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Lekhika Pathak, Sukanya Gayan, Bidisha Pal, Joyeeta Talukdar, Seema Bhuyan, Sorra Sandhya, , ,
The American Journal of Pathology, Volume 191, pp 1255-1268; doi:10.1016/j.ajpath.2021.03.011

Abstract:
We postulate that similar to bacteria, adult stem cells may also exhibit an altruistic defense mechanism to protect their niche against external threat. Herein, we report mesenchymal stem cell (MSC)–based altruistic defense against a mouse model of coronavirus, murine hepatitis virus-1 (MHV-1) infection of lung. MHV-1 infection led to reprogramming of CD271+ MSCs in the lung to an enhanced stemness phenotype that exhibits altruistic behavior, as per previous work in human embryonic stem cells. The reprogrammed MSCs exhibited transient expansion for 2 weeks, followed by apoptosis and expression of stemness genes. The conditioned media of the reprogrammed MSCs exhibited direct antiviral activity in an in vitro model of MHV-1–induced toxicity to type II alveolar epithelial cells by increasing their survival/proliferation and decreasing viral load. Thus, the reprogrammed MSCs can be identified as altruistic stem cells (ASCs), which exert a unique altruistic defense against MHV-1. In a mouse model of MSC-mediated Mycobacterium tuberculosis (MTB) dormancy, MHV-1 infection in the lung exhibited 20-fold lower viral loads than the MTB-free control mice on the third week of viral infection, and exhibited six-fold increase of ASCs, thereby enhancing the altruistic defense. Notably, these ASCs exhibited intracellular replication of MTB, and their extracellular release. Animals showed tuberculosis reactivation, suggesting that dormant MTB may exploit ASCs for disease reactivation.
Yoshiyasu Fukusumi, Hidenori Yasuda, Ying Zhang,
The American Journal of Pathology, Volume 191, pp 1209-1226; doi:10.1016/j.ajpath.2021.04.004

Abstract:
Ephrin-B1 is one of the critical components of the slit diaphragm of kidney glomerular podocyte. However, the precise function of ephrin-B1 is unclear. To clarify the function of ephrin-B1, ephrin-B1–associated molecules were studied. RNA-sequencing analysis suggested that Na+/H+ exchanger regulatory factor 2 (NHERF2), a scaffolding protein, is associated with ephrin-B1. NHERF2 was expressed at the apical area and the slit diaphragm, and interacted with the nephrin–ephrin-B1 complex at the slit diaphragm. The nephrin–ephrin-B1–NHERF2 complex interacted with ezrin bound to F-actin. NHERF2 bound ephrin-B1 via its first postsynaptic density protein-95/disks large/zonula occludens-1 domain, and podocalyxin via its second postsynaptic density protein-95/disks large/zonula occludens-1 domain. Both in vitro analyses with human embryonic kidney 293 cells and in vivo study with rat nephrotic model showed that stimulaiton of the slit diaphragm, phosphorylation of nephrin and ephrin-B1, and dephosphorylation of NHERF2 and ezrin, disrupted the linkages of ephrin-B1–NHERF2 and NHERF2-ezrin. It is conceivable that the linkage of nephrin–ephrin-B1–NHERF2–ezrin–actin is a novel critical axis in the podocytes. Ephrin-B1 phosphorylation also disrupted the linkage of an apical transmembrane protein, podocalyxin, with NHERF2-ezrin-actin. The phosphorylation of ephrin-B1 and the consequent dephosphorylation of NHERF2 are critical initiation events leading to podocyte injury.
Nina Goertzen, Roberto Pappesch, Jana Fassunke, Thomas Brüning, Yon-Dschun Ko, Joachim Schmidt, Frederik Großerueschkamp, Reinhard Buettner,
The American Journal of Pathology, Volume 191, pp 1269-1280; doi:10.1016/j.ajpath.2021.04.013

Abstract:
Therapeutic decisions in lung cancer critically depend on the determination of histologic types and oncogene mutations. Therefore, tumor samples are subjected to standard histologic and immunohistochemical analyses and examined for relevant mutations using comprehensive molecular diagnostics. In this study, an alternative diagnostic approach for automatic and label-free detection of mutations in lung adenocarcinoma tissue using quantum cascade laser–based infrared imaging is presented. For this purpose, a five-step supervised classification algorithm was developed, which was not only able to detect tissue types and tumor lesions, but also the tumor type and mutation status of adenocarcinomas. Tumor detection was verified on a data set of 214 patient samples with a specificity of 97% and a sensitivity of 95%. Furthermore, histology typing was verified on samples from 203 of the 214 patients with a specificity of 97% and a sensitivity of 94% for adenocarcinoma. The most frequently occurring mutations in adenocarcinoma (KRAS, EGFR, and TP53) were differentiated by this technique. Detection of mutations was verified in 60 patient samples from the data set with a sensitivity and specificity of 95% for each mutation. This demonstrates that quantum cascade laser infrared imaging can be used to analyze morphologic differences as well as molecular changes. Therefore, this single, one-step measurement provides comprehensive diagnostics of lung cancer histology types and most frequent mutations.
Chunsun Jiang, Gang Liu, Lu Cai, Jessy Deshane, Veena Antony, Victor J. Thannickal,
The American Journal of Pathology, Volume 191, pp 1227-1239; doi:10.1016/j.ajpath.2021.04.003

Abstract:
Increased apoptosis sensitivity of alveolar type 2 (ATII) cells and increased apoptosis resistance of (myo)fibroblasts, the apoptosis paradox, contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF). The mechanism underlying the apoptosis paradox in IPF lungs, however, is unclear. Aging is the greatest risk factor for IPF. In this study, we show, for the first time, that ATII cells from old mice are more sensitive, whereas fibroblasts from old mice are more resistant, to apoptotic challenges, compared with the corresponding cells from young mice. The expression of plasminogen activator inhibitor 1 (PAI-1), an important profibrogenic mediator, was significantly increased in both ATII cells and lung fibroblasts from aged mice. In vitro studies using PAI-1 siRNA and active PAI-1 protein indicated that PAI-1 promoted ATII cell apoptosis but protected fibroblasts from apoptosis, likely through dichotomous regulation of p53 expression. Deletion of PAI-1 in adult mice led to a reduction in p53, p21, and Bax protein expression, as well as apoptosis sensitivity in ATII cells, and their increase in the lung fibroblasts, as indicated by in vivo studies. This increase was associated with an attenuation of lung fibrosis after bleomycin challenge. Since PAI-1 is up-regulated in both ATII cells and fibroblasts in IPF, the results suggest that increased PAI-1 may underlie the apoptosis paradox of ATII cells and fibroblasts in IPF lungs.
Erratum
The American Journal of Pathology, Volume 191; doi:10.1016/j.ajpath.2021.05.001

