Elevated Myl9 reflects the Myl9-containing microthrombi in SARS-CoV-2-induced lung exudative vasculitis and predicts COVID-19 severity.

The mortality of coronavirus disease 2019 (COVID-19) is strongly correlated with pulmonary vascular pathology accompanied by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-triggered immune dysregulation and aberrant activation of platelets. We combined histological analyses using field emission scanning electron microscopy with energy-dispersive X-ray spectroscopy analyses of the lungs from autopsy samples and single-cell RNA sequencing of peripheral blood mononuclear cells to investigate the pathogenesis of vasculitis and immunothrombosis in COVID-19. We found that SARS-CoV-2 accumulated in the pulmonary vessels, causing exudative vasculitis accompanied by the emergence of thrombospondin-1-expressing noncanonical monocytes and the formation of myosin light chain 9 (Myl9)-containing microthrombi in the lung of COVID-19 patients with fatal disease. The amount of plasma Myl9 in COVID-19 was correlated with the clinical severity, and measuring plasma Myl9 together with other markers allowed us to predict the severity of the disease more accurately. This study provides detailed insight into the pathogenesis of vasculitis and immunothrombosis, which may lead to optimal medical treatment for COVID-19.

Differential bronchial epithelial response regulated by ΔNp63: a functional understanding of the epithelial shedding found in asthma.

Bronchial epithelial cells serve as a physical barrier at the forefront of the immune system. Barrier disruption and an excessive immune response of the bronchial epithelium contribute to the pathophysiology of asthma, a chronic bronchial inflammatory disease. The purpose of this study was to investigate the functional significance of ΔNp63, a p53-like transcription factor expressed by the basal bronchial epithelium. The immunohistochemical expression profile of ΔNp63 was evaluated in human bronchial tissue derived from asthma patients. The role of ΔNp63 in apoptosis inhibition and production of soluble mediators was investigated in vitro with cultured BEAS-2B bronchial epithelial cells using molecular biological analysis. In healthy bronchial tissue, ΔNp63-positive basal epithelial cells were covered with differentiated ΔNp63-negative cells but in the asthmatic airway, ΔNp63-positive cells were directly exposed to the bronchial lumen due to severe epithelial shedding. ΔNp63 regulated bronchial apoptosis in response to Toll-like receptor 3 stimulation. On the other hand, expression of ΔNp63 was modulated by stimulation with trypsin and SLIGKV, protease-activated receptor 2 ligands. Further phenotypic analysis revealed that ΔNp63 controlled the transcriptional expression and protein release of some epithelium-derived proinflammatory cytokines and endogenous protease inhibitors. We conclude that ΔNp63 modulates the bronchial epithelial response to viral infection. At the same time, ΔNp63 expression is influenced by proteases, which are abundant in house dust mites. Therefore, the ΔNp63 axis would be intimately involved in these two major triggers of asthma exacerbations, viral infection and protease overload.

Diagnostic utility of automated SureFISH (Dako Omnis) in the diagnosis of musculoskeletal translocation-related sarcomas.

Fluorescence in situ hybridization (FISH) is an essential tool for genetic diagnosis in daily pathological work. Almost full automation of FISH can be achieved with the recently released automated SureFISH platform (Dako Omnis, Agilent Technologies, Santa Clara, CA, USA). Its utility has been reported in HER2 amplification of breast and gastric carcinoma and ALK-rearranged lung cancer. Here, we examined the utility of automated SureFISH for the identification of rearrangement signals in translocation-related sarcomas (TRSs), including 11 EWSR1-rearranged and 10 synovial sarcoma cases, compared with non-automated conventional FISH using the same specimens. The percentages of EWSR1 or SS18 split signals were higher in automated SureFISH than in conventional FISH in 13 of the 21 cases. On the other hand, 8 of the 21 cases showed the same or lower percentage of split signals in automated SureFISH. Both FISH approaches detected EWSR1 and SS18 split signals in more than 10% of tumor cells in all cases. The strongest advantage of automated SureFISH is its ability to reduce running time without sacrificing quality. Other advantages include improved signal sharpness with oligo probes and reduced ecological toxicity by avoiding formamide use. Automated SureFISH is an excellent tool for the genetic diagnosis of TRSs and contributes to their rapid definitive diagnosis.

