Tracheal Epithelium Research Articles

The combination of infectious bronchitis virus BR1 and Mass vaccines provides broad protection

ABSTRACT Two vaccination-challenge trials were performed using a commercial infectious bronchitis virus (IBV) BR1 vaccine, given alone or combined with a commercial IBV Mass vaccine against challenges with IBV M41, 793B, D388 (QX), Q1, Brasil-1 or Variant 2 challenge viruses, which includes the IB viruses that are dominant in South America. The efficacy of the vaccines against the challenge viruses was investigated by determination of the ciliary activity of the tracheal epithelium after challenge. The level of protection induced by the IBV BR1 vaccine alone against the six IBV challenge strains, of which five were of heterologous genotypes, varied from 50% to 100% with an average of 80%. The level of protection induced by the combination of the IBV BR1 and IBV Mass vaccines against the six IBV challenge strains, of which four were of heterologous genotypes, varied from 80% to 100% with an average of 92%. Vaccination with IBV BR1 alone provided a high level of protection against most tested challenge viruses, though the combination of IBV BR1 and IBV Mass was more consistent, showing less variation and compliance with the criterium mentioned in the European Pharmacopoeia 10th edition (at least 80% protection) for all tested challenge viruses. These trials show that vaccination with a combination of IBV BR1 and IBV Mass vaccines provides high levels of protection against the circulating IBV strains in South America.

Mitochondrial pyruvate carriers control airway basal progenitor cell function through glycolytic-epigenetic reprogramming

Basal cells (BCs) are the progenitor cells responsible for tracheal epithelium integrity. Here, we demonstrate that mitochondrial pyruvate carriers (MPCs) act as metabolic checkpoints that are essential for BC fate decision. Inhibition of MPCs enables long-term expansion of BCs from both mice and humans. Genetic inactivation of Mpc2 in mice leads to BC hyperplasia and reduced ciliated cells during homeostasis, as well as delayed epithelial regeneration and accumulation of intermediate cells following injury. Mechanistically, MPC2 links glycolysis to ATP citrate lyase (ACLY)-dependent cytosolic acetyl-coenzyme A (CoA) generation, which is required for the epigenetic control of differentiation-related gene transcription. Modulating this metabolic-epigenetic axis partially rescues Yes-associated protein (YAP)-dysfunction-induced changes in BCs. Importantly, exogenous citrate promotes the differentiation of BCs from chronic obstructive lung disease (COPD) patients. Thus, beyond demonstrating the role of pyruvate metabolism in BC fate decision, our study suggests that targeting pyruvate-citrate metabolism may serve as a potential strategy to rectify abnormal BC behavior in lung diseases.

A 3-month-old neonatal rhesus macaque HFMD model caused by coxsackievirus B1 infection and viral tissue tropism.

Coxsackievirus B1 (CVB1), an enterovirus with multiple clinical presentations, has been associated with potential long-term consequences, including hand, foot, and mouth disease (HFMD), in some patients. However, the related animal models, transmission dynamics, and long-term tissue tropism of CVB1 have not been systematically characterized. In this study, we established a model of CVB1 respiratory infection in rhesus macaques and evaluated the clinical symptoms, viral load, and immune levels during the acute phase (0-14 days) and long-term recovery phase (15-30 days). We also investigated the distribution, viral clearance, and pathology during the long-term recovery period using 35 postmortem rhesus macaque tissue samples collected at 30 days postinfection (d.p.i.). The results showed that the infected rhesus macaques were susceptible to CVB1 and exhibited HFMD symptoms, viral clearance, altered cytokine levels, and the presence of neutralizing antibodies. Autopsy revealed positive viral loads in the heart, spleen, pancreas, soft palate, and olfactory bulb tissues. HE staining demonstrated pathological damage to the liver, spleen, lung, soft palate, and tracheal epithelium. At 30 d.p.i., viral antigens were detected in visceral, immune, respiratory, and muscle tissues but not in intestinal or neural tissues. Brain tissue examination revealed viral meningitis-like changes, and CVB1 antigen expression was detected in occipital, pontine, cerebellar, and spinal cord tissues at 30 d.p.i. This study provides the first insights into CVB1 pathogenesis in a nonhuman primate model of HFMD and confirms that CVB1 exhibits tissue tropism following long-term infection.

