Probes for INS

Tissue stem cells are generated from a population of embryonic progenitors through organ-specific morphogenetic events1,2. Although tissue stem cells are central to organ homeostasis and regeneration, it remains unclear how they are induced during development, mainly because of the lack of markers that exclusively label prospective stem cells. Here we combine marker-independent long-term 3D live imaging and single-cell transcriptomics to capture a dynamic lineage progression and transcriptome changes in the entire epithelium of the mouse hair follicle as it develops. We found that the precursors of different epithelial lineages were aligned in a 2D concentric manner in the basal layer of the hair placode. Each concentric ring acquired unique transcriptomes and extended to form longitudinally aligned, 3D cylindrical compartments. Prospective bulge stem cells were derived from the peripheral ring of the placode basal layer, but not from suprabasal cells (as was previously suggested3). The fate of placode cells is determined by the cell position, rather than by the orientation of cell division. We also identified 13 gene clusters: the ensemble expression dynamics of these clusters drew the entire transcriptional landscape of epithelial lineage diversification, consistent with cell lineage data. Combining these findings with previous work on the development of appendages in insects4,5, we describe the 'telescope model', a generalized model for the development of ectodermal organs in which 2D concentric zones in the placode telescope out to form 3D longitudinally aligned cylindrical compartments.

The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global health emergency. While most human disease is mild to moderate, some infections lead to a severe disease characterized by acute respiratory distress, hypoxia, anosmia, ageusia, and, in some instances, neurological involvement. Small-animal models reproducing severe disease, including neurological sequela, are needed to characterize the pathophysiological mechanism(s) of disease and to identify medical countermeasures. Transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) viral receptor under the control of the K18 promoter develop severe and lethal respiratory disease subsequent to SARS-CoV-2 intranasal challenge when high viral doses are used. Here, we report on SARS-CoV-2 infection of hamsters engineered to express the hACE2 receptor under the control of the K18 promoter. K18-hACE2 hamsters infected with a relatively low dose of 100 or 1,000 PFU of SARS-CoV-2 developed a severe and lethal disease, with most animals succumbing by day 5 postinfection. Hamsters developed severe lesions and inflammation within the upper and lower respiratory system, including infection of the nasal cavities causing marked destruction of the olfactory epithelium as well as severe bronchopneumonia that extended deep into the alveoli. Additionally, SARS-CoV-2 infection spread to the central nervous system (CNS), including the brain stem and spinal cord. Wild-type (WT) hamsters naturally support SARS-CoV-2 infection, with the primary lesions present in the respiratory tract and nasal cavity. Overall, infection in the K18-hACE2 hamsters is more extensive than that in WT hamsters, with more CNS involvement and a lethal outcome. These findings demonstrate the K18-hACE2 hamster model will be valuable for studying SARS-CoV-2. IMPORTANCE The rapid emergence of SARS-CoV-2 has created a global health emergency. While most human SARS-CoV-2 disease is mild, some people develop severe, life-threatening disease. Small-animal models mimicking the severe aspects of human disease are needed to more clearly understand the pathophysiological processes driving this progression. Here, we studied SARS-CoV-2 infection in hamsters engineered to express the human angiotensin-converting enzyme 2 viral receptor under the control of the K18 promoter. SARS-CoV-2 produces a severe and lethal infection in transgenic hamsters that mirrors the most severe aspects of COVID-19 in humans, including respiratory and neurological injury. In contrast to other animal systems, hamsters manifest disease with levels of input virus more consistent with natural human infection. This system will be useful for the study of SARS-CoV-2 disease and the development of drugs targeting this virus.

Methods: In this proof-of-concept study, we performed bedside ultrasound-guided minimally invasive autopsies (US-MIA) of patients that had died from critical COVID-19 in the intensive care unit (ICU) using a structured protocol to obtain almost autolytic-free tissue. Biopsies were assessed for quality (vitality and length) and for diagnosis. The efficiency of the procedure was monitored in five cases by recording the time of each step and safety issues by swabbing personal protective equipment and devices for viral contamination. 

Coronavirus disease 2019 (COVID-19), initially appreciated as a respiratory illness, is now known to affect many organs in the human body. Significant data has become available on muscle involvement, with creatinine kinase elevations present in a significant percentage of patients. For those with suspected COVID-19-associated myositis, the imaging modality of choice has been gadolinium-enhanced magnetic resonance imaging; however, the use of technitium-99m bone scan has not been previously reported. Here, we report two cases of COVID-19 patients with severe elevation in creatinine kinase who underwent technitium-99m bone scan. The resulting images showed diffuse symmetrical muscle involvement. Both patients developed acute renal injury due to rhabdomyolysis. To our knowledge, this is the first report of bone scan as a diagnostic imaging modality for COVID-19-associated myositis.

The purpose of this review is to analyze the most recent and relevant literature involving lung transplantation for coronavirus disease 2019 (COVID-19) associated acute respiratory distress syndrome (ARDS), the pathological mechanisms of lung injury, selection criteria and outcomes.Pathological analysis of lungs after COVID-19 ARDS has shown architectural distortion similar to that observed in explanted lungs from patients undergoing lung transplantation for end-stage lung diseases such as emphysema. Short-term outcomes after lung transplantation for COVID-19 associated respiratory failure are comparable to those performed for other indications.Lung transplantation after COVID-19 ARDS is a potentially life-saving procedure for appropriately selected patients with no evidence of lung function recovery despite maximal treatment. Lung transplantation should be ideally performed in high-volume centers with expertise.

