Probes for INS

Despite the development and deployment of antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. The recent emergence of B.1.1.529, the Omicron variant1,2, which has more than 30 mutations in the spike protein, has raised concerns for escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in pre-clinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) program of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of multiple B.1.1.529 Omicron isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2) expressing mice and hamsters. Despite modeling and binding data suggesting that B.1.1.529 spike can bind more avidly to murine ACE2, we observed attenuation of infection in 129, C57BL/6, and BALB/c mice as compared with previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. Although K18-hACE2 transgenic mice sustained infection in the lungs, these animals did not lose weight. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease, and pathology with B.1.1.529 also were milder compared to historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from multiple independent laboratories of the SAVE/NIAID network with several different B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

COVID-19 has been associated with cardiac injury and dysfunction. While both myocardial inflammatory cell infiltration and myocarditis with myocyte injury have been reported in patients with fatal COVID-19, clinical-pathologic correlations remain limited. The objective was to determine the relationships between cardiac pathological changes in patients dying from COVID-19 and cardiac infection by SARS-CoV-2, laboratory measurements, clinical features, and treatments. In a retrospective study, 41 consecutive autopsies of patients with fatal COVID-19 were analyzed for the associations between cardiac inflammation, myocarditis, cardiac infection by SARS-CoV-2, clinical features, laboratory measurements, and treatments. Cardiac infection was assessed by in situ hybridization and NanoString transcriptomic profiling. Cardiac infection by SARS-CoV-2 was present in 30/41 cases: virus+ with myocarditis (n = 4), virus+ without myocarditis (n = 26), and virus- without myocarditis (n = 11). In the cases with cardiac infection, SARS-CoV-2+ cells in the myocardium were rare, with a median density of 1 cell/cm2. Virus+ cases showed higher densities of myocardial CD68+ macrophages and CD3+ lymphocytes, as well as more electrocardiographic changes (23/27 vs 4/10; P = 0.01). Myocarditis was more prevalent with IL-6 blockade than with nonbiologic immunosuppression, primarily glucocorticoids (2/3 vs 0/14; P = 0.02). Overall, SARS-CoV-2 cardiac infection was less prevalent in patients treated with nonbiologic immunosuppression (7/14 vs 21/24; P = 0.02). Myocardial macrophage and lymphocyte densities overall were positively correlated with the duration of symptoms but not with underlying comorbidities. In summary, cardiac infection with SARS-CoV-2 is common among patients dying from COVID-19 but often with only rare infected cells. Cardiac infection by SARS-CoV-2 is associated with more cardiac inflammation and electrocardiographic changes. Nonbiologic immunosuppression is associated with lower incidences of myocarditis and cardiac infection by SARS-CoV-2.

Obesity is associated with deficits in reward which have been linked to compensatory overeating. The vagus nerve is a direct neural pathway that conveys post-ingestive feedback from the gut to the brain, including the reward regions, and vagal activation causes stereotypical reward behaviors. Chronic high fat (HF) feeding alters vagal signaling potentially dampening food-associated reward. Microbiota composition changes rapidly with HF feeding, and a HF-type microbiota is sufficient to alter vagal structure and function. However, whether microbiota-driven alterations in vagal signaling affect host appetitive feeding behavior is unknown. Here, we investigate if microbiota composition modulates reward signaling and assess the role of the vagus in mediating microbiota to brain communication. Male germ-free Fisher rats were colonized with gastrointestinal contents from chow (low fat (LF) ConvLF) or HF (ConvHF) fed rats. Following colonization, ConvHF rats consumed significantly more food than ConvLF animals. ConvHF rats displayed lower feeding-induced extracellular DOPAC levels (a metabolite of dopamine) in the Nucleus Accumbens (NAc) as well as reduced motivation for HF foods compared to ConvLF rats. Dopamine receptor 2 (DDR2) expression levels in the NAc were also significantly lower in ConvHF animals. Similar deficits were observed in conventionally raised HF fed rats, showing that diet-driven alteration in reward can be initiated via microbiota. Selective gut to brain deafferentation restored DOPAC levels, DRD2 expression, and motivational drive in ConvHF rats. We concluded from these data that a HF-type microbiota is sufficient to alter appetitive feeding behavior and that bacteria to reward communication is mediated by the vagus nerve.