Abstract:
In the article entitled, “Lack of Annexin A6 Exacerbates Liver Dysfunction and Reduces Lifespan of Niemann-Pick Type C Protein–Deficient Mice” (Volume 191, pages 475-486 of the March 2021 issue of The American Journal of Pathology; DOI: https://doi.org/10.1016/j.ajpath.2020.12.009) the authors have requested an update to the funding information. Grant number RTI2018-098593-B-I00, awarded to Albert Pol is being added to the funding statement:
The American Journal of Pathology, Volume 191; doi:10.1016/s0002-9440(21)00214-5

Steven A. Bloomer,
The American Journal of Pathology, Volume 191, pp 1165-1179; doi:10.1016/j.ajpath.2021.04.005

Abstract:
The liver plays a pivotal role in the regulation of iron metabolism through its ability to sense and respond to iron stores by release of the hormone hepcidin. Under physiologic conditions, regulation of hepcidin expression in response to iron status maintains iron homeostasis. In response to tissue injury, hepcidin expression can be modulated by other factors, such as inflammation and oxidative stress. The resulting dysregulation of hepcidin is proposed to account for alterations in iron homeostasis that are sometimes observed in patients with liver disease. This review describes the effects of experimental forms of liver injury on iron metabolism and hepcidin expression. In general, models of acute liver injury demonstrate increases in hepcidin mRNA and hypoferremia, consistent with hepcidin's role as an acute-phase reactant. Conversely, diverse models of chronic liver injury are associated with decreased hepcidin mRNA but with variable effects on iron status. Elucidating the reasons for the disparate impact of different chronic injuries on iron metabolism is an important research priority, as is a deeper understanding of the interplay among various stimuli, both positive and negative, on hepcidin regulation. Future studies should provide a clearer picture of how dysregulation of hepcidin expression and altered iron homeostasis impact the progression of liver diseases and whether they are a cause or consequence of these pathologies.
Chien-Yu Chen, Yang Li, Ni Zeng, Lina He, Xinwen Zhang, Taojian Tu, Qi Tang, Mario Alba, Sabrina Mir, Eileen X. Stiles, et al.
The American Journal of Pathology, Volume 191, pp 1240-1254; doi:10.1016/j.ajpath.2021.04.007

Abstract:
The estrogen-related receptor (ERR) family of orphan nuclear receptors are transcriptional activators for genes involved in mitochondrial bioenergetics and metabolism. The goal of this study was to explore the role of ERRα in lipid metabolism and the potential effect of inhibiting ERRα on the development of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). In the current study, three experimental mouse models: high-fat diet, high-carbohydrate diet, and a genetic model of hepatic insulin resistance where the liver hyperinsulinemia signal is mimicked via hepatic deletion of Pten (phosphatase and tensin homolog deleted on chromosome 10), the negative regulator of the insulin/phosphatidylinositol 3-kinase signaling pathway, were used. A recently developed small-molecule inhibitor for ERRα was used to demonstrate that inhibiting ERRα blocked NAFLD development induced by either high-carbohydrate diet or high-fat diet feeding. ERRα inhibition also diminished lipid accumulation and attenuated NASH development in the Pten null mice. Glycerolipid synthesis was discovered as an additional mechanism for ERRα-regulated NAFLD/NASH development and glycerophosphate acyltransferase 4 was identified as a novel transcriptional target of ERRα. In summary, these results establish ERRα as a major transcriptional regulator of lipid biosynthesis in addition to its characterized primary function as a regulator for mitochondrial function. This study recognizes ERRα as a potential target for NAFLD/NASH treatment and elucidates novel signaling pathways regulated by ERRα.
, Reshef Tal, Harvey J. Kliman, Hugh S. Taylor
The American Journal of Pathology, Volume 191, pp 1292-1302; doi:10.1016/j.ajpath.2021.04.011

Abstract:
Hyperactivation of the CXCL12-CXCR4 axis occurs in endometriosis; the therapeutic potential of treatments aimed at global inhibition of the axis was recently reported. Because CXCR4 is predominantly expressed on epithelial cells in the uterus, this study explored the effects of targeted disruption of CXCR4 in endometriosis lesions. Uteri derived from adult female mice homozygous for a floxed allele of CXCR4 and co-expressing Cre recombinase under control of progesterone receptor promoter were sutured onto the peritoneum of cycling host mice expressing the green fluorescent protein. Four weeks after endometriosis induction, significantly lower number of lesions developed in Cxcr4-conditional knockout lesions relative to those in controls (37.5% vs. 68.8%, respectively). In lesions that developed in Cxcr4-knockout, reduced epithelial proliferation was associated with a lower ratio of epithelial to total lesion area compared with controls. Furthermore, while CD3+ lymphocytes were largely excluded from the epithelial compartment in control lesions, in Cxcr4-knockout lesions, CD3+ lymphocytes infiltrated the Cxcr4-deficient epithelium in the diestrus and proestrus stages. Current data demonstrate that local CXCR4 expression is necessary for proliferation of the epithelial compartment of endometriosis lesions, that its downregulation compromises lesion numbers, and suggest a role for epithelial CXCR4 in lesion immune evasion.
Erratum
The American Journal of Pathology, Volume 191; doi:10.1016/j.ajpath.2021.04.012

Abstract:
In the article entitled, “Oxidative Phosphorylation Promotes Primary Melanoma Invasion” (Volume 190, pages 1108-1117 of the May 2020 issue of The American Journal of Pathology; DOI: https://doi.org/10.1016/j.ajpath.2020.01.012) the authors have requested an update to the funding information. The NYU Melanoma Specialized Program of Research Excellence (SPORE) grant is being changed from P50 CA16087 to P50 CA225450.
Michael O'Hare, Dhanesh Amarnani, Hannah A.B. Whitmore, Miranda An, , Leslie Ramos, Santiago Delgado-Tirado, Xinyao Hu, Natalia Chmielewska, Anita Chandrahas, et al.
The American Journal of Pathology, Volume 191, pp 1193-1208; doi:10.1016/j.ajpath.2021.04.006

Abstract:
Pulmonary fibrosis (PF) can arise from unknown causes, as in idiopathic PF, or as a consequence of infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current treatments for PF slow, but do not stop, disease progression. We report that treatment with a runt-related transcription factor 1 (RUNX1) inhibitor (Ro24-7429), previously found to be safe, although ineffective, as a Tat inhibitor in patients with HIV, robustly ameliorates lung fibrosis and inflammation in the bleomycin-induced PF mouse model. RUNX1 inhibition blunted fundamental mechanisms downstream pathologic mediators of fibrosis and inflammation, including transforming growth factor-β1 and tumor necrosis factor-α, in cultured lung epithelial cells, fibroblasts, and vascular endothelial cells, indicating pleiotropic effects. RUNX1 inhibition also reduced the expression of angiotensin-converting enzyme 2 and FES Upstream Region (FURIN), host proteins critical for SARS-CoV-2 infection, in mice and in vitro. A subset of human lungs with SARS-CoV-2 infection overexpress RUNX1. These data suggest that RUNX1 inhibition via repurposing of Ro24-7429 may be beneficial for PF and to battle SARS-CoV-2, by reducing expression of viral mediators and by preventing respiratory complications.
, Bruna Amanda da Cruz Rattis, , Mara Rubia Nunes Celes,
The American Journal of Pathology, Volume 191, pp 1154-1164; doi:10.1016/j.ajpath.2021.04.010