The effect of SARS-CoV-2 infection on placental function is not well understood. Analysis of placentas from women who tested positive at delivery showed SARS-CoV-2 genomic and subgenomic RNA in 22 out of 52 placentas. Placentas from two mothers with symptomatic COVID-19 whose pregnancies resulted in adverse outcomes for the fetuses contained high levels of viral Alpha variant RNA. The RNA was localized to the trophoblasts that cover the fetal chorionic villi in direct contact with maternal blood. The intervillous spaces and villi were infiltrated with maternal macrophages and T cells. Transcriptome analysis showed an increased expression of chemokines and pathways associated with viral infection and inflammation. Infection of placental cultures with live SARS-CoV-2 and spike protein-pseudotyped lentivirus showed infection of syncytiotrophoblast and, in rare cases, endothelial cells mediated by ACE2 and Neuropilin-1. Viruses with Alpha, Beta, and Delta variant spikes infected the placental cultures at significantly greater levels.

In some patients, COVID-19 can trigger neurological symptoms with unclear pathogenesis. Here we describe a microphysiological system integrating alveolus and blood-brain barrier (BBB) tissue chips that recapitulates neuropathogenesis associated with infection by SARS-CoV-2. Direct exposure of the BBB chip to SARS-CoV-2 caused mild changes to the BBB, and infusion of medium from the infected alveolus chip led to more severe injuries on the BBB chip, including endothelial dysfunction, pericyte detachment and neuroinflammation. Transcriptomic analyses indicated downregulated expression of the actin cytoskeleton in brain endothelium and upregulated expression of inflammatory genes in glial cells. We also observed early cerebral microvascular damage following lung infection with a low viral load in the brains of transgenic mice expressing human angiotensin-converting enzyme 2. Our findings suggest that systemic inflammation is probably contributing to neuropathogenesis following SARS-CoV-2 infection, and that direct viral neural invasion might not be a prerequisite for this neuropathogenesis. Lung-brain microphysiological systems should aid the further understanding of the systemic effects and neurological complications of viral infection.

The golden Syrian hamster (Mesocricetus auratus) is now commonly used in preclinical research for the study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the assessment of vaccines, drugs and therapeutics. Here, we show that hamsters inoculated via the intranasal route with the same infectious virus dose of prototypical SARS-CoV-2 administered in a different volume present with different clinical signs, weight loss and viral shedding, with a reduced volume resulting in reduced severity of disease similar to that obtained by a 500-fold reduction in the challenge dose. The tissue burden of the virus and the severity of pulmonary pathology were also significantly affected by different challenge inoculum volumes. These findings suggest that a direct comparison between the severity of SARS-CoV-2 variants or studies assessing the efficacy of treatments determined by hamster studies cannot be made unless both the challenge dose and inoculation volume are matched when using the intranasal route. Additionally, analysis of sub-genomic and total genomic RNA PCR data demonstrated no link between sub-genomic and live viral titres and that sub-genomic analyses do not provide any information beyond that provided by more sensitive total genomic PCR.

The COVID-19 pandemic is entering a new era with the approval of many SARS-CoV-2 vaccines. In spite of the restoration of an almost normal way of life thanks to the immune protection elicited by these innovative vaccines, we are still facing high viral circulation, with a significant number of deaths. To further explore alternative vaccination platforms, we developed COVID-eVax-a genetic vaccine based on plasmid DNA encoding the RBD domain of the SARS-CoV-2 spike protein. Here, we describe the correlation between immune responses and the evolution of viral infection in ferrets infected with the live virus. We demonstrate COVID-eVax immunogenicity as means of antibody response and, above all, a significant T-cell response, thus proving the critical role of T-cell immunity, in addition to the neutralizing antibody activity, in controlling viral spread.

The rapid global spread of severe acute respiratory coronavirus 2 (SARS-CoV-2) has resulted in an urgent effort to find efficacious therapeutics. Broad-spectrum therapies which could be used for other respiratory pathogens confer advantages, as do those based on targeting host cells that are not prone to the development of resistance by the pathogen. We tested an intranasally delivered carbohydrate-binding module (CBM) therapy, termed Neumifil, which is based on a CBM that has previously been shown to offer protection against the influenza virus through the binding of sialic acid receptors. Using the recognised hamster model of SARS-CoV-2 infection, we demonstrate that Neumifil significantly reduces clinical disease severity and pathological changes in the nasal cavity. Furthermore, we demonstrate Neumifil binding to the human angiotensin-converting enzyme 2 (ACE2) receptor and spike protein of SARS-CoV-2. This is the first report describing the testing of this type of broad-spectrum antiviral therapy in vivo and provides evidence for the advancement of Neumifil in further preclinical and clinical studies.

Animal models recapitulating COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Intranasally inoculated transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. We evaluated the clinical and virological dynamics of SARS-CoV-2 using two intranasal doses (104 and 106 PFUs), with a detailed spatiotemporal pathologic analysis of the 106 dose cohort. Despite generally mild-to-moderate pneumonia, clinical decline resulting in euthanasia or death was commonly associated with hypothermia and viral neurodissemination independent of inoculation dose. Neuroinvasion was first observed at 4 days post-infection, initially restricted to the olfactory bulb suggesting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. Absence of viremia suggests neuroinvasion occurs independently of transport across the blood-brain barrier. SARS-CoV-2 tropism was neither restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), nor inclusive of some ACE2-positive cell lineages (e.g., bronchiolar epithelium and brain vasculature). Absence of detectable ACE2 protein expression in neurons but overexpression in neuroepithelium suggest this as the most likely portal of neuroinvasion, with subsequent ACE2 independent lethal neurodissemination. A paucity of epidemiological data and contradicting evidence for neuroinvasion and neurodissemination in humans call into question the translational relevance of this model.