The disease severity of coronavirus disease 2019 (COVID-19) varies considerably from asymptomatic to serious, with fatal complications associated with dysregulation of innate and adaptive immunity. Lymphoid depletion in lymphoid tissues and lymphocytopenia have both been associated with poor disease outcomes in patients with COVID-19, but the mechanisms involved remain elusive. In this study, human angiotensin-converting enzyme 2 (hACE2) transgenic mouse models susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were used to investigate the characteristics and determinants of lethality associated with the lymphoid depletion observed in SARS-CoV-2 infection. The lethality of Wuhan SARS-CoV-2 infection in K18-hACE2 mice was characterized by severe lymphoid depletion and apoptosis in lymphoid tissues related to fatal neuroinvasion. The lymphoid depletion was associated with a decreased number of antigen-presenting cells (APCs) and their suppressed functionality below basal levels. Lymphoid depletion with reduced APC function was a specific feature observed in SARS-CoV-2 infection but not in influenza A infection and had the greatest prognostic value for disease severity in murine COVID-19. Comparison of transgenic mouse models resistant and susceptible to SARS-CoV-2 infection revealed that suppressed APC function could be determined by the hACE2 expression pattern and interferon-related signaling. Thus, we demonstrated that lymphoid depletion associated with suppressed APC function characterizes the lethality of COVID-19 mouse models. Our data also suggest a potential therapeutic approach to prevent the severe progression of COVID-19 by enhancing APC functionality.

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson's disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

Kikuchi Fujimoto Disease (KFD) is a rare form of localized lymphadenopathy, commonly affecting young Asian females with a self-limited course. The immunopathogenic mechanisms underlying KFD are still not well understood. KFD and systemic lupus erythematosus (SLE) share several histologic and clinical features, thus posing a diagnostic challenge. The aim of this study was to elucidate the in-situ distribution of immune cells and the cytokine/chemokine milieu of KFD utilizing immunohistochemistry to identify key cellular elements and RNAscope to assess cytokine and chemokine production. This study further compared the clinical, morphologic, and immunologic features of KFD to SLE.18 KFD, 16 SLE and 3 reactive lymph nodes were included. In contrast to KFD and reactive lymph nodes, SLE patients frequently exhibited generalized lymphadenopathy and had significantly higher frequency of systemic manifestations. Both KFD and SLE lymph nodes revealed overlapping morphologic findings with few distinguishing features namely the presence of capsular fibrosis and plasmacytosis in SLE and predominance of CD8-positive T cells in KFD.RNAscope studies in the KFD cohort revealed significantly higher amounts of interferon γ (IFN-γ), CXCL9 and CXCL10 in comparison to the SLE and reactive lymph nodes. These findings suggest a T-helper cell 1 (Th1) response, driven by IFN-γ and IFN-γ induced CXCL9 and CXCL10, is pivotal in the pathogenesis of KFD  and is less evident in lymph nodes from SLE patients. Distinguishing histological features between KFD and SLE are subtle. Studying the cytokine/chemokine environment provides valuable insight into the pathophysiology of KFD. In addition, assessing the production of these cytokines/chemokines may provide further diagnostic help in differentiating KFD from SLE.