Abstract:
Severe acute respiratory syndrome coronavirus 2, the etiologic agent of coronavirus disease 2019 (COVID-19) and the cause of the current pandemic, produces multiform manifestations throughout the body, causing indiscriminate damage to multiple organ systems, particularly the lungs, heart, brain, kidney, and vasculature. The aim of this review is to provide a new assessment of the data already available for COVID-19, exploring it as a transient molecular disease that causes negative regulation of angiotensin-converting enzyme 2, and consequently, deregulates the renin-angiotensin-aldosterone system, promoting important changes in the microcirculatory environment. Another goal of the article is to show how these microcirculatory changes may be responsible for the wide variety of injury mechanisms observed in different organs in this disease. The new concept of COVID-19 provides a unifying pathophysiological picture of this infection and offers fresh insights for a rational treatment strategy to combat this ongoing pandemic.
The American Journal of Pathology, Volume 191; doi:10.1016/s0002-9440(21)00213-3

, Mariana S.L. Praça, Jillian R.H. Wendel, Robert E. Emerson, Francesco J. DeMayo, John P. Lydon,
The American Journal of Pathology, Volume 191, pp 1281-1291; doi:10.1016/j.ajpath.2021.03.013

Abstract:
Oncogenic KRAS mutations are a common finding in endometrial cancers. Recent sequencing studies indicate that loss-of-function mutations in the ARID1A gene are enriched in gynecologic malignant tumors. However, neither of these genetic insults alone are sufficient to develop gynecologic cancer. To determine the role of the combined effects of deletion of Arid1a and oncogenic Kras, Arid1aflox/flox mice were crossed with KrasLox-Stop-Lox-G12D/+ mice using progesterone receptor Cre (PgrCre/+). Histologic analysis and immunohistochemistry of survival studies were used to characterize the mutant mouse phenotype. Hormone dependence was evaluated by ovarian hormone depletion and estradiol replacement. Arid1aflox/flox; KrasLox-Stop-Lox-G12D/+; PgrCre/+ mice were euthanized early because of invasive vaginal squamous cell carcinoma. Younger mice had precancerous intraepithelial lesions. Immunohistochemistry supported the pathological diagnosis with abnormal expression and localization of cytokeratin 5, tumor protein P63, cyclin-dependent kinase inhibitor 2A, and Ki-67, the marker of proliferation. Ovarian hormone deletion in Arid1aflox/flox; KrasLox-Stop-Lox-G12D/+; PgrCre/+ mice resulted in atrophic vaginal epithelium without evidence of vaginal tumors. Estradiol replacement in ovarian hormone–depleted Arid1aflox/flox; KrasLox-Stop-Lox-G12D/+; PgrCre/+ mice resulted in lesions that resembled the squamous cell carcinoma in intact mice. Therefore, this mouse can be used to study the transition from benign precursor lesions into invasive vaginal human papillomavirus–independent squamous cell carcinoma, offering insights into progression and pathogenesis of this rare disease.
, Lea D. Schlieben, , , Robert Stöhr, , Matthias Bieg, Judith V.M.G. Bovée, Philip Ströbel, Naveed Ishaque, et al.
The American Journal of Pathology, Volume 191, pp 1314-1324; doi:10.1016/j.ajpath.2021.03.012

Abstract:
Solitary fibrous tumors (SFTs) harbor activating NAB2-STAT6 gene fusions. Different variants of the NAB2-STAT6 gene fusion have been associated with distinct clinicopathologic features. Lipomatous SFTs are a morphologic variant of SFTs, characterized by a fat-forming tumor component. Our aim was to evaluate NAB2-STAT6 fusion variants and to further study the molecular genetic features in a cohort of lipomatous SFTs. A hybrid-capture–based next-generation sequencing panel was employed to detect NAB2-STAT6 gene fusions at the RNA level. In addition, the RNA expression levels of 507 genes were evaluated using this panel, and were compared with a control cohort of nonlipomatous SFTs. Notably, 5 of 11 (45%) of lipomatous SFTs in the current series harbored the uncommon NAB2 exon 4–STAT6 exon 4 gene fusion variant, which is observed in only 0.9% to 1.4% of nonlipomatous SFTs. Furthermore, lipomatous SFTs displayed significant differences in gene expression compared with their nonlipomatous counterparts, including up-regulation of the gene peroxisome proliferator activated receptor-γ (PPARG). Peroxisome proliferator activated receptor-γ is a nuclear receptor regulating adipocyte differentiation, providing a possible explanation for the fat-forming component in lipomatous SFTs. In summary, the current study provides a possible molecular genetic basis for the distinct morphologic features of lipomatous SFTs.
Tomohisa Akamatsu, , Takuya Oshima, Yoshinori Aoki, Ayumi Mizukami, Keiji Goishi, Hiroyuki Shichino, Norihiro Kato, Naoto Takahashi, Yu-Ichi Goto, et al.
The American Journal of Pathology, Volume 191, pp 1303-1313; doi:10.1016/j.ajpath.2021.04.009

Abstract:
Neonatal hypoxic-ischemic encephalopathy (nHIE) is a major neonatal brain injury. Despite therapeutic hypothermia, mortality and sequelae remain severe. The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is associated with the pathophysiology of nHIE. In this study, morphologic change and microglial activation under the nHIE condition and LOX-1 treatment were investigated. The microglial activity and proliferation were assessed with a novel morphologic method, immunostaining, and quantitative PCR in the rat brains of both nHIE model and anti–LOX-1 treatment. Circumference ratio, the long diameter ratio, the cell area ratio, and the roundness of microglia were calculated. The correlation of the morphologic metrics and microglial activation in nHIE model and anti–LOX-1 treated brains was evaluated. LOX-1 was expressed in activated ameboid and round microglia in the nHIE model rat brain. In the evaluation of microglial activation, the novel morphologic metrics correlated with all scales of the nHIE-damaged and treated brains. While the circumference and long diameter ratios had a positive correlation, the cell area ratio and roundness had a negative correlation. Anti–LOX-1 treatment attenuated morphologic microglial activation and proliferation, and suppressed the subsequent production of inflammatory mediators by microglia. In human nHIE, round microglia and endothelial cells expressed LOX-1. The results indicate that LOX-1 regulates microglial activation in nHIE and anti–LOX-1 treatment attenuates brain injury by suppressing microglial activation.
, Lili Jiang, Lifan Liang, Kelly Koral, Qian Zhang, Lei Zhao, ,
The American Journal of Pathology, Volume 191, pp 1180-1192; doi:10.1016/j.ajpath.2021.04.014

