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Endocrine cell micronests (ECMs) are aggregates of endocrine cells known as enterochromaffin-like cells, typically measuring approximately 50 μm and usually observed in the mucosal layer of atrophic gastric fundic glands associated with hypergastrinemia. Although there are numerous reports on gastric ECMs, reports on duodenal ECMs are exceedingly rare. We report a rare case of Brunner's gland hyperplasia with increased endocrine cells and ECMs. An approximately 40 mm polyp was found in the duodenal bulb of a 57-year-old Japanese male patient during an upper gastrointestinal endoscopy, and a polypectomy was performed. Microscopic examination revealed hyperplasia of Brunner's glands in the duodenal polyp. Compared to normal Brunner's glands, hyperplastic Brunner's glands exhibited more endocrine cells. Additionally, many ECMs were observed in the fibromuscular connective tissue, comprising smooth muscle cells and myofibroblasts, adjacent to the hyperplastic Brunner's glands. The patient presented with hypergastrinemia (2,500 pg/mL; normal range: 30-140 pg/mL), and the ECMs were considered related to this condition. This case represents the first instance of a benign duodenal lesion with an increase in endocrine cells and the presence of ECMs., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. The Ethics Committee at the Affiliated Hospital of Hamamatsu University School of Medicine issued approval 81-004. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Meguro et al.)

Linear nevus sebaceous syndrome (LNSS) is a neurocutaneous syndrome associated with systemic complications that involve multiple organs, including the skin, central nervous system, eyes, and skeleton. LNSS is considered to be caused by mosaic RAS gene mutation. In this report, we present an autopsy case of LNSS in a Japanese boy. The affected neonate had hydrops fetalis and was born at 28 weeks and 4 days of gestation, weighing 2104 g. He had bilateral inverted eyelids, verrucous linear nevus separated along Blaschko's line, myocardial hypertrophy, and pharyngeal constriction, and underwent intensive treatment in NICU for arrhythmia, hydrocephalus, and respiratory distress. The hydrocephalus progressed gradually and he died at the age of 181 days, 12 days after a sudden cardiac arrest and recovery. KRAS G12D mutation was found in a skin biopsy specimen but not in blood cells, suggesting a postzygotic mosaicism. Autopsy revealed novel pathological findings related to LNSS, including intracranial lipomatous hamartoma and mesenteric lymphangioma, in addition to previously reported findings such as multicystic dysplastic kidney. There was the limited expression of mutated KRAS protein in kidneys., (© 2024 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

A double-check process helps prevent errors and ensures quality control. However, it may lead to decreased personal accountability, reduced effort, and declining quality checks. Introducing an artificial intelligence (AI)-based system in such scenarios could effectively address the risk of oversights. This study introduces an innovative AI-integrated workflow for cervical cytology screening that substantially improves efficiency and reduces the burden on cytologists. The AI model prioritizes cases for review based on anomaly scores and streamlines the first screening process to approximately 10 s per case. The model enhances the identification of high-risk cases via detailed microscopic observation, high anomaly scores cases, and a targeted review of low-score cases. The workflow highlights its capability for rapid, accurate, and less labor-intensive evaluations, demonstrating the potential to transform cervical cancer screening. This study highlights the importance of AI in modern medical diagnostics, particularly in areas with a high demand for accuracy and efficiency., (© 2024. The Author(s).)