Deciphering infected cell types, hub gene networks and cell-cell communication in infectious bronchitis virus via single-cell RNA sequencing.

Infectious bronchitis virus (IBV) is a coronavirus that infects chickens, which exhibits a broad tropism for epithelial cells, infecting the tracheal mucosal epithelium, intestinal mucosal epithelium, and renal tubular epithelial cells. Utilizing single-cell RNA sequencing (scRNA-seq), we systematically examined cells in renal, bursal, and tracheal tissues following IBV infection and identified tissue-specific molecular markers expressed in distinct cell types. We evaluated the expression of viral RNA in diverse cellular populations and subsequently ascertained that distal tubules and collecting ducts within the kidney, bursal mucosal epithelial cells, and follicle-associated epithelial cells exhibit susceptibility to IBV infection through immunofluorescence. Furthermore, our findings revealed an upregulation in the transcription of proinflammatory cytokines IL18 and IL1B in renal macrophages as well as increased expression of apoptosis-related gene STAT in distal tubules and collecting duct cells upon IBV infection leading to renal damage. Cell-to-cell communication unveiled potential interactions between diverse cell types, as well as upregulated signaling pathways and key sender-receiver cell populations after IBV infection. Integrating single-cell data from all tissues, we applied weighted gene co-expression network analysis (WGCNA) to identify gene modules that are specifically expressed in different cell populations. Based on the WGCNA results, we identified seven immune-related gene modules and determined the differential expression pattern of module genes, as well as the hub genes within these modules. Our comprehensive data provides valuable insights into the pathogenesis of IBV as well as avian antiviral immunology.

The role of electrogenic and electroneutral monocarboxylate transport in airway clearance

Introduction: Monocarboxylates can be transported by the electrogenic Na+-coupled system (SMCTs/SLC5 family) and the electroneutral H+-coupled system (MCTs/SLC16 family). Previously, MCTs have been described in human bronchial epithelium but there is no evidence of SMCTs activity in airways. We describe here for first time that SMCT1/SCL5A8 is functionally expressed in mouse tracheal epithelium. We hypothesized that monocarboxylate transport activity might downregulate airway clearance in mouse tracheas as increased Na+-absorption reduce airway surface liquid (ASL), and increased H+-secretion acidify ASL. Methods: Short-circuit current ( ISC) recordings were performed in bicarbonate buffer with and without Na+, videomicroscopy of particle track speed (PTS) and mucus transport speed were performed in mouse tracheas. Animals (C57BL6/J) were housed at CECs-Animal facility under controlled temperature, humidity and free access to water and food. All protocols were approved (IACUC, #CECs-2022-04), in accordance with relevant regulations. Results: 10 mM apical L-lactate, D-lactate, pyruvate, propionate or acetate induced a negative current ( ISC~ -35 μA·cm−2; n=4-5 each group) and reduced (p<0.05 rank-sum test) when Na+ was replaced. L-lactate or pyruvate, transported by SMCTs and MCTs increased PTS (2.1±0.1 control vs 3.28±0.4 μm·s−1* for L-lactate and 3.31±0.1 μm·s−1* for pyruvate; n=3 each group; * indicates p<0.05 rank-sum test) and inhibited by 30 μM of the MCT inhibitor AR-C155858 (1.9±0.1 μm·s−1). D-lactate, transported by SMCTs only, did not affected PTS (2.03±0.1 μm·s−1; p>0.05; n=3 each group). We observed a reduction of amiloride-sensitive Na+-absorptive currents in tissues incubated with L-lactate compared to non-incubated ( ISC -17±4 μA·cm−2 vs -5±4 μA·cm−2; p<0.05 rank-sum test; n=4 each group). Finally, analysis of mucus transport determined that L- but not D-lactate increased the speed of mucus, that was prevented when tissues were incubated with AR-C155858 (control 1.06 ± 0.05 μm·s−1 vs L-lactate 1.24 ± 0.08 μm·s−1*, vs L-lactate + AR-C155858 1.02 ± 0.06 μm·s−1; * indicates p<0.05 ANOVA on ranks; n=4 each group). Real time PCR determined that the only SMCT expressed in airway epithelium is SLC5A8. Conclusion: SLC5A8 Na+-dependent D-lactate transport didn't affect mucus clearance. This is explained by the fact that ENaC Na+ absorption competes with SMCTs for basolateral Na+ transport, suggesting that both ENaC and SLC5A8 localize in the same cells. On the other hand, monocarboxylates that increase PTS and mucus speed transport might be explained by MCT-dependent H+ removal (alkalization) from ASL. Since alkalization of the airway surface can restore airway clearance, the use of MCTs transportable substrates might help alleviate mucostasis in muco-obstructive diseases. Funded by FONDECYT 1221257 (C.A.F.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The avian influenza A virus receptor SA-α2,3-Gal is expressed in the porcine nasal mucosa sustaining the pig as a mixing vessel for new influenza viruses