Abstract:
Hepatocellular carcinoma (HCC) is the fifth most common type of cancer and the third leading cause of cancer-related deaths worldwide. Liver resection or liver transplantation is the most effective therapy for HCC because drugs approved by the US Food and Drug Administration to treat patients with unresectable HCC have an unfavorable overall survival rate. Therefore, the development of biomarkers for early diagnosis and effective therapy strategies are still necessary to improve patient outcomes. Fibroblast growth factor (FGF) 19 was amplified in patients with HCC from various studies, including patients from The Cancer Genome Atlas. FGF19 plays a syngeneic function with other signaling pathways in primary liver cancer development, such as epidermal growth factor receptor, Wnt/β-catenin, the endoplasmic reticulum–related signaling pathway, STAT3/IL-6, RAS, and extracellular signal–regulated protein kinase, among others. The current review presents a comprehensive description of the FGF19 signaling pathway involved in liver cancer development. The use of big data and bioinformatic analysis can provide useful clues for further studies of the FGF19 pathway in HCC, including its application as a biomarker, targeted therapy, and combination therapy strategies.
The American Journal of Pathology, Volume 191, pp 1153-1153; doi:10.1016/j.ajpath.2021.05.002

Abstract:
The following highlights summarize research articles that are published in the current issue of The American Journal of Pathology.
Destiny R. Matthews, Honggui Li, Jing Zhou, Qingsheng Li, Shannon Glaser, Heather Francis, ,
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.06.010

Abstract:
Inflammation drives the development and progression of non-alcoholic steatohepatitis (NASH). The present study examined changes in intestinal inflammation during NASH. In male C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in severe hepatic steatosis and inflammation relative to feeding a chow diet (CD). Also, MCD-fed mice revealed characteristics of mucosal and submucosal inflammatory responses and increased CD68-postive cells compared with CD-fed mice. Moreover, intestinal phosphorylation states of c-Jun N-terminal protein kinase (JNK) p46 and mRNA levels of interleukin (IL) 1 beta (IL1B), IL6, tumor necrosis factor alpha (TNF), and monocyte chemoattractant protein-1 were significantly higher and intestinal mRNA levels of IL4 and IL13 were significantly lower in MCD-fed mice compared to their respective levels in CD-fed mice. Surprisingly, upon treatment with MCD-mimicking media, the proinflammatory responses in cultured intestinal epithelial cells (IECs) (CMT-93 cells, a transformed epithelial cell line) did not differ significantly from those in IECs treated with control media. In contrast, in RAW264.7 cells (transformed macrophages), MCD-mimicking media significantly increased the phosphorylation states of JNK p46 and mitogen-activated protein kinases p38 and mRNA levels of IL1B, IL6, IL10, and TNF under either basal or lipopolysaccharide-stimulated conditions. Collectively, these results suggest that increased intestinal inflammation is associated with NASH phenotype. Additionally, elevated proinflammatory responses in macrophages likely contribute to, in large part, increased intestinal inflammation in NASH.
Chhavi Chauhan,
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.06.011

Abstract:
Deep learning has rapidly advanced artificial intelligence (AI) and algorithmic decision-making (ADM) paradigms, impacting many traditional fields of medicine. Pathology is a heavily data-centric specialty of medicine. The structured nature of pathology data repositories makes it highly attractive to AI researchers to train deep learning models to improve healthcare delivery. Equally, there are enormous financial incentives driving adoption of AI and ADM due to promise of increased efficiency of the healthcare delivery process. Unethical use of AI may exacerbate existing inequities of healthcare, especially if not implemented correctly. There is an urgent need to harness the vast power of AI in an ethically and morally justifiable manner. In this mini-review, we explore the key issues involving AI ethics in pathology. Issues related to ethical design of pathology AI studies and the potential risks associated with implementation of AI and ADM within the pathology workflow are discussed. We describe three key foundational principles of ethical AI in the context of pathology: transparency, accountability, and governance. The future practice of pathology must be guided by these principles. Pathologists must be aware of the potential of AI to deliver superlative healthcare and the ethical pitfalls associated with it. Finally, pathologists must have a seat at the table to drive the future implementation of ethical AI in the practice of pathology.
, , Géraldine Dessilly, Pamela Baldin, , Corinne Hubinont, Pierre Sonveaux, Frédéric Debiève
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.05.009

Abstract:
Despite occasional reports of SARS-CoV-2 vertical transmission during pregnancy, the question of placental infection and its consequences for the newborn remain questionable. Here, we analyzed the placentas of 31 COVID-19-positive mothers by RT-PCR, immunohistochemistry and in situ hybridization. We only detected one case of placental infection, which was associated with intrauterine demise of the fetus. We then isolated and differentiated primary trophoblasts from non-pathological human placentas at term, and exposed them to SARS-CoV-2 virions. Unlike for positive control cells Vero E6, we were not able to detect the virus inside cytotrophoblasts and syncytiotrophoblasts or in the supernatant four days after infection. As a mechanism of defense, we hypothesized that trophoblasts at term do not express ACE2 and TMPRSS, the two main host membrane receptors for SARS-CoV-2 entry. The quantification of these proteins in the placenta during pregnancy confirmed the absence of TMPRSS2 at the surface of the syncytium. Surprisingly, a transiently induced experimental expression of TMPRSS2 did not allow the entry or replication of the virus in differentiated trophoblasts. Altogether, these results underline that trophoblasts are not likely to be infected by SARS-CoV-2 at term, but the reported case raises concern about preterm infection.
Shanshan Qin, Dan Predescu, Brandon Carman, Priyam Patel, Jiwang Chen, Miran Kim, Tim Lahm, Mark Geraci, Sanda A. Predescu
The American Journal of Pathology, Volume 191, pp 1135-1150; doi:10.1016/j.ajpath.2021.03.009

Abstract:
Pulmonary arterial hypertension (PAH) is a sex-biased disease. Increased expression and activity of the long-noncoding RNA X-inactive–specific transcript (Xist), essential for X-chromosome inactivation and dosage compensation of X-linked genes, may explain the sex bias of PAH. The present studies used a murine model of plexiform PAH, the intersectin-1s (ITSN) heterozygous knockout (KOITSN+/–) mouse transduced with an ITSN fragment (EHITSN) possessing endothelial cell proliferative activity, in conjunction with molecular, cell biology, biochemical, morphologic, and functional approaches. The data demonstrate significant sex-centered differences with regard to EHITSN-induced alterations in pulmonary artery remodeling, lung hemodynamics, and p38/ETS domain containing protein/c-Fos signaling, altogether leading to a more severe female lung PAH phenotype. Moreover, the long-noncoding RNA–Xist is up-regulated in the lungs of female EHITSN-KOITSN+/– mice compared with that in female wild-type mice, leading to sex-specific modulation of the X-linked gene ETS domain containing protein and its target, two molecular events also characteristic to female human PAH lung. More importantly, cyclin A1 expression in the S and G2/M phases of the cell cycle of synchronized pulmonary artery endothelial cells of female PAH patients is greater versus controls, suggesting functional hyperproliferation. Thus, Xist up-regulation leading to female pulmonary artery endothelial cell sexual dimorphic behavior may provide a better understanding of the origin of sex bias in PAH. Notably, the EHITSN-KOITSN+/– mouse is a unique experimental animal model of PAH that recapitulates most of the sexually dimorphic characteristics of human disease.
, Randall J. Olsen, Paul A. Christensen, Sishir Subedi, Robert Olson, James J. Davis, , Prasanti Yerramilli, Layne Pruitt, Kristina Reppond, et al.
The American Journal of Pathology, Volume 191, pp 983-992; doi:10.1016/j.ajpath.2021.03.004