Aberrant expression of claudin-4 and -7 in hepatocytes in the cirrhotic human liver.

The liver comprises hepatocytes and non-parenchymal cells such as bile duct epithelial cells. Claudin-4 and -7 are not expressed in hepatocytes under physiological conditions. It was reported that claudin-7 increased in human pulmonary fibroses. We therefore investigated claudin-4 and -7 expressions in human cirrhotic livers, in which hepatocyte proliferation is severely delayed. We examined liver tissues from 50 patients with liver tumors. The expression of claudin-4 and -7 in hepatocytes significantly increased with the grade of fibrosis, not with inflammatory activity, in the liver tissues of chronic hepatitis. The number of claudin-4- and -7-positive cells observed was greater than that of alpha-fetoprotein-positive hepatic progenitor cells. In primary cultures of mouse hepatocytes, the expression of claudin-4 and -7 was not induced by treatment with proinflammatory cytokines. In immunohistochemical analysis of liver tissues of 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated mice and primary cultures of mouse hepatocytes, the expression of claudin-4 and -7 increased with proliferation of progenitor cells. However, the claudin-4- and -7-positive cells were not always progenitor cells. Thus, claudin-4 and -7 were observed in hepatocytes of severely damaged mouse and human livers. These findings suggest that claudin-4- and -7-positive hepatocytes may exist during the process of differentiation from progenitor cells into mature hepatocytes.

Evaluating Cellularity Estimation Methods: Comparing AI Counting with Pathologists' Visual Estimates.

The development of next-generation sequencing (NGS) has enabled the discovery of cancer-specific driver gene alternations, making precision medicine possible. However, accurate genetic testing requires a sufficient amount of tumor cells in the specimen. The evaluation of tumor content ratio (TCR) from hematoxylin and eosin (H&E)-stained images has been found to vary between pathologists, making it an important challenge to obtain an accurate TCR. In this study, three pathologists exhaustively labeled all cells in 41 regions from 41 lung cancer cases as either tumor, non-tumor or indistinguishable, thus establishing a "gold standard" TCR. We then compared the accuracy of the TCR estimated by 13 pathologists based on visual assessment and the TCR calculated by an AI model that we have developed. It is a compact and fast model that follows a fully convolutional neural network architecture and produces cell detection maps which can be efficiently post-processed to obtain tumor and non-tumor cell counts from which TCR is calculated. Its raw cell detection accuracy is 92% while its classification accuracy is 84%. The results show that the error between the gold standard TCR and the AI calculation was significantly smaller than that between the gold standard TCR and the pathologist's visual assessment (p<0.05). Additionally, the robustness of AI models across institutions is a key issue and we demonstrate that the variation in AI was smaller than that in the average of pathologists when evaluated by institution. These findings suggest that the accuracy of tumor cellularity assessments in clinical workflows is significantly improved by the introduction of robust AI models, leading to more efficient genetic testing and ultimately to better patient outcomes.

Behavior of tricellulin during destruction and formation of tight junctions under various extracellular calcium conditions.

Tricellulin is an important component of tricellular tight junctions (TJs) and is involved in the formation of tricellular contacts. However, little is known about its regulation during the assembly and disassembly of tricellular TJs. By using the well-differentiated pancreatic cancer cell line HPAC, which highly expresses tricellulin at tricellular contacts, we have investigated changes in the localization, expression and phosphorylation of tricellulin and in its TJ functions as a barrier and fence during the destruction and formation of TJs induced by changes in the extracellular calcium concentration. During both extracellular Ca(2+) depletion caused by EGTA treatment and Ca(2+) repletion after Ca(2+) starvation, the expression of tricellulin increased in whole lysates and in Triton-X-100-insoluble fractions without any change in its mRNA. The increases in immunoreactivity revealed by Western blotting were prevented by alkaline phosphatase treatment. Immunoprecipitation assays showed that tricellulin was phosphorylated on threonine residues when it increased after Ca(2+) depletion and repletion. In the early stage after Ca(2+) repletion, tricellulin was expressed not only at tricellular contacts but also in the cytoplasm and at bicellular borders. In confocal laser microscopy, tricellulin was observed at the apical-most regions and basolateral membranes of tricellular contacts after Ca(2+) repletion. Knockdown of tricellulin delayed the recovery of the barrier and fence functions after Ca(2+) repletion. Thus, the dynamic behavior of tricellulin during the destruction and formation of TJs under various extracellular calcium conditions seems to be closely associated with the barrier and fence functions of TJs.