Cytomegalovirus (CMV), a type of herpes virus, is the predominant cause of congenital anomalies due to intrauterine infections in humans. Adverse outcomes related to intrauterine infections with human cytomegalovirus (HCMV) vary widely, depending on factors such as fetal infection timing, infection route, and viral virulence. The precise mechanism underlying HCMV susceptibility remains unclear. In this study, we compared the susceptibility of neonatal human dermal fibroblast cells (NHDFCs) and human induced pluripotent stem cells (hiPSCs) derived from NHDFCs, which are genetically identical to HCMV, using immunostaining, microarray, in situ hybridization, quantitative PCR, and scanning electron microscopy. These cells were previously used to compare CMV susceptibility, but the underlying mechanisms were not fully elucidated. HCMV susceptibility of hiPSCs was significantly lower in the earliest phase. No shared gene ontologies were observed immediately post-infection between the two cell types using microarray analysis. Early-stage expression of HCMV antigens and the HCMV genome was minimal in immunostaining and in in situ hybridization in hiPSCs. This strongly suggests that HCMV does not readily bind to hiPSC surfaces. Scanning electron microscopy performed using the NanoSuit method confirmed the scarcity of HCMV particles on hiPSC surfaces. The zeta potential and charge mapping of the charged surface in NHDFCs and hiPSCs exhibited minimal differences when assessed using zeta potential analyzer and scanning ion conductance microscopy; however, the expression of heparan sulfate (HS) was significantly lower in hiPSCs compared with that in NHDFCs. Thus, HS expression could be a primary determinant of HCMV resistance in hiPSCs at the attachment level., Importance: Numerous factors such as attachment, virus particle entry, transcription, and virus particle egress can affect viral susceptibility. Since 1984, pluripotent cells are known to be CMV resistant; however, the exact mechanism underlying this resistance remains elusive. Some researchers suggest inhibition in the initial phase of HCMV binding, while others have suggested the possibility of a sufficient amount of HCMV entering the cells to establish latency. This study demonstrates that HCMV particles rarely attach to the surfaces of hiPSCs. This is not due to limitations in the electrostatic interactions between the surface of hiPSCs and HCMV particles, but due to HS expression. Therefore, HS expression should be recognized as a key factor in determining the susceptibility of HCMV in congenital infection in vitro and in vivo . In the future, drugs targeting HS may become crucial for the treatment of congenital CMV infections. Thus, further research in this area is warranted., Competing Interests: The authors declare no conflict of interest.

A 48-year-old woman with advanced ovarian cancer was diagnosed with pulmonary tumor thrombotic microangiopathy (PTTM) by antemortem pulmonary wedge aspiration cytopathology. Despite the initiation of anti-cancer treatment, she unfortunately died due to progressive respiratory failure. Histopathology of the autopsied lung revealed multiple tumor embolization with fibrin-rich clot and fibro-cellular intimal proliferation at the pulmonary arteriole. The embolized tumor showed strong immune-positivity for pro-thrombotic and fibrotic factors (tissue factor and vascular endothelial growth factor), suggesting the underlying mechanisms of PTTM development. This case suggests that a quick antemortem diagnosis and the early induction of specific treatments might ensure a better prognosis of PTTM.

