Long-Term Sequelae of COVID-19 in Experimental Mice.

We recently reported acute COVID-19 symptoms, clinical status, weight loss, multi-organ pathological changes, and animal death in a murine hepatitis virus-1 (MHV-1) coronavirus mouse model of COVID-19, which were similar to that observed in humans with COVID-19. We further examined long-term (12 months post-infection) sequelae of COVID-19 in these mice. Congested blood vessels, perivascular cavitation, pericellular halos, vacuolation of neuropils, pyknotic nuclei, acute eosinophilic necrosis, necrotic neurons with fragmented nuclei, and vacuolation were observed in the brain cortex 12 months post-MHV-1 infection. These changes were associated with increased reactive astrocytes and microglia, hyperphosphorylated TDP-43 and tau, and a decrease in synaptic protein synaptophysin-1, suggesting the possible long-term impact of SARS-CoV-2 infection on defective neuronal integrity. The lungs showed severe inflammation, bronchiolar airway wall thickening due to fibrotic remodeling, bronchioles with increased numbers of goblet cells in the epithelial lining, and bronchiole walls with increased numbers of inflammatory cells. Hearts showed severe interstitial edema, vascular congestion and dilation, nucleated red blood cells (RBCs), RBCs infiltrating between degenerative myocardial fibers, inflammatory cells and apoptotic bodies and acute myocyte necrosis, hypertrophy, and fibrosis. Long-term changes in the liver and kidney were less severe than those observed in the acute phase. Noteworthy, the treatment of infected mice with a small molecule synthetic peptide which prevents the binding of spike protein to its respective receptors significantly attenuated disease progression, as well as the pathological changes observed post-long-term infection. Collectively, these findings suggest that COVID-19 may result in long-term, irreversible changes predominantly in the brain, lung, and heart.

Mechanism of Multi-Organ Injury in Experimental COVID-19 and Its Inhibition by a Small Molecule Peptide.

Severe disease from SARS-CoV-2 infection often progresses to multi-organ failure and results in an increased mortality rate amongst these patients. However, underlying mechanisms of SARS- CoV-2-induced multi-organ failure and subsequent death are still largely unknown. Cytokine storm, increased levels of inflammatory mediators, endothelial dysfunction, coagulation abnormalities, and infiltration of inflammatory cells into the organs contribute to the pathogenesis of COVID-19. One potential consequence of immune/inflammatory events is the acute progression of generalized edema, which may lead to death. We, therefore, examined the involvement of water channels in the development of edema in multiple organs and their contribution to organ dysfunction in a Murine Hepatitis Virus-1 (MHV-1) mouse model of COVID-19. Using this model, we recently reported multi-organ pathological abnormalities and animal death similar to that reported in humans with SARS-CoV-2 infection. We now identified an alteration in protein levels of AQPs 1, 4, 5, and 8 and associated oxidative stress, along with various degrees of tissue edema in multiple organs, which correlate well with animal survival post-MHV-1 infection. Furthermore, our newly created drug (a 15 amino acid synthetic peptide, known as SPIKENET) that was designed to prevent the binding of spike glycoproteins with their receptor(s), angiotensin- converting enzyme 2 (ACE2), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) (SARS-CoV-2 and MHV-1, respectively), ameliorated animal death and reversed altered levels of AQPs and oxidative stress post-MHV-1 infection. Collectively, our findings suggest the possible involvement of altered aquaporins and the subsequent edema, likely mediated by the virus-induced inflammatory and oxidative stress response, in the pathogenesis of COVID- 19 and the potential of SPIKENET as a therapeutic option.

Multi-Organ Histopathological Changes in a Mouse Hepatitis Virus Model of COVID-19.