Abstract:
Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there has been international concern about the emergence of virus variants with mutations that increase transmissibility, enhance escape from the human immune response, or otherwise alter biologically important phenotypes. In late 2020, several variants of concern emerged globally, including the UK variant (B.1.1.7), the South Africa variant (B.1.351), Brazil variants (P.1 and P.2), and two related California variants of interest (B.1.429 and B.1.427). These variants are believed to have enhanced transmissibility. For the South Africa and Brazil variants, there is evidence that mutations in spike protein permit it to escape from some vaccines and therapeutic monoclonal antibodies. On the basis of our extensive genome sequencing program involving 20,453 coronavirus disease 2019 patient samples collected from March 2020 to February 2021, we report identification of all six of these SARS-CoV-2 variants among Houston Methodist Hospital (Houston, TX) patients residing in the greater metropolitan area. Although these variants are currently at relatively low frequency (aggregate of 1.1%) in the population, they are geographically widespread. Houston is the first city in the United States in which active circulation of all six current variants of concern has been documented by genome sequencing. As vaccine deployment accelerates, increased genomic surveillance of SARS-CoV-2 is essential to understanding the presence, frequency, and medical impact of consequential variants and their patterns and trajectory of dissemination.
Wonkyung Cho, Sharad K. Mittal, Elsayed Elbasiony,
The American Journal of Pathology, Volume 191, pp 1108-1117; doi:10.1016/j.ajpath.2021.02.016

Abstract:
Mast cells, historically known for their function as effector cells in the induction of allergic diseases, reside in all vascularized tissues of the body, particularly, in proximity to blood and lymphatic vessels. Despite being neighboring sentinel cells to blood vessels, whether the spatial distribution of mast cells regulates the degree of angiogenesis remains to be investigated. Herein, an asymmetrical distribution of mast cells was shown at the murine ocular surface, with the higher number of mast cells distributed along the nasal limbus of the cornea compared with the temporal side. Using a well-characterized murine model of suture-induced corneal neovascularization, insult to the nasal side was shown to result in more extensive angiogenesis compared with that to the temporal side. To directly assess the impact of the spatial distribution of mast cell on angiogenesis, neovascularization was induced in mast cell–deficient mice (cKitw-sh). Unlike the wild-type (C57BL/6) mice, cKitw-sh mice did not show disproportionate growth of corneal blood vessels following the temporal and nasal insult. Moreover, cromolyn-mediated pharmacologic blockade of mast cells at the ocular surface attenuated the asymmetrical nasal and temporal neovascularization, suggesting that spatial distribution of mast cells significantly contributes to angiogenic response at the ocular surface.
Tobias Furlan, Alexander Kirchmair, Natalie Sampson, Martin Puhr, Martina Gruber, Zlatko Trajanoski, Frédéric R. Santer, Walther Parson, Florian Handle,
The American Journal of Pathology, Volume 191, pp 1094-1107; doi:10.1016/j.ajpath.2021.02.017

Abstract:
Patients with advanced prostate cancer are frequently treated with the antiandrogen enzalutamide. However, resistance eventually develops in virtually all patients, and various mechanisms have been associated with this process. The histone acetyltransferases EP300 and CREBBP are involved in regulation of cellular events in advanced prostate cancer. This study investigated the role of EP300/CREBBP inhibitors in enzalutamide-resistant prostate cancer. EP300/CREBBP inhibitors led to the same inhibition of androgen receptor activity in enzalutamide-resistant and -sensitive cells. However, enzalutamide-resistant cells were more sensitive to these inhibitors in viability assays. As indicated by the RNA-sequencing–based pathway analysis, genes related to the ribosome and MYC activity were significantly altered upon EP300/CREBBP inhibitor treatment. EP300/CREBBP inhibitors led to the down-regulation of ribosomal proteins RPL36 and RPL29. High-level ribosomal proteins amplifications and MYC amplifications were observed in castration-resistant prostate cancer samples of the publicly available Stand Up to Cancer data set. An inhibitor of RNA polymerase I–mediated transcription was used to evaluate the functional implications of these findings. The enzalutamide-resistant cell lines were more sensitive to this treatment. In addition, the migration rate of enzalutamide-resistant cells was strongly inhibited by this treatment. Taken together, the current data show that EP300/CREBBP inhibitors affect the MYC/ribosomal protein axis in enzalutamide-resistant cells and may have promising therapeutic implications.
Mengtao Li, Eva M. McGhee, Lauryn Shinno, Kellie Lee,
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.06.005

Abstract:
Human papillomavirus (HPV) is a ubiquitous human pathogen that can be cleared by host immunity. Nonetheless, a small percentage of the patients develop persistent infection with oncogenic HPV, which poses an increased risk of developing HPV-associated malignancy. While cell-mediated immunity is a known systemic factor, local factors that influence persistent HPV infection have not been fully investigated. HPV-related head/neck cancers have a strong site preference for the oropharynx, suggesting the existence of unique local factors that promote HPV-induced oncogenesis. The human oropharynx often harbors anaerobic bacteria that produce a variety of byproducts, including butyrate. Because butyrate is a potent epigenetic modulator, it could be an environmental factor influencing the development of HPV-positive oropharyngeal malignancy. In this study, we showed that butyrate treatment changed the property of HPV16 E6/E7-immortalized keratinocytes. In vitro, the treatment increased the cells' migration ability, slowed the growth, and increased the genotoxic resistance. When implanted in the syngeneic mice, the treated keratinocytes survived longer and exhibited a different growth pattern. The survival advantage obtained after butyrate exposure potentially can increase the susceptibility of HPV-infected oropharyngeal keratinocytes to further malignant transformation. Our results suggest that tonsillar bacteria's fermentation products may play an important role in the long-term persistence of high risk-HPV infection, which is a critical risk factor for developing HPV-positive oropharyngeal malignancy.
Jinxian Xu, Xinglou Liu, Xinyan Zhang, Brendan Marshall, Zheng Dong, Sylvia B. Smith, Diego G. Espinosa-Heidmann,
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.06.008