Deep learning technology has been used in the medical field to produce devices for clinical practice. Deep learning methods in cytology offer the potential to enhance cancer screening while also providing quantitative, objective, and highly reproducible testing. However, constructing high-accuracy deep learning models necessitates a significant amount of manually labeled data, which takes time. To address this issue, we used the Noisy Student Training technique to create a binary classification deep learning model for cervical cytology screening, which reduces the quantity of labeled data necessary. We used 140 whole-slide images from liquid-based cytology specimens, 50 of which were low-grade squamous intraepithelial lesions, 50 were high-grade squamous intraepithelial lesions, and 40 were negative samples. We extracted 56,996 images from the slides and then used them to train and test the model. We trained the EfficientNet using 2,600 manually labeled images to generate additional pseudo labels for the unlabeled data and then self-trained it within a student-teacher framework. Based on the presence or absence of abnormal cells, the created model was used to classify the images as normal or abnormal. The Grad-CAM approach was used to visualize the image components that contributed to the classification. The model achieved an area under the curve of 0.908, accuracy of 0.873, and F1-score of 0.833 with our test data. We also explored the optimal confidence threshold score and optimal augmentation approaches for low-magnification images. Our model efficiently classified normal and abnormal images at low magnification with high reliability, making it a promising screening tool for cervical cytology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kurita et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: Fetal human cytomegalovirus (HCMV) infection might be involved in fetal growth restriction (FGR). Maternal serostatus and the prevalence of congenital HCMV infection are affected by various factors, such as socioeconomic status and ethnicity. Therefore, the prevalence of congenital HCMV-related FGR should be examined in each region., Methods: Seventy-eight cases of FGR with delivery between January 2012 and January 2017 at Fujita Health University Hospital were studied. Twenty-one non-FGR cases were also included as a control group. Placental sections obtained from the FGR and control cases were immunostained with two primary antibodies for detecting immediate early antigens., Results: Nineteen placental samples from FGR cases with another etiology were excluded. Finally, 59 placental samples from FGR cases of unknown etiology were included in the pathological analysis. Four of 59 (6.8%) placental samples were positive for HCMV antigen. All four positive cases were stained with the M0854 antibody, and there were no positive case with the MAB810R antibody. Neither maternal nor infantile clinical features were different between the HCMV-positive and -negative FGR cases. A pathological examination showed a hematoma in three of four cases and infarction in two of four cases., Conclusions: HCMV antigen was detected in 6.8% of placental samples obtained from FGR cases without an obvious etiology. No remarkable maternal or neonatal clinical features discriminated HCMV-related FGR from FGR due to other causes. Vasculitis and inflammation might play important roles in the pathogenesis of HCMV-related FGR., Competing Interests: The authors have no conflicts of interest relevant to this article to disclose. This work was supported by the Japan Agency for Medical Research and Development (AMED) (Grant number: 20gk0110037s0302).

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus particles in FFPE sections using light microscopy is difficult because of the light diffraction resolution limitations of an optical microscope. In this study, light microscopy and field emission scanning electron microscopy were performed to observe 3-dimensional virus particles in FFPE sections in a nondestructive manner using NanoSuit or osmium conductive treatment methods. The virus particles in FFPE sections were immunostained with specific antibodies against the surface antigens of the viral particles and stained with 3,3'-diaminobenzidine. A metal solution (0.2% gold chloride or 2% osmium tetroxide) was applied to enhance the 3,3'-diaminobenzidine-stained area. This procedure is nondestructive for FFPE sections and is a simpler method than transmission electron microscopy. To validate the applicability of this technique, we performed 3-dimensional imaging of the virus particles of different sizes, such as human papillomavirus, cytomegalovirus, and varicella-zoster virus. Furthermore, ultrathin sections from the FFPE sections that were observed to harbor viral particles using field emission scanning electron microscopy were prepared and assessed using transmission electron microscopy. In the correlative areas, transmission electron microscopy confirmed the presence of large numbers of virus particles. These results indicated that the combination of marking viral particles with 3,3'-diaminobenzidine/metal staining and conductive treatment can identify active progeny virus particles in FFPE sections using scanning electron microscopy. This easy correlative imaging of field emission scanning electron microscopy of the identical area of FFPE in light microscopy may help elucidate new pathological mechanisms of virus-related diseases., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The efficacy of the current influenza vaccines is frequently reduced because of antigenic drift, a trade-off of developing improved vaccines with broad cross-protective activity against influenza A viruses. In this study, we have successfully constructed a chimeric cytokine (CC) comprising the M2 protein, influenza A neuraminidase stalk, and interleukin-12. We produced virus-like particles (VLPs) containing CC and influenza hemagglutinin (HA) proteins using a baculovirus system in Eri silkworm pupae. The protective efficacy of the CCHA-VLP vaccine was evaluated in mice. The CCFkH5HA-VLP vaccine increased the survival rates of BALB/c mice, infected with a lethal dose of PRH1 and HKH5 viruses, to 80% and 100%, respectively. The results suggested that CCHA-VLP successfully induced potent cross-reactive protective immunity against infection with homologous and heterologous subtypes of the influenza A virus. This is the first study to design a CC-containing HA-VLP vaccine and validate its protective efficacy., (© 2022 Nerome et al.)