Infection with SARS-CoV-2, the virus responsible for the global COVID-19 pandemic, causes a respiratory illness that can severely impact other organ systems and is possibly precipitated by cytokine storm, septic shock, thrombosis, and oxidative stress. SARS-CoV-2 infected individuals may be asymptomatic or may experience mild, moderate, or severe symptoms with or without pneumonia. The mechanisms by which SARS-CoV-2 infects humans are largely unknown. Mouse hepatitis virus 1 (MHV-1)-induced infection was used as a highly relevant surrogate animal model for this study. We further characterized this animal model and compared it with SARS-CoV-2 infection in humans. MHV-1 inoculated mice displayed death as well as weight loss, as reported earlier. We showed that MHV-1-infected mice at days 7-8 exhibit severe lung inflammation, peribronchiolar interstitial infiltration, bronchiolar epithelial cell necrosis and intra-alveolar necrotic debris, alveolar exudation (surrounding alveolar walls have capillaries that are dilated and filled with red blood cells), mononuclear cell infiltration, hyaline membrane formation, the presence of hemosiderin-laden macrophages, and interstitial edema. When compared to uninfected mice, the infected mice showed severe liver vascular congestion, luminal thrombosis of portal and sinusoidal vessels, hepatocyte degeneration, cell necrosis, and hemorrhagic changes. Proximal and distal tubular necrosis, hemorrhage in interstitial tissue, and the vacuolation of renal tubules were observed. The heart showed severe interstitial edema, vascular congestion, and dilation, as well as red blood cell extravasation into the interstitium. Upon examination of the MHV-1 infected mice brain, we observed congested blood vessels, perivascular cavitation, cortical pericellular halos, vacuolation of neuropils, darkly stained nuclei, pyknotic nuclei, and associated vacuolation of the neuropil in the cortex, as well as acute eosinophilic necrosis and necrotic neurons with fragmented nuclei and vacuolation in the hippocampus. Our findings suggest that the widespread thrombotic events observed in the surrogate animal model for SARS-CoV-2 mimic the reported findings in SARS-CoV-2 infected humans, representing a highly relevant and safe animal model for the study of the pathophysiologic mechanisms of SARS-CoV-2 for potential therapeutic interventions.

Experimental Models of COVID-19.

COVID-19 is the most consequential pandemic of the 21st century. Since the earliest stage of the 2019-2020 epidemic, animal models have been useful in understanding the etiopathogenesis of SARS-CoV-2 infection and rapid development of vaccines/drugs to prevent, treat or eradicate SARS-CoV-2 infection. Early SARS-CoV-1 research using immortalized in-vitro cell lines have aided in understanding different cells and receptors needed for SARS-CoV-2 infection and, due to their ability to be easily manipulated, continue to broaden our understanding of COVID-19 disease in in-vivo models. The scientific community determined animal models as the most useful models which could demonstrate viral infection, replication, transmission, and spectrum of illness as seen in human populations. Until now, there have not been well-described animal models of SARS-CoV-2 infection although transgenic mouse models (i.e. mice with humanized ACE2 receptors with humanized receptors) have been proposed. Additionally, there are only limited facilities (Biosafety level 3 laboratories) available to contribute research to aid in eventually exterminating SARS-CoV-2 infection around the world. This review summarizes the most successful animal models of SARS-CoV-2 infection including studies in Non-Human Primates (NHPs) which were found to be susceptible to infection and transmitted the virus similarly to humans (e.g., Rhesus macaques, Cynomolgus, and African Green Monkeys), and animal models that do not require Biosafety level 3 laboratories (e.g., Mouse Hepatitis Virus models of COVID-19, Ferret model, Syrian Hamster model). Balancing safety, mimicking human COVID-19 and robustness of the animal model, the Murine Hepatitis Virus-1 Murine model currently represents the most optimal model for SARS-CoV-2/COVID19 research. Exploring future animal models will aid researchers/scientists in discovering the mechanisms of SARS-CoV-2 infection and in identifying therapies to prevent or treat COVID-19.

We Could Use More Tolerance: Role of Intestinal-Allograft-Derived Human Stem Cells.

Mixed chimerism is associated with allograft acceptance and tolerance. In this issue of Cell Stem Cell, Fu et al. (2019) provide evidence that functional, donor-derived hematopoietic stem and progenitor cells in the intestinal allograft can persist long-term, contribute to multi-lineage chimerism in the circulation, and result in T cell tolerance through host lymphoid organ selection.

Mesenchymal stem cell labeling and in vitro MR characterization at 1.5 T of new SPIO contrast agent: Molday ION Rhodamine-B™.