Abstract:
While pathologies associated with acute virus infections have been extensively studied, the effects of long-term latent virus infections are less well understood. Human cytomegalovirus (HCMV), which infects 50 to 80% of humans, is usually acquired during early life and persists in a latent state for the lifetime. The purpose of this study was to determine if systemic murine cytomegalovirus (MCMV) infection acquired early in life disseminates to and becomes latent in the eye and if ocular MCMV can trigger in situ inflammation and occurrence of ocular pathology. Here we show that neonatal infection of BALB/c mice with MCMV resulted in dissemination of virus to the eye where it localized principally to choroidal endothelia and pericytes while less frequently, to The retinal pigment epithelium (RPE) cells. MCMV underwent ocular latency which was associated with expression of multiple virus genes and from which MCMV could be reactivated by immunosuppression. Latent ocular infection was associated with significant upregulation of several inflammatory/angiogenic factors. Retinal and choroidal pathologies developed in a progressive manner with deposits appearing at both basal and apical aspects of the RPE, RPE/choroidal atrophy, photoreceptor degeneration and neovascularization. The pathologies induced by long term ocular MCMV latency share features of previously described human ocular diseases such as age-related macular degeneration (AMD).
Felipe Castro-Martinez, Aurora Candelario-Martinez, Maria Del Rocio Encarnacion-Garcia, Zayda Piedra-Quintero, Raul Bonilla-Moreno, , Rocio Perez-Orozco, Maria De Los Angeles Hernandez-Cueto, Jose Esteban Muñoz-Medina, Genaro Patiño-Lopez, et al.
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.06.004

Abstract:
Epithelial barrier impairment is a hallmark of several pathological processes in the gut, including inflammatory bowel diseases. Several intracellular signals prevent apoptosis in intestinal epithelial cells. Here we shown that in colonocytes, rictor/mTORC2 signaling is a pro-survival stimulus. Mechanistically, mTORC2 activates Akt which in turns inhibits apoptosis by phosphorylating Bad and preventing caspase-3 activation. Nevertheless, during inflammation, rictor/mTORC2 signaling declines and Akt activity is reduced. Consequently, active caspase-3 increases in surface colonocytes undergoing apoptosis/anoikis and causes epithelial barrier breakdown. Likewise, Rictor ablation in intestinal epithelial cells interrupts mTORC2/Akt signaling and increases apoptosis/anoikis of surface colonocytes without affecting the crypt architecture. The increase in epithelial permeability induced by Rictor ablation produces a mild inflammatory response in the colonic mucosa, but minimally affects the development/establishment of colitis. The data identify a previously unknown mechanism by which rictor/mTORC2 signaling regulates apoptosis/anoikis in intestinal epithelial cells during colitis and clarifies its role in the maintenance of the intestinal epithelial barrier.
, , Tanner J. Wetzel, Troy Suwondo, Gage P. Rensch, Jane M. DeVasure, Deanna D. Mosley, , , , et al.
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.06.007

Abstract:
Alcohol misuse and smoking are risk factors for pneumonia, yet the impact of combined cigarette smoke and alcohol on pneumonia remains understudied. Smokers who misuse alcohol form lung malondialdehyde-acetaldehyde (MAA) protein adducts and have decreased levels of anti-MAA secretory IgA (sIgA). Transforming growth factor beta (TGFβ) downregulates polymeric Ig receptor (pIgR) on mucosal epithelium, resulting in decreased sIgA transcytosis to the mucosa. It is hypothesized that MAA-adducted lung protein increases TGFβ, preventing expression of epithelial cell pIgR and decreasing sIgA. Cigarette smoke and alcohol co-exposure on sIgA and TGFβ in human bronchoalveolar lavage (BAL) fluid and in mice instilled with MAA-adducted surfactant protein D (SPD-MAA) were studied herein. Human (HBEC) and mouse (MTEC) lung epithelial cells were treated with SPD-MAA and sIgA and TGFβ measured. Decreased sIgA and increased TGFβ were observed in BAL from combined alcohol and smoking groups in humans and mice. CD204 (MAA receptor) knockout mice showed no changes in sIgA. SPD-MAA decreased pIgR in HBEC. Conversely, SPD-MAA stimulated TGFβ release in both HBEC and MTEC, but not in CD204 knockout mice. SPD-MAA stimulated TGFβ in alveolar macrophage cells. These data demonstrate that MAA-adducted surfactant protein stimulates lung epithelial cell TGFβ, downregulates pIgR, and decreases sIgA transcytosis. These data provide a mechanism for the decreased levels of sIgA observed in smokers who misuse alcohol.
The American Journal of Pathology, Volume 191; doi:10.1016/s0002-9440(21)00168-1

The American Journal of Pathology, Volume 191; doi:10.1016/s0002-9440(21)00169-3

Jacqueline T. Hecht, Alka C. Veerisetty, Juliana Wu, Francoise Coustry, Mohammad G. Hossain, Frankie Chiu, Francis H. Gannon,
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.05.016

Abstract:
Given the increasing number of people living with OA, due to population aging and rising rates of obesity, there is an urgent need to identify effective treatment and preventions. Two top risk factors for OA, age and obesity, are associated with ER stress. The I-ERS mouse, an ER stress driven model of primary OA, was developed to study the role of ER stress in primary OA susceptibility. The I-ERS mouse has the unique ability to induce ER stress in healthy adult I-ERS mouse articular chondrocytes and cartilage, driving joint degeneration that mimics early primary OA. ER stress-induced damage occurred gradually and stimulated joint degeneration with OA characteristics including increased matrix metalloproteinase activity, inflammation, senescence, chondrocyte death, decreased proteoglycans, autophagy block and gait dysfunction. Consistent with human OA, intense exercise hastened and increased the level of ER stress-induced joint damage. Notably, loss of a critical ER stress response protein (CHOP), largely ameliorated ER stress-stimulated OA outcomes including preserving proteoglycan content, reducing inflammation and relieving autophagy block. Resveratrol diminished ER stress-induced joint degeneration by decreasing CHOP, TNFα, IL-1β, MMP-13, pS6, number of TUNEL positive chondrocytes and senescence marker p16 INK4a. The finding that a dietary supplement can prevent ER stressed-induced joint degeneration in mice, provides a preclinical foundation to potentially develop a prevention strategy for those at high risk to develop OA.
, Rebecca Abraham, Marcus R. Clark,
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.05.022

Abstract:
With applications in object detection, image feature extraction, image classification, and image segmentation, artificial intelligence is enabling high-throughput analysis of image data in a variety of biomedical imaging disciplines, ranging from radiology and pathology to cancer biology and immunology. Specifically, a growth in research surrounding deep learning has led to widespread application of computer vision techniques to analyze and mine data from biomedical images. The availability of open-source software packages and the development of novel, trainable deep neural network architectures has led to an increase in accuracy of cell detection and segmentation algorithms. By automating cellular segmentations, it is now possible to mine quantifiable cellular and spatio-cellular features from microscopy images, providing insight into the organization of cells in various pathologies. This mini-review provides an overview of the current state-of-the-art deep learning and artificial intelligence methods for segmentation and data mining of cells in microscopy images of tissue.
Catharina Müller, , Emma Åhrman, Hans Brunnström, , Annika Nybom, Barbora Michaliková, Hillevi Larsson, Leif T. Eriksson, Hans Henrik Schultz, et al.
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.05.014