Influenza A viruses (IAVs) originate from wild birds but have on several occasions jumped host barriers and are now also circulating in humans and mammals. The IAV host receptors (glycans with galactose linked to a sialic acid (SA) in an α2,3 or α2,6 linkage) are crucial host factors restricting inter-species transmission. In general, avian-origin IAVs show a preference for SA-α2,3 (avian receptor), whereas IAVs isolated from humans and pigs prefer SA-α2,6 (human receptor). N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two major SAs. Neu5Ac is expressed in all species, whereas Neu5Gc is only expressed in a limited number of domestic species such as pigs and horses, but not in humans. Despite that previous studies have shown that the IAV host receptor distribution appears to be similar in pigs and humans, none of these studies have investigated the expression of Neu5Gc-α2,6 in situ in porcine tissues. Thus, the aim of this study was to elucidate the distribution of IAV host receptors expressed in the porcine respiratory tract and relate the expression to the viral tropism of diverse host-adapted IAVs.The IAV receptor (SA-α2,3 and SA-α2,6) distribution and the presence of specifically Neu5Gc-α2,6 in the porcine nasal, tracheal, and lung tissues was investigated by lectin histochemistry. Furthermore, IAV immunohistochemistry was performed on tissues from pigs experimentally infected with IAVs, either adapted to pigs or humans, to investigate the significance of the IAV host receptors and the tropism of the diverse host-adapted IAVs. We document for the first time the expression of the avian receptor on the surface of the porcine nasal mucosa and an equal expression of Neu5Ac- α2,6 and Neu5Gc-α2,6 on the surface of the tracheal epithelium and alveoli. In all IAV-infected pigs, we found a low amount of IAV-positive cells in the trachea despite a high expression of the human receptor. Cumulatively, these findings suggest that optimal IAV replication involves a complex interplay between the viruses and their host receptors and that there might be other less clearly defined host factors that determine the site of replication.

Effects of Dietary Supplementation of Essential Oils, Lysozyme, and Vitamins' Blend on Layer Hen Performance, Viral Vaccinal Response, and Egg Quality Characteristics.

Maintaining respiratory tract health is crucial for layers, impacting gut health, laying performance, and egg quality. Viral diseases and standard vaccinations can compromise tracheal epithelium function, leading to oxidative stress. This study assessed the impact of a blend of feed additives, predominantly lysozyme (L), essential oils (EO), and vitamins (VIT) (referred to as L + EO + VIT), on young layers during an oral vaccination schedule. The supplementation significantly enhanced antibody titers for Newcastle Disease Virus (NDV) and Infectious Bronchitis Virus (IBV) after vaccination, trachea functionality and intestinal health in the jejunum, increased egg production, and exhibited a trend toward higher egg weight. Although feed intake showed no significant difference, egg quality remained consistent across experimental groups. Moreover, L + EO + VIT supplementation elevated total phenolic content in eggs, improving oxidative stability in both fresh and stored eggs, particularly under iron-induced oxidation. Notably, it substantially reduced yolk lipid peroxidation and albumen protein carbonyls. In conclusion, water supplementation with L + EO + VIT may enhance humoral immune response to IBV and NDV, positively impacting hen productivity. These findings indicate improved tracheal function and enhanced oxidative stability, emphasizing the potential of this blend in promoting overall health and performance in layers.

Short Term Exposure of Sheep Tracheal Epithelium to Cigarette Smoke Extract Reduces ENaC Current: A Pilot Study.

