Probes for INS

Ameloblastoma (AB) is the most common benign, epithelial odontogenic tumor that occurs in the jawbone. AB is a slow-growing, benign epithelial tumor but shows locally invasive growth, with bone resorption or recurrence if not adequately resected. From these points of view, understanding the mechanism of AB-induced bone resorption is necessary for better clinical therapy and improving patients’ quality of life. In bone resorption, osteoclasts play critical roles, and RANKL is a pivotal regulator of osteoclastogenesis. However, the source of RANKL-expressing cells in the AB tumor microenvironment is controversial, and the mechanism of osteoclastogenesis in AB progression is not fully understood. In this study, we investigated the distribution of the RNA expression of RANKL in AB specimens. We found that PDGFRα- and S100A4-positive stromal fibroblasts expressed RANKL in the AB tumor microenvironment. Moreover, we analyzed the mechanisms of osteoclastogenesis in the AB tumor microenvironment using the human AB cell line AM-1 and a human primary periodontal ligament fibroblast cells. The results of histopathologic and in vitro studies clarified that the interaction between AB cells and stromal fibroblasts upregulated IL-6 expression and that AB cells induced RANKL expression in stromal fibroblasts and consequent osteoclastogenesis in AB progression.

B cell follicles (BCFs) in lymph nodes (LNs) are generally exempt of CD8+ T and NK cells. African green monkeys (AGMs), a natural host of simian immunodeficiency virus (SIV), display NK cell-mediated viral control in BCF. NK cell migration into BCF in chronically SIVagm-infected AGM is associated with CXCR5+ NK cells. We aimed to identify the mechanism leading to CXCR5 expression on NK cells. We show that CXCR5+ NK cells in LN were induced following SIVagm infection. CXCR5+ NK cells accumulated preferentially in BCF with proliferating B cells. Autologous NK-B cell co-cultures in transwell chambers induced CXCR5+ NK cells. Transcriptome analysis of CXCR5+ NK cells revealed expression of bcl6 and IL6R. IL-6 induced CXCR5 on AGM and human NK cells. IL6 mRNA was detected in LN at higher levels during SIVagm than SIVmac infection and often produced by plasma cells. Our study reveals a mechanism of B cell-dependent NK cell regulation.

CXCL8 is an inflammatory chemokine elevated in the colorectal cancer (CRC) tumour microenvironment. CXCR2, the major receptor for CXCL8, is predominantly expressed by neutrophils. In the cancer setting, CXCL8 plays important roles in neutrophil chemotaxis, facilitating angiogenesis, invasion, and metastasis. This study aimed to assess the spatial distribution of CXCL8 mRNA expression in CRC specimens, explore associations with clinical characteristics, and investigate the underlying biology of aberrant CXCL8 levels. CXCR2 expression was also assessed in a second cohort of unique CRC primary tumours and synchronously resected matched liver metastases. A previously constructed tissue microarray consisting of a cohort of stage I-IV CRC patients undergoing surgical resection with curative intent (n = 438) was probed for CXCL8 via RNAscope . Analysis was performed using HALO digital pathology software to quantify expression in the tumour and stromal compartments. Scores were assessed for association with clinical characteristics. Mutational analyses were performed on a subset of these patients to determine genomic differences in patients with high CXCL8 expression. A second cohort of stage IV CRC patients with primary and matched metastatic liver tumours was stained via immunohistochemistry for CXCR2, and scores were assessed for clinical significance. CXCL8 expression within the stromal compartment was associated with reduced cancer-specific survival in the first cohort (p = 0.035), and this relationship was potentiated in right-sided colon cancer cases (p = 0.009). High CXCL8 within the stroma was associated with driving a more stromal-rich phenotype and the presence of metastases. When stromal CXCL8 scores were combined with tumour-infiltrating macrophage counts or systemic neutrophil counts, patients classified as high for both markers had significantly poorer prognosis. CXCR2+ immune cell infiltration was associated with increased stromal invasion in liver metastases (p = 0.037). These data indicate a role for CXCL8 in driving unfavourable tumour histological features and promoting metastases. This study suggests that inhibiting CXCL8/CXCR2 should be investigated in patients with right-sided colonic disease and stroma-rich tumours.