Changes of Tight Junction Protein Claudins in Small Intestine and Kidney Tissues of Mice Fed a DDC Diet.

DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine)-fed mice are widely used as a model for cholestatic liver disease. We examined the expression of tight junction protein claudin subspecies by immunofluorescent histochemistry in small intestine and kidney tissues of mice fed a DDC diet for 12 weeks. In the small intestine, decreases in claudin-3, claudin-7 and claudin-15 were observed in villous epithelial cells corresponding to the severity of histological changes while leaving the abundance of these claudin subspecies unchanged in crypt cells. Nevertheless, the proliferative activity of intestinal crypt cells measured by immunohistochemistry for Ki-67 decreased in the mice fed the DDC diet compared with that of control mice. These results suggest the possibility that DDC feeding affects the barrier function of villous epithelial cells and thus inhibits the proliferative activity of crypt epithelial cells. On the other hand, in the kidney, remarkable changes were found in the subcellular localization of claudin subspecies in a segment-specific manner, although histological changes of renal epithelial cells were quite minimal. These results indicate that immunohistochemistry for claudin subspecies can serve as a useful tool for detecting minute functional alterations of intestinal and renal epithelial cells.

Downregulation of angulin-1/LSR induces malignancy via upregulation of EGF-dependent claudin-2 and TGF-ß-dependent cell metabolism in human lung adenocarcinoma A549 cells.

Abnormal expression of bicellular tight junction claudins, including claudin-2 are observed during carcinogenesis in human lung adenocarcinoma. However, little is known about the role of tricellular tight junction molecule angulin-1/lipolysis-stimulated lipoprotein receptor (LSR). In the lung adenocarcinoma tissues examined in the present study, expression of claudin-2 was higher than in normal lung tissues, while angulin-1/LSR was poorly or faintly expressed. We investigated how loss of angulin-1/LSR affects the malignancy of lung adenocarcinoma cell line A549 and normal human lung epithelial (HLE) cells. The EGF receptor tyrosine kinase inhibitor AG1478 prevented the increase of claudin-2 expression induced by EGF in A549 cells. Knockdown of LSR induced expression of claudin-2 at the protein and mRNA levels and AG1478 prevented the upregulation of claudin-2 in A549 cells. Knockdown of LSR induced cell proliferation, cell migration and cell metabolism in A549 cells. Knockdown of claudin-2 inhibited the cell proliferation but did not affect the cell migration or cell metabolism of A549 cells. The TGF-ß type I receptor inhibitor EW-7197 prevented the decrease of LSR and claudin-2 induced by TGF-ß1 in A549 cells and 2D culture of normal HLE cells. EW-7197 prevented the increase of cell migration and cell metabolism induced by TGF-ß1 in A549 cells. EW-7197 prevented the increase of epithelial permeability of FITC-4kD dextran induced by TGF-ß1 in 2.5D culture of normal HLE cells. In conclusion, downregulation of angulin-1/LSR induces malignancy via EGF-dependent claudin-2 and TGF-ß-dependent cell metabolism in human lung adenocarcinoma.

Downregulation of tight junction-associated MARVEL protein marvelD3 during epithelial-mesenchymal transition in human pancreatic cancer cells.