Introduction: COVID-19 (Coronavirus disease 2019) appeared in Wuhan, China, at the ending of 2019. The SARS-CoV-2 virus which causes the illness has spread all over the world and caused a pandemic. The first target of the virus is the respiratory tract; however, the COVID-19 may present different types of course. It is known that the SARS-CoV-2 affects multiple organs, including the heart. Cardiac manifestations of COVID-19 include myocarditis, myocardial infarction, heart failure, acute coronar... Morey syndrome, arrhythmia. The authors know about the patients who had only cardiovascular complications due to the COVID-19. Several mechanisms of heart injury are considered and so is the direct infection. Aim of the study: The present review aimed to find out if the SARS-CoV-2 may infect the heart directly and in which mechanism. The review is an information collection considering the SARS-CoV-2 impact on the heart. Material and methods: The authors have made research using the PubMed search engine to find studies and case reports considering the cardiovascular implications of COVID-19. The signs and symptoms in patients with cardiac implications were studied. The authors have also checked if studies explaining does the SARS-CoV-2 affects the heart directly were conducted. Results: SARS-CoV-2 brings several cardiovascular signs such as changes in imaging tests and elevation of several laboratory markers. The changes may suggest myocarditis or mimic cardiac infarction. The SARS-CoV-2 may affect cardiomyocytes indirectly by causing hypoxia and cytokine storm. As the heart tissue presents a high level of ACE2 which is the target of the virus, the SARS-CoV may infect cardiomyocytes directly. The hypothesis was confirmed in endomyocardial biopsies, autopsy, and in vitro studies. Conclusions: The SARS-CoV-2 impacts several organs. The heart may be injured indirectly (hypoxia and cytokine storm) and directly (ACE2 present in the heart), which gives consequences in a clinical course. The direct injury was confirmed in a variety of ways. Less

SARS-CoV-2 is a single-stranded positive-sense RNA virus. Several negative-sense SARS-CoV-2 RNA species, both full-length genomic and subgenomic, are produced transiently during viral replication. Methodologies for rigorously characterising cell tropism and visualising ongoing viral replication at single-cell resolution in histological sections are needed to assess the virological and pathological phenotypes of future SARS-CoV-2 variants. We aimed to provide a robust methodology for examining the human lung, the major target organ of this RNA virus.A prospective cohort study took place at the University Hospitals Leuven in Leuven, Belgium. Lung samples were procured postmortem from 22 patients who died from or with COVID-19. Tissue sections were fluorescently stained with the ultrasensitive single-molecule RNA in situ hybridisation platform of RNAscope combined with immunohistochemistry followed by confocal imaging.We visualised perinuclear RNAscope signal for negative-sense SARS-CoV-2 RNA species in ciliated cells of the bronchiolar epithelium of a patient who died with COVID-19 in the hyperacute phase of the infection, and in ciliated cells of a primary culture of human airway epithelium that had been infected experimentally with SARS-CoV-2. In patients who died between 5 and 13 days after diagnosis of the infection, we detected RNAscope signal for positive-sense but not for negative-sense SARS-CoV-2 RNA species in pneumocytes, macrophages, and among debris in the alveoli. SARS-CoV-2 RNA levels decreased after a disease course of 2-3 weeks, concomitant with a histopathological change from exudative to fibroproliferative diffuse alveolar damage. Taken together, our confocal images illustrate the complexities stemming from traditional approaches in the literature to characterise cell tropism and visualise ongoing viral replication solely by the surrogate parameters of nucleocapsid-immunoreactive signal or in situ hybridisation for positive-sense SARS-CoV-2 RNA species.Confocal imaging of human lung sections stained fluorescently with commercially available RNAscope probes for negative-sense SARS-CoV-2 RNA species enables the visualisation of viral replication at single-cell resolution during the acute phase of the infection in COVID-19. This methodology will be valuable for research on future SARS-CoV-2 variants and other respiratory viruses.Max Planck Society, Coronafonds UZ/KU Leuven, European Society for Organ Transplantation.