Cigarette smoke has been shown to induce a phenotype in humans known as "acquired cystic fibrosis". This occurs because the cystic fibrosis transmembrane conductance regulator (CFTR) functions are impaired systemically due to the deleterious effects of smoke components. Elucidation of cigarette smoke effects on the tracheal epithelium is important. The aim of this study was to develop an ex vivo sheep tracheal model to investigate tracheal ion function. In this model, the epithelial sodium channel (ENaC) is inhibited after exposure to cigarette smoke extract (CSE) as a proof of principle. Tracheas were isolated from healthy sheep and the tracheal epithelium was surgically excised. Tissues were mounted in Ussing chambers and the short circuit current (Isc) was measured after incubation with 5% CSE in PBS or PBS alone for 30 min. The function of ENaC was investigated by the addition of amiloride (10-5M) apically. Western blot analysis was performed to assess differences in ENaC quantity after CSE exposure. Some specimens were stained with H&E for detection of histological alterations. The amiloride effect on normal epithelium led to a significant decrease in Isc [ΔI=33±5.92 μA/cm2; p<0.001 versus control experiments (ΔI=1.44±0.71 μA/cm2)]. After incubation with CSE, ENaC Isc was significantly reduced (ΔI=14.80±1.96 μA/cm2; p<0.001). No differences in αENaC expression were observed between CSE-exposed and normal tracheal epithelium. Histological images post CSE incubation revealed decreases in the height of the epithelium, with basal cell hyperplasia and loss of ciliated cells. Reduced ENaC inhibition by amiloride after CSE incubation could be due to alterations in the tracheal epithelium.

Dopamine, norepinephrine, and vasopressin accelerate particle transport velocity in murine tracheal epithelium via substance-specific receptor pathways: dependency on intra- and extracellular Ca2+ sources.

The unique ability of the respiratory tract to protect the integrity of the airways by removing potentially harmful substances is defined as mucociliary clearance. This complex physiological mechanism protects the lower airways by ridding them of pollutants and pathogens. This study aimed to evaluate the potential influence of clinically relevant vasopressors on mucociliary clearance. The particle transport velocity (PTV) of isolated murine tracheae was measured as a surrogate for mucociliary clearance under the influence of dopamine, norepinephrine, and vasopressin. Inhibitory substances were applied to elucidate relevant signal transduction cascades and the value and origin of calcium ions. Reverse-transcription polymerase chain reactions (RT-PCR) were performed to identify the expression of vasopressin receptor subtypes. Dopamine, norepinephrine, and vasopressin significantly increased the PTV in a dose-dependent manner with half maximal effective concentrations of 0.58µM, 1.21µM, and 0.10µM, respectively. Each substance increased the PTV via separate receptor pathways. While dopamine acted on D1-like receptors to increase the PTV, norepinephrine acted on β-adrenergic receptors, and vasopressin acted on V1a receptors. RT-PCR revealed the expression of V1a in the murine whole trachea and tracheal epithelium. PTV increased when protein kinase A was inhibited and norepinephrine or vasopressin were applied, but not when dopamine was applied. Phospholipase C inhibition decreased the PTV when vasopressin was applied. In general, maximum PTV was significantly reduced when extracellular calcium entry was inhibited. When intracellular calcium stores were depleted, no increase in PTV was observed after administering all three substances. Inositol trisphosphate receptor activation was found to be pivotal in the increase in murine PTV after applying dopamine and vasopressin. Dopamine, norepinephrine, and vasopressin accelerate the murine PTV via substance-specific receptor pathways. Further investigations should assess the value and interaction of these substances on mucociliary clearance in clinical practice.

Caracterização molecular e patologia de isolados de campo do vírus da febre aftosa em camundongos albinos suíços

Foot-and-mouth disease is responsible for severe economic losses to the livestock industry of Pakistan. This study aimed to use Swiss albino mice as a cost-effective experimental animal model to study different immunological and histopathological aspects of FMDV instead of natural targeted species like cattle. After isolation of field isolates FMDV on BHK-21 cell line, biological titer of the virus and mice infectious dose50 was calculated. Virus was injected in 45 Swiss albino mice (group A) through intraperitoneal route. The gross, histopathological and immunopathological lesions in heart, trachea and lungs were recorded at different day's intervals. Histopathologically, the heart showed congestion, hemorrhages and necrosis of cardiac muscles. Trachea showed deciliated epithelium and lungs showed hemorrhages, bronchial edema and alveolar emphysema. Immunohistochemical studies revealed the presence of virus in cardiac muscles, tracheal and bronchial epithelium and alveolar lumen. The findings evoked a thought that laboratory animals could be an alternative to large animals to meet budget limitations for further research on foot-and-mouth-disease.