Objective: This study aimed to examine the prevalence of deep vein thrombosis (DVT) among evacuees in Minamiaso, a village which was temporarily isolated after the earthquakes, from the acute to recovery phase after the 2016 Kumamoto Earthquakes (GLIDE no: EQ-2016-000033-JPN)., Methods: This prospective study, which was approved by Fukui University Medical Research Ethics Committee (approval no. 20160024 and 20160089), enrolled 181 evacuees (73.9 ± 11.6 y) who participated in a series of 3 DVT screenings using portable ultrasound machines conducted over 19 mo. All participants completed a questionnaire before the screenings, and none of the participants attended all 3 screenings. Data analysis was performed using EZR version 1.41., Results: The DVT prevalence was 14.3% (79.4 ± 8.2 y) at first screening of evacuees staying in shelters and 18.5% (71.5 ± 13.1 y) and 12.2% (72.8 ± 10.9 y) in second and third screenings of evacuees staying in temporary housing, respectively. Multivariate analysis revealed age ≥75 y and alcohol consumption as independent risk factors in the entire cohort and in patients aged ≤74 y, respectively., Conclusions: A high DVT prevalence over a long time period of 19 mo was observed where survivors were temporarily isolated after the disaster.

We herein report a case of Behçet's disease with renal infarction due to mucormycosis. A 76-year-old man with entero-Behçet's disease had been treated with glucocorticoid and tumor necrosis factor (TNF) inhibitors. His entero-Behçet's disease was refractory to these treatments, and ileocecal resection was performed. After the operation, renal infarction that was unresponsive to anticoagulation therapy developed. He ultimately died of renal failure due to renal infarction. At the autopsy, histopathology of abundant hyphae in the renal vessel wall revealed mucormycosis. Renal mucormycosis is an important cause of renal failure with renal infarction in immunocompromised patients.

We have previously reported that swellings caused by haptens, such as 2,4,6-trinitrochlorobenzene (TNCB), may be associated with the extracellular signal-regulated kinase (ERK)-induced proliferation pathway. However, the involvement of the Spred/Sprouty family as critical negative regulators of the Ras/Raf/ERK signaling pathway at disease sites is not well-established. Thus, in the present study, the effects of hapten-challenge on the expression levels of genes and proteins associated with the Spred/Sprouty family in the ear of mice were investigated. The activation of ERK and epidermal growth factor receptor (EGFR) tyrosine kinase was inhibited by their selective inhibitors, namely, U0126 and PD168393, respectively. Twenty-four hours after the final challenge by the haptens TNCB, 2,4-dinitrofluorobenzene, or oxazolone, ear thickness was augmented by challenge with all haptens and the gene expression levels of Spred1, Spred2, Sprouty1, and Sprouty2 in swelling induced by all haptens were significantly decreased. Furthermore, Spred2, Sprouty1, and Sprouty2 genes were decreased in the epidermis and dermis of the TNCB-challenged ear. In conclusion, it is possible that the mechanism of hapten-challenge-induced skin thickening involves not only the enhancement of cell proliferative functions via the activation of ERK by EGFR tyrosine kinase activation but also the decreases expression of Spred/Sprouty family members.