The novel tight junction protein marvelD3 contains a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain like occludin and tricellulin. However, little is yet known about the detailed role and regulation of marvelD3 in normal epithelial cells and cancer cells, including pancreatic cancer. In the present study, we investigated marvelD3 expression in well and poorly differentiated human pancreatic cancer cell lines and normal pancreatic duct epithelial cells in which the hTERT gene was introduced into human pancreatic duct epithelial cells in primary culture, and the changes of marvelD3 during Snail-induced epithelial-mesenchymal transition (EMT) under hypoxia, TGF-ß treatment and knockdown of FOXA2 in well differentiated pancreatic cancer HPAC cells. MarvelD3 was transcriptionally downregulated in poorly differentiated pancreatic cancer cells and during Snail-induced EMT of pancreatic cancer cells in which Snail was highly expressed and the fence function downregulated, whereas it was maintained in well differentiated human pancreatic cancer cells and normal pancreatic duct epithelial cells. Depletion of marvelD3 by siRNAs in HPAC cells resulted in downregulation of barrier functions indicated as a decrease in transepithelial electric resistance and an increase of permeability to fluorescent dextran tracers, whereas it did not affect fence function of tight junctions. In conclusion, marvelD3 is transcriptionally downregulated in Snail-induced EMT during the progression for the pancreatic cancer.

Kikuchi-Fujimoto Disease Following COVID-19 Infection in a 7-Year-Old Girl: A Case Report and Literature Review.

The coronavirus disease 2019 (COVID-19) symptoms in children are relatively mild and often do not require treatment. Nonetheless, complications caused by the immune response to COVID-19 in children are possible and diverse. We present the case of a 7-year-old girl with persistent fever and lymphadenopathy arising from SARS-CoV-2 infection, diagnosed with Kikuchi-Fujimoto Disease (KFD) on lymph node biopsy. KFD is a rare benign disease, clinically characterized by fever and tender cervical lymphadenopathy affecting posterior cervical lymph nodes. We also reviewed six previously reported cases of COVID-19-associated KFD that occurred in school-aged children and compared them with the present case. The clinical course of COVID-19-associated KFD was similar to that of previous reports of KFD with a favorable prognosis. This is the first report of a school-aged child developing KFD following SARS-CoV-2 infection. KFD should be considered when approaching patients with hyperinflammatory states who present with prolonged fever and cervical lymphadenopathy after COVID-19.

Spindle cell tumor with histiocytic and myogenic marker expression in the lymph node of a human T-cell leukemia virus type 1 carrier.

Carriers of oncogenic human T-cell leukemia virus type 1 (HTLV-1) can develop adult T-cell leukemia/lymphoma (ATLL). While an increasing number of animal models of HTLV-1 infection have revealed that malignant tumors with a histiocytic phenotype can arise, they have not been reported in humans. Here, we present a 79-year-old female HTLV-1 carrier who presented with a swollen lymph node. Histological examination revealed that the lymph node was replaced with a malignant spindle cell tumor, but not ATLL. Immunohistochemical analysis indicated that the tumor was positive for histiocytic (CD68 and CD163) and myogenic (α-smooth muscle actin, desmin, and caldesmon) markers, suggesting some differential diagnoses. We could not reach a definitive diagnosis under the current notion of the disease entity. In addition, we could not provide an exact causal relationship between HTLV-1 infection and the development of the current tumor. Nevertheless, this tumor may be a human counterpart of murine HTLV-1-related histiocytic tumors. Curiously, the tumor showed a good response to chemotherapy with the combination of cyclophosphamide, vincristine, and prednisone, a standard approach for ATLL. This case might represent a novel entity of an HTLV-1-related malignant tumor. Further accumulation of case reports will certainly contribute to our understanding of human HTLV-1-related disease and the mechanism of viral oncogenesis.

Inhibition of HDAC and Signal Transduction Pathways Induces Tight Junctions and Promotes Differentiation in p63-Positive Salivary Duct Adenocarcinoma.

BACKGROUND: The p53 family p63 is essential for the proliferation and differentiation of various epithelial basal cells. It is overexpressed in several cancers, including salivary gland neoplasia. Histone deacetylases (HDACs) are thought to play a crucial role in carcinogenesis, and HDAC inhibitors downregulate p63 expression in cancers. METHODS: In the present study, to investigate the roles and regulation of p63 in salivary duct adenocarcinoma (SDC), human SDC cell line A253 was transfected with siRNA-p63 or treated with the HDAC inhibitors trichostatin A (TSA) and quisinostat (JNJ-26481585). RESULTS: In a DNA array, the knockdown of p63 markedly induced mRNAs of the tight junction (TJ) proteins cingulin (CGN) and zonula occuludin-3 (ZO-3). The knockdown of p63 resulted in the recruitment of the TJ proteins, the angulin-1/lipolysis-stimulated lipoprotein receptor (LSR), occludin (OCLN), CGN, and ZO-3 at the membranes, preventing cell proliferation, and leading to increased cell metabolism. Treatment with HDAC inhibitors downregulated the expression of p63, induced TJ structures, recruited the TJ proteins, increased the epithelial barrier function, and prevented cell proliferation and migration. CONCLUSIONS: p63 is not only a diagnostic marker of salivary gland neoplasia, but it also promotes the malignancy. Inhibition of HDAC and signal transduction pathways is, therefore, useful in therapy for p63-positive SDC cells.