The severe acute respiratory distress syndrome-associated coronavirus-2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 (COVID-19), is a single-stranded RNA virus whose sequence is known. COVID-19 is associated with a heterogeneous clinical phenotype ranging from asymptomatic to fatal disease. It appears that access to nasopharyngeal respiratory epithelia expressing angiotensin-converting enzyme (ACE) 2, the receptor for SARS CoV-2, is followed by viral replication in the pulmonary alveolar septal capillary bed. We have shown in prior studies that incomplete viral particles, termed pseudovirions, dock to deep subcutaneous and other vascular beds potentially contributing to the prothrombotic state and systemic complement activation that characterizes severe and critical COVID-19. A variety of skin rashes have been described in the setting of SARS-CoV-2 infection and more recently, following COVID-19 vaccination. The vaccines deliver a laboratory synthesized mRNA that encodes a protein that is identical to the spike glycoprotein of SARS-COV-2 allowing the production of immunogenic spike glycoprotein that will then elicit T cell and B cell adaptive immune responses. In this paper we review an array of cutaneous manifestations of COVID-19 that provide an opportunity to study critical pathophysiologic mechanisms that underlie all clinical facets of COVID-19 ranging from asymptomatic/mild to severe and critical COVID-19. We classify cutaneous COVID-19 according to underlying pathophysiologic principles. In this regard we propose two main pathways: 1) complement mediated thrombotic vascular injury syndromes deploying the alternative and mannan binding lectin pathways in the setting of severe and critical COVID-19 and 2) the robust T cell and type I interferon driven inflammatory and humoral driven immune complex mediated vasculitic cutaneous reactions seen with mild and moderate COVID-19. Novel data on cutaneous vaccine reactions are presented that manifest a clinical and morphologic parallel with similar eruptions seen in patients suffering from mild and moderate COVID-19 and in most cases represent systemic eczematoid hypersensitivity reactions to a putative vaccine based antigen. Finally, we show for the first time the localization of human synthesized spike glycoprotein following the COVID-19 vaccine to the cutaneous and subcutaneous vasculature confirming the ability of SARS CoV-2 spike glycoprotein to bind endothelium in the absence of intact virus.

Simultaneous nanoscale imaging of mRNAs and proteins of the same specimen can provide better information on the translational regulation, molecular trafficking, and molecular interaction of both normal and diseased biological systems. Expansion microscopy (ExM) is an attractive option to achieve such imaging; however, simultaneous ExM imaging of proteins and mRNAs has not been demonstrated. Here, a technique for simultaneous ExM imaging of proteins and mRNAs in cultured cells and tissue slices, which we termed dual-expansion microscopy (dual-ExM), is demonstrated. First, we verified a protocol for the simultaneous labeling of proteins and mRNAs. Second, we combined the simultaneous labeling protocol with ExM to enable the simultaneous ExM imaging of proteins and mRNAs in cultured cells and mouse brain slices and quantitatively study the degree of signal retention after expansion. After expansion, both proteins and mRNAs can be visualized with a resolution beyond the diffraction limit of light in three dimensions. Dual-ExM is a versatile tool to study complex biological systems, such as the brain or tumor microenvironments, at a nanoscale resolution.

To detail early tissue distribution and innate immune response to rabbit hemorrhagic disease virus 2 (RHDV2), 13 rabbits were orally ( Oryctolagus cuniculus ) inoculated with liver homogenate made from a feral rabbit that succumbed to RHDV2 during the 2020 outbreak in Oregon, USA. Rabbits were monitored regularly, with euthanasia and collection of tissues and swabs, at 12, 24, 36, 48, 96, and 144 hours post inoculation. Livers from these rabbits were positive by RT-rtPCR for presence of the virus. Using RNAscope for viral and replicative intermediates, rabbits had detectable viral genomic RNA at each time point, initially within the gastrointestinal tract, then in the liver by 36 hours post inoculation. Also using RNAscope, there were increasing amounts of mRNA coding for TNF-α, IL-6, and IL-1β within the liver and spleen through 48 hours post inoculation. The results of this study aided our understanding of the local innate immune response to RHDV2, as well as aspects of pathogenesis.