Probes for INS

RATIONALE: Patients with idiopathic pulmonary fibrosis (IPF) have worse outcomes following COVID-19. SARSCoV-2 (2019-nCoV) spike protein (S1) harbors an RGD motif in its receptor-binding domain (RBD). Although SARS-CoV-2 is to exploit human Angiotensin Converting Enzyme-2 (ACE2) receptors for cell entry. Single Cell RNA-seq showed that normal lung expresses low levels of ACE2 with very low expression (1.5%) in Alveolar type 2 epithelial cells. It is possible that SARS-CoV-2 needs a cellular co-receptor, which could include integrins, to promote alveolar cell internalization and pneumonitis.METHODS: Solid-phase binding assays were used to investigate S1 binding to ACE2 or αv containing integrins. Pseudovirus entry assays were used to measure the internalization of SARS-CoV-2 into Human embryonic kidney 293T cells expressing different combinations of potential receptors. RNAscope was used to visualize the co-localization of SARS-CoV-2, ACE2, and integrin mRNAs. Immunohistochemistry was used to evaluate the expression of αvβ6 integrins and ACE2 in lung tissue.RESULTS: Binding assays demonstrated that the RGD containing αvβ3 and αvβ6 integrins bound robustly to the SARS-CoV-2 S1 subunit of Spike protein and overexpression of the αvβ6 integrin modestly augments ACE2 mediated SARS-CoV-2 pseudoviral entry into epithelial cells. In COVID-19 damaged lung ACE2 levels are low but the αvβ6 integrin levels are increased in alveolar epithelium whereas both ACE2 and αvβ6 integrin are increased in lung sections from idiopathic pulmonary fibrosis compared with normal lung samples. CONCLUSION: The SARS-CoV-2 S1 subunit can bind αvβ6 integrins augmenting ACE2-dependent internalization of pseudovirus. In IPF patients, ACE2 levels and αvβ6 integrin levels are increased. Increased binding of the SARS-CoV-2 to ACE2 and the αvβ6 integrin within fibrotic lung may explain the increased risk of severe COVID in patients with IPF.

SARS-CoV-2, the etiological agent of COVID-19, causes life-threatening disease. This novel coronavirus enters host cells via the respiratory tract, promoting the formation of severe pulmonary lesions and systemic disease. Few animal models can simulate the clinical signs and pathology of COVID-19 patients. Diverse preclinical studies using K18-hACE2 mice and Syrian golden hamsters, which are highly permissive to SARS-CoV-2 in the respiratory tract, are emerging; however, the systemic pathogenesis and cellular tropism of these models remain obscure. We intranasally infected K18-hACE2 mice and Syrian golden hamsters with SARS-CoV-2, and compared the clinical features, pathogenesis, cellular tropism, and infiltrated immune-cell subsets. In K18-hACE2 mice, SARS-CoV-2 persistently replicated in alveolar cells and caused pulmonary and extra-pulmonary disease, resulting in fatal outcomes. Conversely, in Syrian golden hamsters, transient SARS-CoV-2 infection in bronchial cells caused reversible pulmonary disease, without mortality. Our findings provide comprehensive insights into the pathogenic spectrum of COVID-19 using pre-clinical models.

Sarcoidosis is an idiopathic inflammatory disorder that is commonly treated with glucocorticoids. An imprecise understanding of the immunologic changes underlying sarcoidosis has limited therapeutic progress. Here in this open-label trial (NCT03910543), 10 patients with cutaneous sarcoidosis are treated with tofacitinib, a Janus kinase inhibitor. The primary outcome is the change in the cutaneous sarcoidosis activity and morphology instrument (CSAMI) activity score after 6 months of treatment. Secondary outcomes included change in internal organ involvement, molecular parameters, and safety. All patients experience improvement in their skin with 6 patients showing a complete response. Improvement in internal organ involvement is also observed. CD4+ T cell-derived IFN-γ is identified as a central cytokine mediator of macrophage activation in sarcoidosis. Additional type 1 cytokines produced by distinct cell types, including IL-6, IL-12, IL-15 and GM-CSF, also associate with pathogenesis. Suppression of the activity of these cytokines, especially IFN-γ, correlates with clinical improvement. Our results thus show that tofacitinib treatment is associated with improved sarcoidosis symptoms, and predominantly acts by inhibiting type 1 immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has led to more than 3.25 million recorded deaths worldwide as of May 2021. COVID-19 is known to be clinically heterogeneous, and whether the reported oral signs and symptoms in COVID-19 are related to the direct infection of oral tissues has remained unknown. Here, we review and summarize the evidence for the primary infection of the glands, oral mucosae, and saliva by SARS-CoV-2. Not only were the entry factors for SARS-CoV-2 found in all oral tissues, but these were also sites of SARS-CoV-2 infection and replication. Furthermore, saliva from asymptomatic individuals contained free virus and SARS-CoV-2-infected oral epithelial cells, both of which were found to transmit the virus. Collectively, these studies support an active role of the oral cavity in the spread and transmission of SARS-CoV-2 infection. In addition to maintaining the appropriate use of personal protective equipment and regimens to limit microbial spread via aerosol or droplet generation, the dental community will also be involved in co-managing COVID-19 'long haulers'-now termed Post-Acute COVID-19 Syndrome. Consequently, we propose that, as SARS-CoV-2 continues to spread and as new clinical challenges related to COVID-19 are documented, oral symptoms should be included in diagnostic and prognostic classifications as well as plans for multidisciplinary care. This article is protected by

