ABSTRACT
Purpose of Study: COVID pneumonia caused by SARS-CoV-2 can result in a depletion of surfactant & lung injury, which resembles neonatal respiratory distress syndrome. Exogenous surfactant has shown promise as a therapeutic option in intubated hospitalized patients. Our preliminary data in human lung organoids (LOs) with a deficiency of surfactant protein B (SP-B) showed an increased viral load compared to normal LOs. Single cell RNA sequencing (scRNAseq) revealed that SP-B-deficient cells showed increased viral entry genes (ACE2 receptor) & dysregulated inflammatory markers emanating from the lung cells themselves. Our objective was to determine: (1) cell-specific transcriptional differences between normal & SP-B deficient human lung cells after infection with SARS-CoV-2 and (2) a therapeutic role of SP-B protein & surfactant in COVID-19 pneumonia. Methods Used: We used normal and SP-B mutant (homozygous, frameshift, loss of function mutation p.Pro133GlnfsTer95, previously known as 121ins2) human induced pluripotent stem cells (hiPSC) and differentiated them into 3D proximal lung organoids. The organoids were infected with the delta variant of SARS-CoV-2 for 24 hours at an MOI of 1. Infected and uninfected organoids were fixed in trizol in triplicate and underwent processing for bulk RNA sequencing. We tested for differentially expressed genes using the program DEseq. We also plated normal iPSC derived lung organoids as a monolayer and pre-treated them with 1mg/ml of Poractant alfa or 5 uM of recombinant SP-B protein. The delta strain of SARS-CoV-2 was added to the 96 wells at an MOI of 0.1 for one hour with shaking, then an overlay with DMEM/CMC/FBS was added and left on for 23 hours. The plate was fixed and stained for nucleocapsid (NC) protein. Summary of Results: Bioinformatic analysis of the bulk RNA sequencing data showed an increase in the multiple cytokines and chemokines in the SP-B mutant LOs compared to control. We also saw differential gene expression patterns in the SP-B mutant LOs including a reduction in SFTPC, FOXA2, and NKX2-1 and an increase in IL1A, VEGFA, PPARG and SMAD3. In the exogenous surfactant experiments, there was a decrease in total expression of viral NC in the Poractant alfa & rSP-B-treated cells compared to SARS-CoV-2 infection alone (p<0.001). Conclusion(s): Surfactant modulates the viral load of SARS-CoV-2 infection in the human lung. Deficiency in SP-B results in the dysregulation of the lung epithelial inflammatory signaling pathways resulting in worsening infections.
ABSTRACT
Once believed to be sterile, recent studies now show microbes inhabiting healthy lungs that are dysregulated in patients with chronic obstructive pulmonary disease (COPD), asthma, tuberculosis (TB), and SARS-CoV-2 infection. Other studies have shown an increase in pulmonary disease and recurrent respiratory infections in malnourished patients. According to the World Health Organization, vitamin A deficiency (VAD) is now a major public health issue in low-income communities and many developing countries. While VAD has been shown to alter gene expression and tissue morphology in humans and mice, research suggests the lung microbiome plays an intimate role in the metabolic regulation, pathogen inhibition, and inflammatory responses in the lung. Whether dysbiosis is a cause or consequence of chronic respiratory conditions, or whether retinoic acid (RA) - the bioactive metabolite of Vitamin A - is essential for lung microbiome homeostasis, remains unknown. Therefore, we hypothesize that dietary VAD leads to epithelial remodeling which promotes microbial dysbiosis;the dysbiosis then perpetuates epithelial remodeling via host-microbe interactions. Our preliminary results show anatomical/pathological changes to the epithelium in VAD adult mouse lungs compared to controls (VAS). Using our Nkx2- 1creERT2/dnRAR Rosa26 tdTomato transgenic mouse model that selectively induces VAD in the adult lung epithelium following tamoxifen injections, our data supports the hypothesis that host epithelial aberration associated with dietary VAD is induced locally in the lung and not via distal or systemic mechanisms. Our data also indicates the onset of dysbiosis in adult mouse lungs as early as three weeks post-diet modulation as observed through changes in microbial composition in VAD mice compared to controls. Finally, our bulk RNAseq analysis of host and microbial gene signatures has uncovered mechanisms associated with microbial metabolic functions, ciliopathy, host cellular polarity, and immune response to infection, that are dysregulated in the absence of vitamin A. Further, we have also identified altered transcriptional activity of microbes that are traditionally symbiotic or pathobiotic under normal homeostasis. This work indicates the presence of specific host-microbe interactions that are essential for lung homeostasis and protection against lung infection and disease that are dysregulated or lost in the absence of dietary vitamin A.
ABSTRACT
Background: Neuroendocrine cancer of the prostate can present in diverse clinical pictures, potentially hampering the diagnosis and probably leading to underdiagnosis. Methods: Two cases are presented corresponding respectively to two forms of the disease: de novo neuroendocrine cancer and dedifferenciation of an adenocarcinoma of the prostate to neuroendocrine cancer under long term luteinising hormone releasing hormone (LHRH) agonist treatment. Results: Suspicion of neuroendocrine cancer may be raised in prostate cancer patients presenting either clinical or radiological metastatic progression without prostate specific antigen (PSA) rise, or relatively extended metastatic disease right at diagnosis associated to relatively low PSA, yet any non-pulmonary visceral metastases. Neuroendocrine cancer of the prostate can also turn out to be the origin of an adenocarcinoma of unknown primary. Conclusion: In case these considerations are respected the risk of missing the correct diagnosis of a neuroendocrine cancer of the prostate may be minimised.