COVID-19-associated disseminated mucormycosis: An autopsy case report.

BACKGROUND: Reports of mucormycosis, an infectious disease that commonly affects immunocompromised individuals, have increased during the ongoing coronavirus disease 2019 (COVID-19) pandemic. Disseminated mucormycosis associated with COVID-19 is rare but fatal and is characterized by an aggressive clinical course and delayed diagnosis. Our report documents a case of disseminated mucormycosis after COVID-19 infection. This is a rare pathological autopsy report on COVID-19-associated mucormycosis. CASE SUMMARY: A 58-year-old man was transferred to our hospital with severe COVID-19 pneumonia. During treatment for acute respiratory distress syndrome, he developed intra-abdominal bleeding that required a right hemicolectomy and ileostomy for hemostasis. The ileostoma and surgical wound developed necrosis followed by sepsis and multi-organ failure, which led to death. An autopsy revealed multiple thrombi associated with Rhizopus oryzae infection, which led to the necrosis of multiple infected organs. CONCLUSION: Early suspicion and diagnosis followed by treatment are keys to better outcomes of mucormycosis in patients with severe COVID-19.

Dysfunction of epithelial permeability barrier induced by HMGB1 in 2.5D cultures of human epithelial cells.

Airway and intestinal epithelial permeability barriers are crucial in epithelial homeostasis. High mobility group box 1 (HMGB1), increased by various stimuli, is involved in the induction of airway inflammation, as well as the pathogenesis of inflammatory bowel disease. HMGB1 enhances epithelial hyperpermeability. Two-and-a-half dimensional (2.5D) culture assays are experimentally convenient and induce cells to form a more physiological tissue architecture than 2D culture assays for molecular transfer mechanism analysis. In 2.5D culture, treatment with HMGB1 induced permeability of FITC-dextran into the lumen formed by human lung, nasal and intestinal epithelial cells. The tricellular tight junction molecule angulin-1/LSR is responsible for the epithelial permeability barrier at tricellular contacts and contributes to various human airway and intestinal inflammatory diseases. In this review, we indicate the mechanisms including angulin-1/LSR and multiple signaling in dysfunction of the epithelial permeability barrier induced by HMGB1 in 2.5D culture of human airway and intestinal epithelial cells.

Extensive mucosal sloughing of the small intestine and colon in a patient with severe COVID-19.

Patients with coronavirus disease 2019 (COVID-19) primarily cause respiratory symptoms. However, gastrointestinal (GI) symptoms can also occur. The endoscopic characteristics of the GI tract in COVID-19 patients remain unclear. We herein report a 62-year-old male with severe COVID-19 who needed multidisciplinary treatment, including extracorporeal membrane oxygenation (ECMO). Despite the improvement in his respiratory status, GI bleeding developed. Capsule endoscopy and colonoscopy revealed extensive mucosal sloughing in the lower intestinal tract. Additionally, we performed a comprehensive analysis of the mRNA expression levels of various proinflammatory cytokines in the intestinal mucosal tissues. The results suggested a significant elevation of IL-6, which could be involved in the pathophysiology of the GI involvement in COVID-19. Further investigation with more clinical data, including endoscopic findings and molecular analyses, will contribute to a comprehensive understanding of COVID-19-associated GI injury.

Protein kinase Cα inhibitor enhances the sensitivity of human pancreatic cancer HPAC cells to Clostridium perfringens enterotoxin via claudin-4.