Pathology and molecular detection of influenza Asubtype H9N2 virus in commercial poultry in Nigeria, 2024

Background: After the first Avian Influenza H5N1 outbreak in Nigerian poultry in 2006, subsequent waves of outbreaks occurred, causing substantial losses. Despite effective control measures by 2008, a resurgence in 2015 led to further losses and required depopulation efforts. Aim: The aim of this study was to do Pathology and molecular detection of influenza A subtype H9N2 virus in commercial poultry in Nigeria during 2024. Methods: In February 2024, a poultry farmer reported high mortality in his mixed commercial poultry flock in Ibadan, Nigeria, submitting carcasses to the University of Ibadan's V.T.H. and the FAO Regional Laboratory at NVRI, Vom. Results: Necropsy of nine Isa Brown layers and three Abor Acre broilers revealed cyanosis of comb and wattles, generalized petechial and ecchymotic haemorrhages including shank haemorrhages with sinusitis, pneumonia, and severe greenish faecal pasting also observed. At histopathology, denudation of the tracheal epithelia and parabronchial epithelial necrosis, obliteration, with airsac oedema and emphysema were observed. At NVRI, qPCR detected an Influenza A H9N2 virus in several pooled organ samples of layers, and broilers and eliminating the avian infectious bronchitis and Newcastle disease viruses. Conclusion: This is the first report of H9N2 outbreak in commercial poultry in Southern Nigeria. The high pathogenicity shown in commercial poultry in this outbreak and the risk of dispersal of infected live poultry in Nigeria as previously seen in H5N1 require stakeholders’ intervention.

Porcine circovirus type 2 and Glaesserella parasuis serotype 4 co-infection activates Snail1 to disrupt the intercellular junctions and facilitate bacteria translocation across the tracheal epithelium

Clinically, Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality. However, the mechanism of PCV2 and G. parasuis serotype 4 (GPS4) co-infection is still not fully understood. In this study, swine tracheal epithelial cells (STEC) were used as a barrier model, and our results showed that PCV2 infection increased the adhesion of GPS4 to STEC, while decreasing the levels of ZO-1, Occludin and increasing tracheal epithelial permeability, and ultimately facilitated GPS4 translocation. Snail1 is a transcriptional repressor, and has been known to induce epithelial-to-mesenchymal transition (EMT) during development or in cancer metastasis. Importantly, we found that Snail1, as a transcriptional repressor, was crucial in destroying the tracheal epithelial barrier induced by PCV2, GPS4, PCV2 and GPS4 coinfection. For the first time, we found that PCV2, GPS4, PCV2 and GPS4 coinfection cross-activates TGF-β and p38/MAPK signaling pathways to upregulate the expression of Snail1, down-regulate the levels of ZO-1 and Occludin, and thus disrupt the integrity of tracheal epithelial barrier then promoting GPS4 translocation. Finally, PCV2 and GPS4 co-infection also can activate TGF-β and p38/MAPK signaling pathways in vivo and upregulate Snail1, ultimately down-regulating the expression of ZO-1 and Occludin. Our study elucidates how PCV2 infection promotes GPS4 to breach the tracheal epithelial barrier and aggravate clinical manifestations.

Molecular and Pathological Investigation of Infectious Bronchitis in Broiler Flocks in Sulaimani Province, Iraq

This research's goal was to examine the molecular analysis and pathological lesions linked to IBV infection in flocks of naturally infected broiler chickens from non-vaccinated and vaccinated (H120, MA5+IB 4-91) flocks. One hundred sixteen birds were collected in Sulaimani provinces between November 2021 and July 2022 from various broiler chicken flocks of vaccinated (H120, MA5+IB 4-91) and non-vaccinated birds who suffered from respiratory illness. Viral isolation and PCR were used to confirm the infection. The majority of the necropsied birds showed congested trachea and the presence of mucus with caseated debris at the bifurcation. There was a white precipitate in the ureters, also the kidneys were enlarged and congested. Sequencing of the S1 glycoprotein gene revealed that four isolates found and after phylogenetic analysis of the strains' deduced amino acid sequences indicated that they had a close relationship with the Iranian strains MG013972 and MG013973 as well as the Iraqi strain MH747093. The non-vaccinated chickens' histopathological evaluation of all organs was more severe than that of the vaccinated chicks, with catarrhal exudate obstructing the tracheal lumen and tracheal epithelium undergoing hyperplasia to squamous metaplasia. In lung sections, catarrhal exudate partially to completely blocked the parabronchial lumen and the intrabronchial septa thickened due to alveolar capillary congestion, and an inflammatory reaction. Mild to severe glomerulonephritis with considerable collecting epithelial cell degeneration was seen in the kidney segment. Finally, it can be said that the infectious bronchitis virus is a significant contributor to the respiratory and/or renal issues affecting the flocks of broiler chickens in Sulaimani Province.

