Protocol for primary human lung organoid-derived air-liquid interface in vitro model to study response to SARS-CoV-2 (preprint)

This article presents a comprehensive protocol for establishing primary human lung organoid-derived air-liquid interface (ALI) cultures from cryopreserved human lung tissue. These cultures serve as a physiologically relevant model to study human airway epithelium in vitro. The protocol encompasses lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. It also demonstrates SARS-CoV-2 infection in these cultures as an example of their utility. Quality control steps, ALI characterization, and technical readouts for monitoring virus response are included in the study. For additional details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (https://doi.org/10.1016/j.isci.2023.107374).

Spatiotemporally organized immunomodulatory response to SARS-CoV-2 virus in primary human broncho-alveolar epithelia (preprint)

The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression towards severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4-6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an early inflammatory/immune signature preceding a late type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.

‘Psychoanalysis is one more way of taking people seriously’: Adam Phillips in conversation with Emma Williams

Adam Phillips is a leading psychoanalyst and author. Phillips was educated at Clifton College and studied English Literature at Oxford University. He trained to be a psychoanalyst at the Institute of Child Psychology. Across the course of his professional career, he has worked at Guys Hospital, with a school for ‘maladjusted children’, at Camberwell Child Guidance Clinic and at Charing Cross Hospital in the Department of Child Psychiatry. He now works in private practice. Phillips is the author of many works, including Terrors and Experts (1997), In Writing: Essays on Literature (2016), Attention Seeking (2019) and his most recent book, The Cure for Psychoanalysis (2021). He also served as the General Editor of the New Penguin Classics Translations of the works of Sigmund Freud.The conversation begins by exploring the way mental health has become a topic of public interest as a result of the COVID‐19 pandemic. The opportunities and challenges in Phillips's experience working with schools and for young people's mental health services during the 1970s, 1980s and 1990s are then discussed. Questions about the nature of psychoanalysis are introduced, and the discussion turns towards the relationship between philosophy, literature and psychoanalysis. There is a brief discussion of connections between Phillips's work and the philosophy of Stanley Cavell. Phillips's essays on schools and education are explored in connection with ideas of omniscience, sadomasochism and ‘experiments in living’. The conversation ends with a glimpse of school as a place to cultivate one's interest and one's sociability with others.

Single-cell longitudinal analysis of SARS-CoV-2 infection in human bronchial epithelial cells (preprint)

SARS-CoV-2, the causative agent of COVID-19, has tragically burdened individuals and institutions around the world. There are currently no approved drugs or vaccines for the treatment or prevention of COVID-19. Enhanced understanding of SARS-CoV-2 infection and pathogenesis is critical for the development of therapeutics. To reveal insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2 we performed single-cell RNA sequencing of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface cultures over a time-course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target of infection, which we confirmed by electron microscopy. Over the course of infection, cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III IFNs and IL6 but not IL1. This results in expression of interferon-stimulated genes in both infected and bystander cells. We observe similar gene expression changes from a COVID-19 patient ex vivo. In addition, we developed a new computational method termed CONditional DENSity Embedding (CONDENSE) to characterize and compare temporal gene dynamics in response to infection, which revealed genes relating to endothelin, angiogenesis, interferon, and inflammation-causing signaling pathways. In this study, we conducted an in-depth analysis of SARS-CoV-2 infection in HBECs and a COVID-19 patient and revealed genes, cell types, and cell state changes associated with infection.