Complex tissues such as tumors are comprised of multiple cells types and extracellular matrix. These cells include heterogenous populations of immune cells that infiltrate the tumors. Understanding the composition of these immune infiltrates in the tumor microenvironment (TME) can provide key insights to guide therapeutic intervention and predict treatment response. Thorough understanding of complex tissue dynamics and immune cell characterization requires a multi-omics approach. Simultaneous detection of RNA and protein using in situ hybridization (ISH) and immunohistochemistry/immunofluorescence (IHC/IF) can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. However, a sequential workflow of ISH followed by IHC/IF frequently yields suboptimal protein detection because the protease digestion step in the ISH protocol resulting in poor antibody signal. Here we demonstrate a newly developed integrated ISH/IHC workflow that can substantially improve RNA-protein co-detection, enabling the visualization and characterization of tumor immune infiltrates at single-cell resolution with spatial and morphological context. To characterize tumor-infiltrating immune cells in a tumor TMA (tumor microarray), we utilized the RNAscope Multiplex Fluorescence assay in combination with the RNA-Protein Co-detection Kit to detect multiple immune cell populations. Immune cells such as macrophages, T cells and NK cells were detected using specific antibodies against CD68, CD8, CD4 and CD56, respectively. Precise characterization of these immune cells was achieved by using probes against targets such as CCL5, IFNG, GNZB, IL-12, NCR1 etc. that not only help in identifying specific immune cells but also assist in determining their activation states. We identified subsets of T cells such as CD4+ regulatory T cells and CD8+ cytotoxic T lymphocytes. Additionally, we were able to determine the activation states of CD8+ T cells by visualizing the expression of IFNG and GZMB. Furthermore, infiltrating macrophages were identified by detecting the CD68 protein expression while the M1 and M2 subsets were differentiated by detecting the M2-specific target RNA for CD163. Similarly, NK cells were identified by detecting CD56 protein in combination with CCL5 and NCR1 RNA expression. Interestingly, the degree of infiltration of the different immune cell populations varied based on the tumor type. In conclusion, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies - including many previously incompatible antibodies - with RNAscope. This new workflow provides a powerful new approach to identifying and characterizing tumor infiltrating populations of immune cells.

Chimeric antigen receptor (CAR) T cell therapy has limited efficacy against solid tumors, and one major challenge is T cell exhaustion. To address this challenge, we performed a candidate gene screen using a hypofunction CAR-T cell model and found that depletion of basic leucine zipper ATF-like transcription factor (BATF) improved the antitumor performance of CAR-T cells. In different types of CAR-T cells and mouse OT-1 cells, loss of BATF endows T cells with improved resistance to exhaustion and superior tumor eradication efficacy. Mechanistically, we found that BATF binds to and up-regulates a subset of exhaustion-related genes in human CAR-T cells. BATF regulates the expression of genes involved in development of effector and memory T cells, and knocking out BATF shifts the population toward a more central memory subset. We demonstrate that BATF is a key factor limiting CAR-T cell function and that its depletion enhances the antitumor activity of CAR-T cells against solid tumors.

Cutaneous leishmaniasis is a parasitic infection that causes significant maternal morbidity, and even fetal mortality, during pregnancy, yet there are limited therapeutic options. Here, we report a case of leishmaniasis in a pregnant immigrant with exuberant mucocutaneous lesions with favorable response to liposomal amphotericin B.