where observed, were scarce and endotheliitis was not identified. Although other non-pulmonary organs showed susceptibility to infection, their contribution to the pathogenesis of SARS-CoV-2 infection requires further examination.FundingNone.

Caspofungin induces the release of Ca2+ ions from internal stores by activating ryanodine receptor-dependent pathways in human tracheal epithelial cells

The antimycotic drug caspofungin is known to alter the cell function of cardiomyocytes and the cilia-bearing cells of the tracheal epithelium. The objective of this study was to investigate the homeostasis of intracellular Ca2+ concentration ([Ca2+]i) after exposure to caspofungin in isolated human tracheal epithelial cells. The [Ca2+]i was measured using the ratiometric fluoroprobe FURA-2 AM. We recorded two groups of epithelial cells with distinct responses to caspofungin exposure, which demonstrated either a rapid transient rise in [Ca2+]i or a sustained elevation of [Ca2+]i. Both patterns of Ca2+ kinetics were still observed when an influx of transmembraneous Ca2+ ions was pharmacologically inhibited. Furthermore, in extracellular buffer solutions without Ca2+ ions, caspofungin exposure still evoked this characteristic rise in [Ca2+]i. To shed light on the origin of the Ca2+ ions responsible for the elevation in [Ca2+]i we investigated the possible intracellular storage of Ca2+ ions. The depletion of mitochondrial Ca2+ stores using 25 µM 2,4-dinitrophenol (DNP) did not prevent the caspofungin-induced rise in [Ca2+]i, which was rapid and transient. However, the application of caffeine (30 mM) to discharge Ca2+ ions that were presumably stored in the endoplasmic reticulum (ER) prior to caspofungin exposure completely inhibited the caspofungin-induced changes in [Ca2+]i levels. When the ER-bound IP3 receptors were blocked by 2-APB (40 µM), we observed a delayed transient rise in [Ca2+]i as a response to the caspofungin. Inhibition of the ryanodine receptors (RyR) using 40 µM ryanodine completely prevented the caspofungin-induced elevation of [Ca2+]i. In summary, caspofungin has been shown to trigger an increase in [Ca2+]i independent from extracellular Ca2+ ions by liberating the Ca2+ ions stored in the ER, mainly via a RyR pathway.

Hcp2a of type VI secretion system contributes to IL8 and IL1β expression of chicken tracheal epithelium by affecting APEC colonization

Avian pathogenic Escherichia coli (APEC) is an important pathogen that causes avian colibacillosis in poultry. APEC infection can lead to pathological changes in chicken trachea. The type VI secretion system (T6SS) of APEC contribute to the pathogenicity of APEC. However, whether T6SS plays a role in infection of the trachea remains unclear. We constructed mutant strain Δhcp2a by the Red recombination method system. The role of hcp2a (the structural secretion components and secretory protein of the T6SS) in the infection of trachea was investigated. The mutation strain displayed a significant increase in biofilm formation and a decrease in resistance to chicken serum. Moreover, RNA sequencing analyses showed that infection of chicken tracheal epithelium by the mutant strain Δhcp2a induced differential expression of genes. The result also showed that 14 genes (13 genes were downregulated) were enriched in cytokine-cytokine receptor interaction signalling pathway at 12 and 24 h post infection. The mutation Δhcp2a resulted in significant decreases in the bacterial loads in trachea at 6 and 12 h post infection. Real-time PCR analyses showed that the hcp2a mutation downregulated the expression of IL8 and IL1β at mRNA level in chicken tracheal epithelium. Our results indicate that mutation of hcp2a influenced genes expression of the cytokine-cytokine receptor interaction pathway by decreasing APEC colonization in the trachea.