CIGARETTE SMOKE-INDUCED VITAMIN A DEFICIENCY CONTRIBUTES TO EMPHYSEMA PROGRESSION IN RATS

Emphysema and lung inflammation can be accelerated by a lack of vitamin A, an important nutrient. Vitamin A levels were shown to be lower in rats that were given benzopyrene, a component of cigarette smoke. Two separate groups of rats served as experimental subjects for the length of the study. The first group, known as the experimental group, was subjected to daily, unfiltered cigarette smoke exposure for fifteen sessions, while the second group, called the control group, was exposed just to outside air. An assessment was conducted to measure the levels of retinol and vitamin A in the blood, liver, and lungs six weeks after the investigation began. Vitamin A levels were found to be lower in the group of rats that were exposed to cigarette smoke. Additionally, amounts of pulmonary vitamin A were positively correlated with emphysema severity, indicating a negative connection. Hyperplasia of the tracheal epithelium and the development of hepatic vacuoles were also noted.

Reconstruction of tracheal window-shape defect by 3D printed polycaprolatone scaffold coated with Silk Fibroin Methacryloyl.

In this study, we aimed to utilize autologous tracheal epithelia and BMSCs as the seeding cells, utilize PCL coated with SilMA as the hybrid scaffold to carry the cells and KGN, which can selectively stimulate chondrogenic differentiation of BMSCs. This hybrid tracheal substitution was carried out to repair the tracheal partial window-shape defect. Firstly, SilMA with the concentration of 10%, 15% and 20% was prepared, and the experiment of swelling and degradation was performed. With the increase of the concentration, the swelling ratio of SilMA decreased, and the degradation progress slowed down. Upon the result of CCK-8 test and HE staining of 3D co-culture, the SilMA with concentration of 20% was selected. Next, SilMA and the cells attached to SilMA were characterized by SEM. Furthermore, in vitro cytotoxicity test shows that 20% SilMA has good cytocompatibility. The hybrid scaffold was then made by PCL coated with 20% SilMA. The mechanical test shows this hybrid scaffold has better biomechanical properties than native trachea. In vivo tracheal defect repair assays were conducted to evaluate the effect of the hybrid substitution. H&E staining, IHC staining and IF staining showed that this hybrid substitution ensured the viability, proliferation and migration of epithelium. However, it is sad that the results of chondrogenesis were not obvious. This study is expected to provide new strategies for the fields of tracheal replacement therapy needing mechanical properties and epithelization.

Modified Guo-Min decoction ameliorates PM2.5-induced lung injury by inhibition of PI3K-AKT and MAPK signaling pathways