Objectives: Up to 20% and 15% of critically ill influenza and coronavirus disease 2019 (COVID-19) patients are affected by influenza- and COVID-19-associated pulmonary aspergillosis (IAPA and CAPA) respectively. These viral-fungal coinfections are difficult to diagnose and are associated with increased mortality. Mechanistic insights into the development of IAPA and CAPA are a prerequisite for the development of new biomarkers and novel immunomodulatory therapeutic targets. However, data on the pathophysiology are scarce. With this study, we aimed at expanding our knowledge of IAPA and CAPA pathophysiology in an explorative way, resorting to lower respiratory tract samples and focusing on the epithelial and myeloid innate immunity components of the antifungal host response. Methods: We performed nCounter gene expression analyses of 755 genes linked to innate immunity, and determined protein levels of 47 cytokines, chemokines, growth factors, and other inflammatory mediators on bronchoalveolar lavage (BAL) fluid samples from 166 ICU-admitted influenza and COVID-19-patients with or without aspergillosis. Additionally, we performed spatial transcriptomics and RNAscope on in vivo tracheobronchial biopsies from four IAPA and CAPA patients. Results: Several genes encoding proteins with important effector functions in antifungal immunity are downregulated in BAL fluid of IAPA and CAPA patients compared with influenza-only or COVID-19-only patients. Cellular deconvolution of the gene expression data reveals a significantly lower BAL neutrophil fraction in CAPA patients compared to COVID-19-only patients. IAPA and CAPA patients have high BAL fluid levels of pro-inflammatory cytokines, but these are not significantly different from the levels seen in influenza-only and COVID-19-only patients. By integrating the BAL fluid cytokine levels with their respective transcriptional responses, we show that IAPA patients, and to a lesser extent CAPA patients, have an aberrant transcriptional response to pro-inflammatory cytokines as well as type I and type II interferons, which may result in poor cellular effector functions (Fig. 1a). Interferon-gamma signaling is abrogated in both IAPA and CAPA patients when compared with influenza-only and COVID-19-only patients. We observe significantly higher levels of growth factors associated with lung fibrosis in both IAPA and CAPA BAL fluid, which may contribute to the higher mortality seen in these coinfections (Fig. 1b). Using spatial transcriptomics, we show that different epithelial defense mechanisms are at play in IAPA and CAPA (Fig. 2a). Finally, using RNAscope ultrasensitive single-molecule RNA in situ hybridization, we visualize fungal and viral colocalization in CAPA tracheobronchial tissue, proving that virus-induced epithelial barrier disruption paves the way for tissueinvasive aspergillosis (Fig. 2b). Conclusion: Using state-of-the-art techniques in lower respiratory tract samples obtained from a large representative patient cohort, we provide arguments for a three-level breach in antifungal immunity in IAPA and CAPA. A hampered ability to phagocytize and kill fungal spores enables Aspergillus germination and growth, leading to hyphae that are not contained because of restrained extracellular defense mechanisms. These hyphae may easily become tissue-invasive through an epithelium that is weakened by the viral infection, causing detrimental damage to the respiratory system. Functional studies will be necessary to further unravel the pathophysiology of IAPA and CAPA.

Models of heart disease and drug responses are increasingly based on human pluripotent stem cells (hPSCs) since their ability to capture human heart (dys-)function is often better than animal models. Simple monolayer cultures of hPSC-derived cardiomyocytes, however, have shortcomings. Some of these can be overcome using more complex, multi cell-type models in 3D. Here we review modalities that address this, describe efforts to tailor readouts and sensors for monitoring tissue- and cell physiology (exogenously and in situ) and discuss perspectives for implementation in industry and academia.

Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver macrophages. However, also hepatocytes, the parenchymal cells of the liver, possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct anti-viral mechanisms employed by hepatocytes. METHODS: Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-?B signaling (IKK??Hep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-?/? signaling-(IFNAR?Hep), or interferon-?/? signaling in myeloid cells-(IFNAR?Myel) were infected. RESULTS: Here, we demonstrate that LCMV activates NF-?B signaling in hepatocytes. LCMV-triggered NF-?B activation in hepatocytes did not depend on Kupffer cells or TNFR1- but rather on TLR-signaling. LCMV-infected IKK??Hep livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8+ T-cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKK?, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IKK??Hep mice, enhanced hepatocytic LCMV accumulation was observed in livers of IFNAR?Hep, whereas IFNAR?Myel mice were able to control LCMV-infection. Hepatocytic NF-?B signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-?/?-mediated inhibition of HBV replication in vitro. CONCLUSIONS: Together, these data show that hepatocyte-intrinsic NF-?B is a vital amplifier of interferon-?/? signaling pivotal for early, strong ISG responses, influx of immune cells and hepatic viral clearance.