Metformin, an AMP-activated protein kinase activator used to treat diabetes mellitus, has recently attracted attention as a promising anti-fibrotic agent. However, its anti-fibrotic effects on pleural fibroelastosis remain unknown. We induced mouse pleural fibroelastosis by intra-pleural coadministration of bleomycin and carbon and evaluated its validity as a preclinical model for human pleural fibrosis. We assessed the expression of the myofibroblast surface marker CD90 in the fibrotic pleura and the effects of metformin in vivo and in vitro . Finally, we evaluated the effects of metformin on human pleural mesothelial cells stimulated by transforming growth factor β1 (TGFβ1). The fibrotic pleura in mice had collagen and elastin fiber deposition similar to that seen in human fibrotic pleura. Moreover, CD90-positive myofibroblasts were detected in and successfully isolated from the fibrotic pleura. Metformin significantly suppressed the deposition of collagen and elastic fibers in the fibrotic pleura and decreased the expression of extracellular matrix (ECM)-related genes, including Col1a1 , Col3a1 , Fn1 , and Eln , in pleural CD90-positive myofibroblasts. In human pleural mesothelial cells, metformin decreased TGFβ1-induced upregulation of ECM-related genes and SNAI1 . Overall, metformin suppresses pleural fibroelastosis by inhibition of ECM production by pleural myofibroblasts, suggesting that this drug has therapeutic potential against human pleural fibrosis, including pleuroparenchymal fibroelastosis., Competing Interests: None., (AJTR Copyright © 2021.)

Micronodular thymoma with lymphoid stroma (MNT) is a rare thymic epithelial neoplasm subtype characterized by a micronodular tumor cell growth pattern and abundant lymphoid stroma. Micronodular thymic carcinoma with lymphoid stroma (MNCA) is considered as a malignant counterpart of MNT and exhibits a growth pattern similar to that of MNT but has histologic features reminiscent of thymic squamous cell carcinoma, such as cytologic atypia and CD5 and CD117 immunoexpression. Although both MNT and MNCA are characterized by abundant lymphoid stroma, it remains unknown whether there are differences in infiltrating lymphocytes between MNT and MNCA. We analyzed the immune microenvironment profile in eight MNT and three MNCA cases. The cell density of CD8-positive T cells was significantly higher in MNT than in MNCA, whereas that of FOXP3-positive T cells was significantly higher in MNCA than in MNT. There was no significant difference in the cell density of programmed death protein 1-positive T cells and programmed death ligand 1 expression between the MNT and MNCA cases. Our findings indicated that the immune microenvironment of MNCA differed from that of MNT and, compared with the T-cell profile of MNT, that of MNCA was more suppressive to patients' antitumor immune response., (© 2021 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Cytomegalovirus (CMV) reactivation in the colon is common in patients with severe ulcerative colitis (UC). Ganciclovir (GCV) resistance conferring CMV UL97 gene mutations have been reported in recent years. However, the prevalence of UL97 gene mutations in GCV-naive CMV infection in the colon remains unknown. We investigated the prevalence of CMV UL97 gene mutations in patients with colonic CMV infection associated with or without UC. Twenty-two GCV-naive patients with colonic CMV infection, 15 with UC and 7 with other diseases, were enrolled. Frozen biopsy samples or formalin-fixed paraffin-embedded samples were used for nested polymerase chain reaction (PCR) amplification of the UL97 gene. Sanger DNA sequencing was performed. In comparison with AD169 reference strain, natural polymorphisms were frequently detected in codons N68D (100%), I244V (100%), and D605E (86.4%). Seven polymorphisms were detected infrequently (< 10%) outside the kinase domain. However, no known GCV resistance mutations were found. There seemed to be no difference between the ratio of polymorphisms in patients with and without UC. In conclusions, we did not detect UL97 gene mutations associated with GCV resistance in GCV-naive patients with or without UC. Consistent with previous reports, D605E polymorphism may be used as a genetic marker for CMV in East Asian countries.

Ion-exchange resins are commonly used to treat complications such as hyperkalemia, hyperphosphatemia, and hypercholesterolemia. Gastrointestinal complications may occur as side effects of such treatments. Sodium and calcium polystyrene sulfonate (PS-Ca) are cation-exchange resins comprising an insoluble structure that binds to potassium ions in the digestive tract and exchanges them with sodium and calcium ions, respectively, to promote their elimination. PS crystals are rhomboid, refractive, and basophilic in hematoxylin and eosin staining. To differentiate PS crystals from other ion-exchange resin crystals such as sevelamer and cholestyramine, periodic acid-Schiff, Ziehl-Neelsen, and Congo red staining are usually performed. Here, correlative light and electron microscopy (CLEM)-energy-dispersive X-ray spectroscopy and the NanoSuit method (CENM) was applied to perform a definitive identification of ion-exchange resins. CENM could detect sulfur in PS crystals without destroying the glass slides. Notably, PS retained its ion-exchange ability to bind potassium in paraffin sections. Differential diagnosis of anion-exchange resins, such as sevelamer and cholestyramine, was possible using these characteristics. The phosphorus:carbon ratio was higher in sevelamer than in cholestyramine after soaking paraffin sections in a phosphate solution. Therefore, CENM may be used for the differential pathological diagnosis of ion-exchange resins in paraffin sections.

