air liquid interface
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2022 ◽  
Vol 23 (2) ◽  
pp. 835
Author(s):  
Bang M. Tran ◽  
Samantha L. Grimley ◽  
Julie L. McAuley ◽  
Abderrahman Hachani ◽  
Linda Earnest ◽  
...  

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air–liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.


mSphere ◽  
2021 ◽  
Vol 6 (6) ◽  
Author(s):  
Rahul K. Nelli ◽  
Juan Carlos Mora-Díaz ◽  
Luis G. Giménez-Lirola

The neurotropic betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) primarily infects and replicates in the swine upper respiratory tract, causing vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study investigated the modulation of key early innate immune genes at the respiratory epithelia in vivo, on tracheal tissue sections from experimentally infected pigs, and in vitro , on air-liquid interface porcine respiratory cell cultures.


2021 ◽  
Author(s):  
Majlinda Lako ◽  
Robert M Jackson ◽  
Catherine F Hatton ◽  
Jarmila S Spegarova ◽  
Maria Georgiou ◽  
...  

Although tropism of SARS-CoV-2 for respiratory tract epithelial cells is well established, an open question is whether the conjunctival epithelium is also a target for SARS-CoV-2. Conjunctival epithelial cells, which express viral entry receptors ACE2 and TMPRSS2, constitute the largest exposed epithelium of the ocular surface tissue, and may represent a relevant viral entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of progenitor, basal and superficial epithelial cells and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA Seq, with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to SARS-CoV-2 genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-Kβ activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplants.


PLoS Biology ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. e3001065
Author(s):  
Vanessa Herder ◽  
Kieran Dee ◽  
Joanna K. Wojtus ◽  
Ilaria Epifano ◽  
Daniel Goldfarb ◽  
...  

The pandemic spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19), represents an ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41°C. Fever is an evolutionarily conserved host response to microbial infection that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 replication. Utilizing a three-dimensional (3D) air–liquid interface (ALI) model that closely mimics the natural tissue physiology of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. Respiratory tissue incubated at 40°C remained permissive to SARS-CoV-2 entry but refractory to viral transcription, leading to significantly reduced levels of viral RNA replication and apical shedding of infectious virus. We identify tissue temperature to play an important role in the differential regulation of epithelial host responses to SARS-CoV-2 infection that impact upon multiple pathways, including intracellular immune regulation, without disruption to general transcription or epithelium integrity. We present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication in respiratory epithelia. Our data identify an important role for tissue temperature in the epithelial restriction of SARS-CoV-2 independently of canonical interferon (IFN)-mediated antiviral immune defenses.


Author(s):  
Pei Thing Chang ◽  
Ismat Muhsin Baharuddin ◽  
Qi Hwa Ng ◽  
Guang Hui Teoh ◽  
Abdul Latif Ahmad ◽  
...  

Nanomaterials ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3225
Author(s):  
Maria João Bessa ◽  
Fátima Brandão ◽  
Paul H. B. Fokkens ◽  
Daan L. A. C. Leseman ◽  
A. John F. Boere ◽  
...  

Diverse industries have already incorporated within their production processes engineered nanoparticles (ENP), increasing the potential risk of worker inhalation exposure. In vitro models have been widely used to investigate ENP toxicity. Air–liquid interface (ALI) cell cultures have been emerging as a valuable alternative to submerged cultures as they are more representative of the inhalation exposure to airborne nano-sized particles. We compared the in vitro toxicity of four ENP used as raw materials in the advanced ceramics sector in human alveolar epithelial-like cells cultured under submerged or ALI conditions. Submerged cultures were exposed to ENP liquid suspensions or to aerosolised ENP at ALI. Toxicity was assessed by determining LDH release, WST-1 metabolisation and DNA damage. Overall, cells were more sensitive to ENP cytotoxic effects when cultured and exposed under ALI. No significant cytotoxicity was observed after 24 h exposure to ENP liquid suspensions, although aerosolised ENP clearly affected cell viability and LDH release. In general, all ENP increased primary DNA damage regardless of the exposure mode, where an increase in DNA strand-breaks was only detected under submerged conditions. Our data show that at relevant occupational concentrations, the selected ENP exert mild toxicity to alveolar epithelial cells and exposure at ALI might be the most suitable choice when assessing ENP toxicity in respiratory models under realistic exposure conditions.