Abstract:
Bronchiolitis obliterans syndrome, a common form of chronic lung allograft dysfunction, is the major limitation to long-term survival after lung transplantation. The histologic correlate is progressive, fibrotic occlusion of small airways, obliterative bronchiolitis lesions, which ultimately lead to organ failure. The molecular composition of these lesions is unknown. In this sutdy, the protein composition of the lesions in explanted lungs from four end-stage bronchiolitis obliterans syndrome patients was analyzed using laser-capture microdissection and optimized sample preparation protocols for mass spectrometry. Immunohistochemistry and immunofluorescence were used to determine the spatial distribution of commonly identified proteins on the tissue level, and protein signatures for 14 obliterative bronchiolitis lesions were established. A set of 39 proteins, identified in >75% of lesions, included distinct structural proteins (collagen types IV and VI) and cellular components (actins, vimentin, and tryptase). Each respective lesion exhibited a unique composition of proteins (on average, n = 66 proteins), thereby mirroring the morphologic variation of the lesions. Antibody-based staining confirmed these mass spectrometry–based findings. The 14 analyzed obliterative bronchiolitis lesions showed variations in their protein content, but also common features. This study provides molecular and morphologic insights into the development of chronic rejection after lung transplantation. The protein patterns in the lesions were correlated to pathways of extracellular matrix organization, tissue development, and wound healing processes.
Sabine Dieleman, Romy Aarnoutse, , , ,
The American Journal of Pathology, Volume 191, pp 968-982; doi:10.1016/j.ajpath.2021.02.020

Abstract:
Breast cancer tissue contains its own unique microbiota. Emerging preclinical data indicates that breast microbiota dysbiosis contributes to breast cancer initiation and progression. Furthermore, the breast microbiota may be a promising biomarker for treatment selection and prognosis. Differences in breast microbiota composition have been found between breast cancer subtypes and disease severities that may contribute to immunosuppression, enabling tumor cells to evade immune destruction. Interactions between breast microbiota, gut microbiota, and immune system are proposed, all forming potential targets to increase therapeutic efficacy. In addition, because the gut microbiota affects the host immune system and systemic availability of estrogen and bile acids known to influence tumor biology, gut microbiota modulation could be used to manipulate breast microbiota composition. Identifying breast and gut microbial compositions that respond positively to certain anticancer therapeutics could significantly reduce cancer burden. Additional research is needed to unravel the complexity of breast microbiota functioning and its interactions with the gut and the immune system. In this review, developments in the understanding of breast microbiota and its interaction with the immune system and the gut microbiota are discussed. Furthermore, the biomarker potential of breast microbiota is evaluated in conjunction with possible strategies to target microbiota in order to improve breast cancer treatment.
Brian E. Sansbury, Xiaofeng Li, Blenda Wong, Colin O. Riley, Ashley E. Shay, Robert Nshimiyimana, , ,
The American Journal of Pathology, Volume 191, pp 1049-1063; doi:10.1016/j.ajpath.2021.02.015

Abstract:
Tissue injury elicits an inflammatory response that facilitates host defense. Resolution of inflammation promotes the transition to tissue repair and is governed, in part, by specialized pro-resolving mediators (SPM). The complete structures of a novel series of cysteinyl-SPM (cys-SPM) were recently elucidated, and proved to stimulate tissue regeneration in planaria and resolve acute inflammation in mice. Their functions in mammalian tissue repair are of interest. Here, nine structurally distinct cys-SPM were screened and PCTR1 uniquely enhanced human keratinocyte migration with efficacy similar to epidermal growth factor. In skin wounds of mice, PCTR1 accelerated closure. Wound infection increased PCTR1 that coincided with decreased bacterial burden. Addition of PCTR1 reduced wound bacteria levels and decreased inflammatory monocytes/macrophages, which was coupled with increased expression of genes involved in host defense and tissue repair. These results suggest that PCTR1 is a novel regulator of host defense and tissue repair, which could inform new approaches for therapeutic management of delayed tissue repair and infection.
, Yoshihiko Usui, Takaaki Hattori, Masaru Takeuchi, Kei Takayama, Yoko Karasawa, Yoshiaki Nishio, Naoyuki Yamakawa, Daizoh Saitoh, Hiroshi Goto, et al.
The American Journal of Pathology, Volume 191, pp 1077-1093; doi:10.1016/j.ajpath.2021.02.014

Abstract:
Programmed cell death protein (PD)-1 is a coinhibitory molecule that suppresses immune response and maintains immune homeostasis. Moreover, the PD-1 pathway blocks cancers from being attacked by immune cells. Anti–PD-1 antibody therapy such as nivolumab improves survival in cancer patients. However, the occurrence of autoimmune inflammatory disorders in various organs has been increasingly reported as an adverse effect of nivolumab. Of the disorders associated with nivolumab, Sicca syndrome occurs in 3% to 11% of cases and has unknown pathologic mechanisms. Whether the absence of the PD-1 pathway causes functional and morphologic disorders in lacrimal glands was determined by analyzing PD-1 gene–knockout (Pdcd1−/−) mice. Histopathologic analysis showed that Pdcd1−/− mice developed dacryoadenitis beginning at 3 to 4 months of age, and deteriorated with age. Flow-cytometric analysis confirmed that cells infiltrating the affected lacrimal glands consisted mainly of CD3+ T cells and only a small proportion of CD19+ B cells. Among infiltrating T cells, the CD4+ Th-cell subset consisted of Th1 cells producing interferon-γ in an early stage of dacryoadenitis in Pdcd1−/− mice. Experiments of lymphocyte transfer from Pdcd1−/− into irradiated wild-type mice confirmed that CD4+ T cells from Pdcd1−/− mice induced dacryoadenitis. These results indicate that PD-1 plays an important role in the prevention of autoimmune inflammatory disorders in lacrimal glands caused by activated CD4+ Th1 cells.
Chenna Kesavulu Sugali, Naga Pradeep Rayana, Jiannong Dai, Michael Peng, Sherri L. Harris, Hannah C. Webber, Shaohui Liu, Stephan G. Dixon, Priyanka H. Parekh, Elizabeth A. Martin, et al.
The American Journal of Pathology, Volume 191, pp 1020-1035; doi:10.1016/j.ajpath.2021.02.018