In vivo detection of transplanted stem cells is requisite for improving stem cell-based treatments by developing a thorough understanding of their therapeutic mechanisms. MRI tracking of magnetically labeled cells is non-invasive and is suitable for longitudinal studies. Molday ION Rhodamine-B™ (MIRB) is a new superparamagnetic iron oxide (SPIO) contrast agent specifically formulated for cell labeling and is readily internalized by non-phagocytic cells. This investigation characterizes mesenchymal stem cell (MSC) labeling and MR imaging properties of this new SPIO agent. Effects of MIRB on MSC viability and differentiation as well as cellular loading properties were assessed for MSC labeled with MIRB at concentrations from 5 to 100 µg Fe/ml. Labeled MSC were evaluated, in vitro, on a clinical 1.5 T MRI. Optimal scanning sequences and imaging parameters were determined based on contrast-to-noise ratio and contrast modulation. Relaxation rates (1/T(2)*) for gradient-echo sequences were approximated and an idealized limit of detection was established. MIRB labeling did not affect MSC viability or the ability to differentiate into either bone or fat. Labeling efficiency was found to be approximately 95% for labeling concentrations at or above 20 µg Fe/ml. Average MIRB per MSC ranged from 0.7 pg Fe for labeling MIRB concentration of 5 µg Fe/ml and asymptotically approached a value of 20-25 pg Fe/MSC as labeling concentration increased to 100 µg Fe/ml. MRI analysis of MIRB MSC revealed long echo time, gradient echo sequences to provide the most sensitivity. Limit of detection for gradient echo sequences was determined to be less than 1000 MSC, with approximately 15 pg Fe/MSC (labeled at 20 µg Fe/ml). These investigations have laid the groundwork and established feasibility for the use of this contrast agent for in vivo MRI detection of MSC. Properties evaluated in this study will be used as a reference for tracking labeled MSC for in vivo studies.

Loss of IL-4 secretion from human type 1a diabetic pancreatic draining lymph node NKT cells.

Altered frequency and function of peripheral invariant NKT (iNKT) cells have been implicated in the regulation of murine and human type 1a diabetes. To examine regulatory cells from the site of drainage of autoinflammatory tissue and autoantigenic T cell priming in diabetes, we directly cloned iNKT cells from human pancreatic draining lymph nodes (PLN). From 451 T cell clones from control and diabetic PLN, we derived 55 iNKT cells by two methods and analyzed function by cytokine secretion. iNKT cell clones isolated from control PLN secreted IL-4 and IFN-gamma upon TCR stimulation. For type 1a diabetic subjects, PLN iNKT cell clones from three samples secreted IFN-gamma and no IL-4. In a rare recent onset diabetic sample with islet-infiltrating CD4+ T cells, the phenotype of PLN iNKT cell clones was mixed. From normal and diabetic PLN, one-third of CD1d tetramer+-sorted T cell clones were reactive with CD1d transfectants or proliferated/secreted cytokine in response to alpha-galactosylceramide-pulsed PBMCs; tetramer-staining T cell clones from diabetic PLN did not secrete IL-4. This is the first report directly examining iNKT cells from lymph nodes draining the site of autoimmunological attack in humans; iNKT cells were altered in cytokine secretion as previously reported for circulating iNKT cells in human type 1a diabetes.

Expanded T cells from pancreatic lymph nodes of type 1 diabetic subjects recognize an insulin epitope.

In autoimmune type 1 diabetes, pathogenic T lymphocytes are associated with the specific destruction of insulin-producing beta-islet cells. Identification of the autoantigens involved in triggering this process is a central question. Here we examined T cells from pancreatic draining lymph nodes, the site of islet-cell-specific self-antigen presentation. We cloned single T cells in a non-biased manner from pancreatic draining lymph nodes of subjects with type 1 diabetes and from non-diabetic controls. A high degree of T-cell clonal expansion was observed in pancreatic lymph nodes from long-term diabetic patients but not from control subjects. The oligoclonally expanded T cells from diabetic subjects with DR4, a susceptibility allele for type 1 diabetes, recognized the insulin A 1-15 epitope restricted by DR4. These results identify insulin-reactive, clonally expanded T cells from the site of autoinflammatory drainage in long-term type 1 diabetics, indicating that insulin may indeed be the target antigen causing autoimmune diabetes.

Alternatives to immunosuppressive drugs in human islet transplantation.

Although intensive insulin therapy has resulted in improved metabolic control and decreases in the incidence of complications, the occurrence of severe hypoglycemia remains an issue, as does the continued potential for complications. Islet transplantation, a promising treatment for type I diabetes, has been shown to improve blood sugar levels and decrease or even abrogate the incidence of hypoglycemia. The lack of tissue availability and the toxic effects of immunosuppressants, however, limit the application of islet transplantation as a cure for diabetes. This article discusses possible alternatives to immunosuppressive drugs in human islet transplantations.