Nanomaterials ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3226
Author(s):  
Sivakumar Murugadoss ◽  
Sonja Mülhopt ◽  
Silvia Diabaté ◽  
Manosij Ghosh ◽  
Hanns-Rudolf Paur ◽  
...  

Extensive production and use of nanomaterials (NMs), such as titanium dioxide (TiO2), raises concern regarding their potential adverse effects to humans. While considerable efforts have been made to assess the safety of TiO2 NMs using in vitro and in vivo studies, results obtained to date are unreliable, possibly due to the dynamic agglomeration behavior of TiO2 NMs. Moreover, agglomerates are of prime importance in occupational exposure scenarios, but their toxicological relevance remains poorly understood. Therefore, the aim of this study was to investigate the potential pulmonary effects induced by TiO2 agglomerates of different sizes at the air–liquid interface (ALI), which is more realistic in terms of inhalation exposure, and compare it to results previously obtained under submerged conditions. A nano-TiO2 (17 nm) and a non-nano TiO2 (117 nm) was selected for this study. Stable stock dispersions of small agglomerates and their respective larger counterparts of each TiO2 particles were prepared, and human bronchial epithelial (HBE) cells were exposed to different doses of aerosolized TiO2 agglomerates at the ALI. At the end of 4h exposure, cytotoxicity, glutathione depletion, and DNA damage were evaluated. Our results indicate that dose deposition and the toxic potential in HBE cells are influenced by agglomeration and exposure via the ALI induces different cellular responses than in submerged systems. We conclude that the agglomeration state is crucial in the assessment of pulmonary effects of NMs.


2021 ◽  
pp. 026119292110618
Author(s):  
Vivek Patel ◽  
Khalid Amin ◽  
David Allen ◽  
Lindsey Ukishima ◽  
Adam Wahab ◽  
...  

As non-animal alternatives gain acceptance, a need for harmonised testing strategies has emerged. Arguably the most physiologically-relevant model for assessing potential respiratory toxicants, that based on human precision-cut lung slices (hPCLS) has been utilised in many laboratories, but a variety of culture methodologies are employed. In this pilot study, combinations of three different hPCLS culture methods (dynamic organ roller culture (DOC), air–liquid interface (ALI) and submersion) and various media (based on E-199, DMEM/F12 and RPMI-1640) were compared. The hPCLS were assessed in terms of their viability and responsiveness to challenge. The endpoints selected to compare the medium–method (M–M) combinations, which included histological features and viability, were evaluated at day 14 (D14) and day 28 (D28); protein and adenylate kinase (AK) content, and cytokine response to immunostimulants (lipopolysaccharide (LPS) at 5 μg/ml; polyinosinic:polycytidylic acid (Poly I:C) at 15 μg/ml) were evaluated at D28 only. Based on the set of endpoints assessed at D28, it was clear that certain culture conditions significantly affected the hPCLS, with the tissue retaining more of its native features and functionality (in terms of cytokine response) in some of the M–M combinations tested more than others. This pilot study indicates that the use of appropriate M–M combinations can help maintain the health and functional responses of hPCLS, and highlights the need for the standardisation of culture conditions in order to facilitate effective inter-laboratory comparisons and encourage greater acceptance by the regulatory community.


PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259914
Author(s):  
Joana Candeias ◽  
Carsten B. Schmidt-Weber ◽  
Jeroen Buters

In real life, humans are exposed to whole pollen grains at the air epithelial barrier. We developed a system for in vitro dosing of whole pollen grains at the Air-Liquid Interface (ALI) and studied their effect on the immortalized human bronchial epithelial cell line BEAS-2B. Pollen are sticky and large particles. Dosing pollen needs resuspension of single particles rather than clusters, and subsequent transportation to the cells with little loss to the walls of the instrumentation i.e. in a straight line. To avoid high speed impacting insults to cells we chose sedimentation by gravity as a delivery step. Pollen was resuspended into single particles by pressured air. A pollen dispersion unit including PTFE coating of the walls and reduced air pressure limited impaction loss to the walls. The loss of pollen to the system was still about 40%. A linear dose effect curve resulted in 327-2834 pollen/cm2 (± 6.1%), the latter concentration being calculated as the amount deposited on epithelial cells on high pollen days. After whole pollen exposure, the largest differential gene expression at the transcriptomic level was late, about 7 hours after exposure. Inflammatory and response to stimulus related genes were up-regulated. We developed a whole pollen exposure air-liquid interface system (Pollen-ALI), in which cells can be gently and reliably dosed.


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