Purpose: The differential diagnosis of interstitial lung diseases (ILDs), particularly idiopathic pulmonary fibrosis (IPF) versus other non-IPF ILDs, is important for selecting the appropriate treatment. This retrospective study aimed to explore the utility of gremlin-1 for the differential diagnosis., Methods: Serum gremlin-1 concentrations were measured using an ELISA in 50 patients with IPF, 42 patients with non-IPF ILD, and 30 healthy controls. The baseline clinical data, including pulmonary functions, prognosis, and three serum biomarkers (Krebs von den Lungen-6 [KL6], surfactant protein-D [SP-D], and lactate dehydrogenase [LDH]), were obtained through a medical record review for analyzing their associations with serum gremlin-1 concentrations. To evaluate the origin of gremlin-1, we performed immunostaining on lung sections., Results: Serum gremlin-1 concentrations were significantly higher in patients with IPF (mean concentration, 14.4 ng/mL), followed by those with non-IPF ILD (8.8 ng/mL) and healthy controls (1.6 ng/mL). The area under the curve for IPF versus non-IPF ILDs was 0.759 (95% confidence interval, 0.661-0.857), which was superior to that of KL6/SP-D/LDH. The sensitivity and specificity for gremlin-1 (cutoff, 10.4 ng/mL) was 72 and 69%, respectively. By contrast, serum gremlin-1 concentrations were not associated with the pulmonary functions nor the prognosis in all patients with ILDs. In immunostaining, the gremlin-1 was broadly upregulated in IPF lungs, particularly at myofibroblasts, bronchiolar/alveolar epithelium, and CD163-positive M2-like macrophages., Conclusions: Gremlin-1 may be a useful biomarker to improve the diagnostic accuracy for IPF compared to non-IPF ILDs, suggesting a role of this molecule in the pathogenesis of IPF.

The brain is the major target of congenital cytomegalovirus (CMV) infection. It is possible that neuron disorder in the developing brain is a critical factor in the development of neuropsychiatric diseases in later life. Previous studies using mouse model of murine CMV (MCMV) infection demonstrated that the viral early antigen (E1 as a product of e1 gene) persists in the postnatal neurons of the hippocampus (HP) and cerebral cortex (CX) after the disappearance of lytic infection from non-neuronal cells in the periventricular (PV) region. Furthermore, neuron-specific activation of the MCMV-e1-promoter (e1-pro) was found in the cerebrum of transgenic mice carrying the e1-pro-lacZ reporter construct. In this study, in order to elucidate the mechanisms of e1-pro activation in cerebral neurons during actual MCMV infection, we have generated the recombinant MCMV (rMCMV) carrying long e1-pro1373- or short e1-pro448-EGFP reporter constructs. The length of the former, 1373 nucleotides (nt), is similar to that of transgenic mice. rMCMVs and wild type MCMV did not significantly differed in terms of viral replication or E1 expression. rMCMV-infected mouse embryonic fibroblasts showed lytic infection and activation of both promoters, while virus-infected cerebral neurons in primary neuronal cultures demonstrated the non-lytic and persistent infection as well as the activation of e1-pro-1373, but not -448. In the rMCMV-infected postnatal cerebrum, lytic infection and the activation of both promoters were found in non-neuronal cells of the PV region until postnatal 8 days (P8), but these disappeared at P12, while the activation of e1-pro-1373, but not -448 appeared in HP and CX neurons at P8 and were prolonged exclusively in these neurons at P12, with preservation of the neuronal morphology. Therefore, e1-pro-448 is sufficient to activate E1 expression in non-neuronal cells, however, the upstream sequence from nt -449 to -1373 in e1-pro-1373 is supposed to work as an enhancer necessary for the neuron-specific activation of e1-pro, particularly around the second postnatal week. This unique activation of e1-pro in developing cerebral neurons may be an important factor in the neurodevelopmental disorders induced by congenital CMV infection.