Protein kinase C (PKC) is overexpressed in cancer, including pancreatic cancer, compared with normal tissue. Moreover, PKCα is considered one of the biomarkers for the diagnosis of cancers. In several human cancers, the claudin tight junction molecules are abnormally regulated and are thus promising molecular targets for diagnosis and therapy with Clostridium perfringens enterotoxin (CPE). In order to investigate the changes of tight junction functions of claudins via PKCα activation in pancreatic cancer cells, the well-differentiated human pancreatic cancer cell line HPAC, with its highly expressed tight junction molecules and well-developed barrier function, was treated with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment with TPA modified the activity of phosphoPKCα and caused an increase of the Snail family members Snail, Slug and Smad-interacting protein 1 and a decrease of E-cadherin. In HPAC cells treated with TPA, downregulation of claudin-1 and mislocalization of claudin-4 and occludin around the nuclei were observed, together with a decrease in the numbers of tight junction strands and an increase in phosphorylation of claudin-4. The barrier function and the cytotoxicity of CPE were significantly decreased on TPA treatment. All such changes after TPA treatment were prevented by inhibitors of panPKC and PKCα. These findings suggest that, in human pancreatic cancer cells, PKCα activation downregulates tight junction functions as a barrier and as a receptor of CPE via the modification of claudin-1 and -4 during epithelial to mesenchymal transition-like changes. PKCα inhibitors might represent potential therapeutic agents against human pancreatic cancer cells by use of CPE cytotoxicity via claudin-4.

Assessment of H3K27me3 immunohistochemistry and combination of NF1 and p16 deletions by fluorescence in situ hybridization in the differential diagnosis of malignant peripheral nerve sheath tumor and its histological mimics.

BACKGROUND: A definitive diagnosis of malignant peripheral nerve sheath tumor (MPNST) is challenging, especially in cases without neurofibromatosis 1 (NF1), because MPNST lacks specific markers on immunohistochemistry (IHC). METHODS: We performed IHC for histone 3 trimethylated on lysine 27 (H3K27me3) and evaluated the percentage of cells with H3K27me3 loss using measured values at 10% intervals, categorized as complete loss (100% of tumor cells lost staining), partial loss (10% to 90% of tumor cells lost staining), and intact (no tumor cells lost staining). We conducted fluorescence in situ hybridization (FISH) for NF1 and p16 deletions comparing 55 MPNSTs and 35 non-MPNSTs, consisting of 9 synovial sarcomas (SSs), 8 leiomyosarcomas (LMSs), 10 myxofibrosarcomas (MFSs), and 8 undifferentiated pleomorphic sarcomas (UPSs). We assessed the percentage of cells with homozygous and heterozygous deletions and defined "deletion" if the percentage of either the NF1 or p16 deletion signals was greater than 50% of tumor cells. RESULTS: Among the 55 MPNSTs, 23 (42%) showed complete H3K27me3 loss and 32 (58%) exhibited partial loss or intact. One each of the 9 SSs (11%), 8 LMSs (12%), and 8 UPSs (12%) showed complete H3K27me3 loss and many non-MPNSTs exhibited intact or partial H3K27me3 loss. Among the 55 MPNSTs, 33 (60%) and 44 (80%) showed NF1 or p16 deletion, respectively. Co-deletion of NF1 and p16 was observed in 29 (53%) MPNSTs. Among the 23 MPNTSs showing H3K27me3 complete loss, 18 (78%) and 20 (87%) exhibited NF1 or p16 deletion, respectively. Among the 32 MPNSTs with H3K27me3 partial loss or intact, 15 (47%) and 24 (75%) exhibited NF1 or p16 deletion, respectively. The frequency of NF1 and/or p16 deletion tended to be lower in non-MPNSTs than in MPNSTs. Approximately 90% of MPNSTs included cases with H3K27me3 complete loss and cases showing H3K27me3 partial loss or intact with NF1 and/or p16 deletion. Approximately 50% of MPNSTs showed co-deletion of NF1 and p16 regardless of H3K27me3 loss. CONCLUSIONS: FISH for NF1 and p16 deletions, frequently observed in high-grade MPNSTs, might be a useful ancillary diagnostic tool for differentiating MPNST from other mimicking spindle cell and pleomorphic sarcomas.