Background/PurposeExposure to particles with an aerodynamic diameter of ≤2.5 μm (PM2.5) increased various lung diseases, which lack effective treatment. Massive evidence links PM2.5 to the development of allergic lung diseases like asthma. Modified Guo-Min Decoction (MGMD) is a traditional Chinese formula for allergic diseases. However, whether MGMD could improve PM2.5-induced lung injury and the underlying mechanism remain unclear and we aimed to explore. Study Design/MethodsMale Wistar rats (200–220 g) were intratracheally instilled of PM2.5 suspension daily for 4 weeks to establish PM2.5-induced lung injury model. MGMD (2.1 g/kg) treatment by gavage was started 1 week before, at the same time or 1 week after the instillation of PM2.5 suspension, namely the pre-, sync- or post-administration groups. HE and Masson staining were used to observe morphological changes. Immunohistochemistry staining was used to detect macrophage and neutrophil infiltration. The levels of inflammatory cytokines in the bronchoalveolar lavage fluid were detected by ELISA. The main components of MGMD were detected by UHPLC-LTQ-Orbitrap MSn. Network pharmacology was used to identify the key targets mediating the effect of MGMD in treating PM2.5-induced lung injury. Changes in the expression of target proteins were examined by western blot. In-vitro experiments were carried out in Beas2b cells to evaluate the protective effect and mechanism of MGMD against PM2.5 induced injury. ResultsExposure to PM2.5 suspension resulted in disarrangement of tracheal epithelium, neutrophil and M1 macrophage infiltration and collagen deposition, and significantly increased IgE, IL-1β and IL-17 secretion and NLRP3 expression, which were inhibited by MDMD treatment and pre-MGMD treatment showed the best effect. By UHPLC-LTQ-Orbitrap MSn, 46 main compounds were identified in MGMD. Using network pharmacology approach, we found MGMD attenuate PM2.5-induced lung damage by targeting 216 genes, and PPI network, GO and KEGG analysis all indicated that PI3K-AKT and MAPK pathways were important. Western blot showed that PM2.5 suspension exposure increased PI3K, AKT, ERK and JNK phosphorylation, which were reversed by MGMD intervention significantly. In vitro, the viability of Beas2b cells was significantly decreased after PM2.5 suspension exposure, and was obviously upregulated after MGMD-containing serum or LY294002 treatment. Conclusion: The present study demonstrated that MGMD could improve PM2.5-induced lung injury through reducing inflammation and pulmonary fibrosis, which was probably mediated by inhibition of the PI3K-AKT and MAPK signaling pathways, and NLRP3 inflammasome. The results of this study support and provide scientific evidence for the clinical application of MGMD on PM2.5-induced lung injury. Pre-treatment, sync-treatment, and post-treatment is the highlight of this study.

Prevalence, associated factors and predictors of post stroke pneumonia in a Nigerian population: A retrospective study

ObjectivesStroke can trigger an immune response that can raise the risk of infection, alter tracheal epithelium, reduce pulmonary clearance and impair secretions drainage capacity. Infection, altered tracheal epithelium, reduced pulmonary clearance, impaired secretions drainage capacity and aspiration can cause pneumonia after stroke. The aim of this study is to find out the prevalence of post stroke pneumonia in a Nigerian population and factors that are associated with it. Materials and MethodStudy data was extracted from the case files of patients with stroke who were managed between 1st January, 2011 and 1st February, 2021 in the study setting. ResultsThe result showed that, there was a record of only 591 patients with stroke (mean age, 62.78 ± 14.86 years) who were managed in the two hospitals during the period of the study. Out of this number, only 102 (17.3 %) had pneumonia. Presence of the pneumonia was only significantly (p < 0.05) associated with sex, type of stroke, lower limb muscle power, and outcome (died or alive). However, only those with ischaemic stroke are less likely to have pneumonia (Odds ratio= 0.467; CI: 0.275 to 0.791, p= 0.005), and patients who survived the stroke and are alive are less likely to develop pneumonia (Odds ratio= 0.150; CI: 0.092 to 0.245, p < 0.001). ConclusionsPneumonia occurs to a large extent after stroke. Therefore, it is important measures are taken to prevent it or complications arising from it especially in those with a hemorrhagic stroke.

A fixed 20:1 combination of cafedrine/theodrenaline increases cytosolic Ca2+ concentration in human tracheal epithelial cells via ryanodine receptor-mediated Ca2+ release