Viral cell-to-cell spread, a method employed by several viral families for entrance via cell junctions, is highly relevant to the pathogenesis of various viral infections. Cell-to-cell spread of herpes simplex virus 1 (HSV-1) is known to depend greatly on envelope glycoprotein E (gE). However, the molecular mechanism by which gE acts in HSV-1 cell-to-cell spread and the mechanisms of cell-to-cell spread by other herpesviruses remain poorly understood. Here, we describe our identification of prohibitin-1 as a novel gE-interacting host cell protein. Ectopic expression of prohibitin-1 increased gE-dependent HSV-1 cell-to-cell spread. As observed with the gE-null mutation, decreased expression or pharmacological inhibition of prohibitin-1 reduced HSV-1 cell-to-cell spread without affecting the yield of virus progeny. Similar effects were produced by pharmacological inhibition of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, wherein prohibitin-1 acts as a protein scaffold and is required for induction of this pathway. Furthermore, artificial activation of the MAPK/ERK pathway restored HSV-1 cell-to-cell spread impaired by the gE-null mutation. Notably, pharmacological inhibition of prohibitins or the MAPK/ERK pathway reduced viral cell-to-cell spread of representative members in all herpesvirus subfamilies. Our results suggest that prohibitin-1 contributes to gE-dependent HSV-1 cell-to-cell spread via the MAPK/ERK pathway and that this mechanism is conserved throughout the Herpesviridae , whereas gE is conserved only in the Alphaherpesvirinae subfamily. IMPORTANCE Herpesviruses are ubiquitous pathogens of various animals, including humans. These viruses primarily pass through cell junctions to spread to uninfected cells. This method of cell-to-cell spread is an important pathogenic characteristic of these viruses. Here, we show that the host cell protein prohibitin-1 contributes to HSV-1 cell-to-cell spread via a downstream intracellular signaling cascade, the MAPK/ERK pathway. We also demonstrate that the role of the prohibitin-1-mediated MAPK/ERK pathway in viral cell-to-cell spread is conserved in representative members of every herpesvirus subfamily. This study has revealed a common molecular mechanism of the cell-to-cell spread of herpesviruses., (Copyright © 2021 American Society for Microbiology.)

Background: Lung fibrosis is a serious life-threatening condition whose manifestation varies according to the localization and characteristics of fibroblasts, which are considered heterogeneous. Therefore, to better understand the pathology and improve diagnosis and treatment of this disease, it is necessary to elucidate the nature of this heterogeneity and identify markers for the accurate classification of human lung fibroblast subtypes., Methods: We characterized distinct mouse lung fibroblast subpopulations isolated by fluorescence-activated cell sorting (FACS) and performed microarray analysis to identify molecular markers that could be useful for human lung fibroblast classification. Based on the expression of these markers, we evaluated the fibroblast-like cell subtype localization in normal human lung samples and lung samples from patients with idiopathic pulmonary fibrosis (IPF)., Results: Mouse lung fibroblasts were classified into Sca-1 high fibroblasts and Sca-1 low fibroblasts by in vitro biological analyses. Through microarray analysis, we demonstrated CD248 and integrin alpha-8 (ITGA8) as cell surface markers for Sca-1 high fibroblasts and Sca-1 low fibroblasts, respectively. In mouse lungs, Sca-1 high fibroblasts and Sca-1 low fibroblasts were localized in the collagen fiber-rich connective tissue and elastic fiber-rich connective tissue, respectively. In normal human lungs and IPF lungs, two corresponding major fibroblast-like cell subtypes were identified: CD248 high ITGA8 low fibroblast-like cells and CD248 low ITGA8 high fibroblast-like cells, localized in the collagen fiber-rich connective tissue and in the elastic fiber-rich connective tissue, respectively., Conclusion: CD248 high ITGA8 low fibroblast-like cells and CD248 low ITGA8 high fibroblast-like cells were localized in an almost exclusive manner in human lung specimens. This human lung fibroblast classification using two cell surface markers may be helpful for further detailed investigations of the functions of lung fibroblast subtypes, which can provide new insights into lung development and the pathological processes underlying fibrotic lung diseases.

Histological examination using the light microscopy is currently the gold standard for life science research and diagnostics. However, magnified observations are limited because of the limitations intrinsic to light microscopy. Thus, a dual approach, known as correlative light and electron microscopy (CLEM), has emerged, although several technical challenges remain in terms of observing myriad stored paraffin sections. Previously, we developed the NanoSuit method, which enabled us to keep multicellular organisms alive/wet in the high vacuum of a scanning electron microscope by encasing the sample in a thin, vacuum-proof membrane. The approach uses the native extracellular substance (ECS) or an ECS-mimicking substance to polymerize a membrane by plasma or electron beam irradiation. Since the resulting NanoSuit is flexible and dense enough to prevent a living organism's bodily gas and liquids from evaporating (which we refer to as the "surface shield enhancer" (SSE) effect), it works like a miniature spacesuit with sufficient electron conductivity for an SEM observation. Here, we apply the NanoSuit method to CLEM analysis of paraffin sections. Accordingly, the NanoSuit method permits the study of paraffin sections using CLEM at low and high magnification, with the following features: (i) the integrity of the glass slide is maintained, (ii) three-dimensional microstructures of tissue and pathogens are visualized, (iii) nuclei and 3,3'-diaminobenzidine-stained areas are distinct because of gold chloride usage, (iv) immunohistochemical staining is quantitative, and (v) contained elements can be analyzed. Removal of the SSE solution after observation is a further advantage, as this allows slides to be restained and stored. Thus, the NanoSuit method represents a novel approach that will advance the field of histology.

Purpose: Human cytomegalovirus (HCMV) infections can cause endotheliitis which is associated with an elevation in the intraocular pressure (IOP). However, the mechanism of the IOP elevation has not been established. The purpose of this study was to determine whether HCMV strains which are capable of infecting corneal endothelial cells can also replicate and induce anti-viral responses, and can reorganize the actin cytoskeleton in trabecular meshwork cells., Study Design: Experimental study design., Methods: Cultured primary human trabecular meshwork cells (HTMCs) were infected with the Towne or TB40/E strains of HCMV. TB40/E is trophic for vascular endothelial and corneal endothelial cells. Real-time PCR, western blot, and fluorescent immunostaining have been used to determine whether HCMV-infected HTMCs will support the expression of viral mRNA and protein, allow viral replication, and elicit anti-viral host responses. We also determined whether lytic replication was present after HCMV infection., Results: HCMV infection led to the expression of viral mRNA and proteins of IE1, glycoprotein B(gB), and pp65. TB40/E infection induced interferon-β, a sign of host anti-viral immune response and monocyte chemotactic protein-1 (MCP-1) as IOP-related chemokine. Together with the induction of the regulators of actin cytoskeleton, myosin phosphatase Rho interacting protein (MPRIP) and MCP-1, TB40/E induced a high level of expression of viral proteins, including IE1, gB, and pp65 as well as actin stress fiber formation, and achieved pathogenically high viral titers., Conclusions: Human trabecular meshwork cells support the replication of endotheliotropic TB40/E strain of HCMV which indicates that this strain may have high virulence for trabecular meshwork.