Mucociliary clearance is a pivotal physiological mechanism that protects the lung by cleaning the airways from pollution and colonization, thereby preventing infection. Ciliary function is influenced by various signal transduction cascades, and Ca2+ represents a key second messenger. A fixed 20:1 combination of cafedrine and theodrenaline has been widely used to treat perioperative hypotension and emergency hypotensive states since the 1960s; however, its effect on the intracellular Ca2+ concentration ([Ca2+]i) of respiratory epithelium remains unknown. Therefore, human tracheal epithelial cells were exposed to the clinically applied 20:1 mixture of cafedrine/theodrenaline and the individual substances separately. [Ca2+]i was assessed by FURA-2 340/380 fluorescence ratio. Pharmacological inhibitors were applied to elucidate relevant signal transduction cascades, and reverse transcription polymerase chain reaction (RT-PCR) was performed on murine tracheal epithelium to analyze ryanodine receptor (RyR) subtype expression. All three pharmacological preparations instantaneously induced a steep increase in [Ca2+]i that quickly returned to its baseline value despite the persistence of each substance. Peak [Ca2+]i following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone increased in a dose-dependent manner, with median effective concentrations of 0.35 mM (7.32 mM cafedrine and 0.35 mM theodrenaline), 3.14 mM, and 3.45 mM, respectively. When extracellular Ca2+ influx was inhibited using a Ca2+-free buffer solution, the peak [Ca2+]i following the administration of cafedrine alone and theodrenaline alone were reduced but not abolished. No alteration in [Ca2+]i compared with baseline [Ca2+]i was observed during β-adrenergic receptor inhibition. Depletion of caffeine-sensitive stores and inhibition of RyR, but not IP3 receptors, completely abolished any increase in [Ca2+]i. However, [Ca2+]i still increased following the depletion of mitochondrial Ca2+ stores using 2,4-dinitrophenol. RT-PCR revealed RyR-2 and RyR-3 expression on murine tracheal epithelium. Although our experiments showed that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone release Ca2+ from intracellular stores through mechanisms that are exclusively triggered by β-adrenergic receptor stimulation, which most probably lead to RyR activation, clinical plasma concentrations are considerably lower than those used in our experiments to elicit an increase in [Ca2+]i; therefore, further studies are needed to evaluate the ability of cafedrine/theodrenaline to alter mucociliary clearance in clinical practice.

Airway stem cell reconstitution by the transplantation of primary or pluripotent stem cell-derived basal cells.

Life-long reconstitution of a tissue's resident stem cell compartment with engrafted cells has the potential to durably replenish organ function. Here, we demonstrate the engraftment of the airway epithelial stem cell compartment via intra-airway transplantation of mouse or human primary and pluripotent stem cell (PSC)-derived airway basal cells (BCs). Murine primary or PSC-derived BCs transplanted into polidocanol-injured syngeneic recipients give rise for at least two years to progeny that stably display the morphologic, molecular, and functional phenotypes of airway epithelia. The engrafted basal-like cells retain extensive self-renewal potential, evident by the capacity to reconstitute the tracheal epithelium through seven generations of secondary transplantation. Using the same approach, human primary or PSC-derived BCs transplanted into NOD scid gamma (NSG) recipient mice similarly display multilineage airway epithelial differentiation invivo. Our results may provide a step toward potential future syngeneic cell-based therapy for patients with diseases resulting from airway epithelial cell damage or dysfunction.

Histological and immunohistochemical characterization of mucosa-associated lymphoid tissue and antigen-presenting cells in trachea and lung of cattle.

The presence of bronchus-associated lymphoid tissue (BALT) and its structural components has been described in different healthy animal species and in animals with diseases of the respiratory tract. In contrast to normal mammals, BALT is absent in healthy human adult lungs, but has been found in the lungs of children. The histological characteristics of organized mucosa-associated lymphoid tissue (MALT), its subsets of immune cells and their in situ distribution in the lung of healthy subadult and adult cattle shows close similarities with BALT in humans and other animal species such as sheep, horses and pigs. This study clearly demonstrates that organized MALT also occurs in the tracheal mucosa of cattle. The absence of tracheal MALT and BALT in calves suggest that these structures are not constitutive. In the mucosa of bovine trachea, bronchi and bronchioli, MHC II+ and CD11c+ dendritic cells (DCs) are located in the epithelium and in the lamina propria mucosae. These DCs are already present in calves soon after birth. Examination of tangential epithelial sheets shows that in the bovine tracheal epithelium, like in man and rat, a dense network of MHC II+ and CD11c+ DCs exists and that their number is considerably higher than in conventional transverse sections. In the bovine tracheal and bronchial epithelium, MHC II+ DCs are extending their dendrites towards the lumen indicating that these DCs possibly are involved in sampling of luminal antigens. The presence of significantly higher numbers of MHC II+ DCs in the tracheal and bronchial/bronchiolar mucosa of older cattle in than in calves possibly results from local stimulation with exogenous antigens during postnatal life. Detection of DCs expressing the costimulatory molecules CD80 and CD86 in calves and cattle suggests maturation of DCs, which is most likely induced by stimulation with exogenous antigens.