Abstract:
Glucocorticoid-induced glaucoma is a secondary open-angle glaucoma. About 40% of the general population may develop elevated intraocular pressure on prolonged glucocorticoid treatment secondary to damages in the trabecular meshwork (TM), a tissue that regulates intraocular pressure. Therefore, identifying the key molecules responsible for glucocorticoid-induced ocular hypertension is crucial. In this study, Dickkopf-related protein 1 (Dkk1), a canonical Wnt signaling inhibitor, was found to be elevated in the aqueous humor and TM of glaucoma patients. At the signaling level, Dkk1 enhanced glucocorticoid receptor (GR) signaling, whereas Dkk1 knockdown or Wnt signaling activators decreased GR signaling in human TM cells as indicated by luciferase assays. Similarly, activation of the GR signaling inhibited Wnt signaling. At the protein level, glucocorticoid-induced extracellular matrix was inhibited by Wnt activation using Wnt activators or Dkk1 knockdown in primary human TM cells. In contrast, inhibition of canonical Wnt signaling by β-catenin knockdown increased glucocorticoid-induced extracellular matrix proteins. At the physiological level, adenovirus-mediated Wnt3a expression decreased glucocorticoid-induced ocular hypertension in mouse eyes. In summary, Wnt and GR signaling inhibit each other in the TM, and canonical Wnt signaling activators may prevent the adverse effect of glucocorticoids in the eye.
Ming-Sian Wu, Yi-Ping Kuo, Yin-Chiu Lo, De-Jiun Tsai, Chao-Yang Lai, Tsung-Hsien Chuang, , Wan-Ting Tsai, Pei-Jung Chung,
The American Journal of Pathology, Volume 191, pp 1036-1048; doi:10.1016/j.ajpath.2021.03.006

Abstract:
Type I interferon (IFN-I) has a well-known function in controlling viral infections, but its contribution in hepatocyte proliferation and hepatocellular carcinoma (HCC) formation remains unclear. Mice deficient in IFN-α receptor expression in whole mice or only in hepatocytes (Ifnar−/− and IfnarΔliver) were used to investigate the role of IFN-I signaling in cell proliferation and cancer formation in the liver. Ifnar−/− mice were resistant to chemical-induced HCC formation in the absence of infection. The results show that low grade of IFN-I and interferon-stimulated gene were expressed substantially in naïve mouse liver. The low level of IFN-I activation is constantly present in mouse liver after weaning and negatively modulates forkhead box O hepatic expression. The IFN-I signaling can be partially blocked by the clearance of lipopolysaccharide. Mice lacking IFN-I signaling have lower basal proliferation activity and delayed liver regeneration processes after two-thirds partial hepatectomy. The activation of IFN-I signaling on hepatocyte controls glucose homeostasis and lipid metabolism to support proliferation potency and long-term tumorigenesis. Our results reveal a positive role of low-grade IFN-I singling to hepatocyte proliferation and HCC formation by modulating glucose homeostasis and lipid metabolism.
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.06.003

Yannan Wang, Fan Yan, Abu Nasar, Zhe-Sheng Chen, Nasser Khaled Altorki, Brendon Stiles, Navneet Narula,
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.05.018

Abstract:
Cullin 4A and 4B ubiquitin ligases are often highly accumulated in human malignancies, and are believed to possess oncogenic properties. However, the underlying mechanisms by which CUL4A and CUL4B promote pulmonary tumorigenesis remain largely elusive. In this study, we report that both CUL4A and CUL4B are highly expressed in non-small cell lung cancer (NSCLC) patients, and high expression of both is associated with disease progression, chemotherapy resistance, and poor survival in adenocarcinomas. Depletion of CUL4A (CUL4Ak/d) or CUL4B (CUL4Bk/d) leads to cell cycle arrest at G1 and loss of proliferation and viability of NSCLC cells both in culture and in a lung cancer xenograft model, suggesting that CUL4A and 4B are oncoproteins required for tumor maintenance of certain NSCLC. Mechanistically, increased accumulation of the cell cycle-dependent kinase inhibitor p21/Cip1/WAF1 was observed in lung cancer cells upon CUL4 silencing. Knockdown of p21 rescued the G1 arrest of CUL4Ak/d or CUL4Bk/d NSCLC cells and allowed proliferation to resume. These findings reveal that p21 is the primary downstream effector of lung adenocarcinoma dependence on CUL4, highlight the notion that not all substrates respond equally to abrogation of the CUL4 ubiquitin ligase in NSCLCs, and imply that CUL4Ahigh CUL4Bhigh may serve as a prognostic marker and therapeutic target for NSCLC patients.
Lingtong Meng, Masanori Goto, Hiroki Tanaka, Yuki Kamikokura, Yumiko Fujii, Yoko Okada, Hiroyuki Furukawa,
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.06.001

Abstract:
Nonalcoholic fatty liver disease (NAFLD) often progresses to cirrhosis and causes liver cancer, but mechanisms of its progression have not been elucidated. Although NAFLD is often associated with abnormal portal circulation, there have not been any experimental studies to test its pathogenic role. Here, we examined whether decreased portal circulation affected the pathology of nonalcoholic steatohepatitis (NASH) using congenital portosystemic shunt (PSS) in C57BL/6J mice. Whereas PSS significantly attenuated free radical-mediated CCl4 injury, it augmented pericellular fibrosis in the centrilobular area induced by a 0.1% methionine choline-deficient L-amino acid-defined high-fat diet (CDAHFD). PSS aggravated ductular reaction and increased the expression of connective tissue growth factor. Pimonidazole immunohistochemistry of the liver revealed that the centrilobular area of PSS-harboring mice was more hypoxic than that of control mice. While tissue hypoxia was observed in the fibrotic area in CDAHFD-induced NASH in both control and PSS-harboring mice, it was more profound in the latter, which was associated with higher carbonic anhydrase 9 and vascular endothelial growth factor expression and neovascularization in the fibrotic area. Furthermore, partial ligation of the portal vein also augmented pericellular fibrosis and ductular reaction induced by a CDAHFD. These results demonstrate that decreased portal circulation, which induces hypoxia due to disrupted intralobular perfusion, is an important aggravating factor of liver fibrosis in NASH.
, Yunpeng Du, , , Emma M. Lessieur, Jianying Kiser, Xiangyi Wen, Bruce A. Berkowitz, Timothy S. Kern
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.06.006

Abstract:
In this study, we test the hypothesis that diabetes promotes a greater than normal cytosolic calcium level in rod cells that activates a Ca2+-sensitive protease, calpain, resulting in oxidative stress and inflammation, two pathogenic factors of early diabetic retinopathy (DR). Nondiabetic and 2-months diabetic C57Bl/6J and calpain1 knockout (Capn1-/-) mice were studied; subgroups were treated with a calpain inhibitor (CI). Ca2+ content was measured in photoreceptors using Fura-2. Retinal calpain expression was studied by qTR-PCR and immunohistochemistry. Superoxide and expression of inflammatory proteins were measured using published methods. Proteomic analysis was conducted on photoreceptors isolated from diabetic mice untreated or treated daily with CI for 2-months. Cytosolic Ca+2 content was increased two-fold in photoreceptors of diabetic mice as compared to nondiabetic mice. Capn1 expression increased 5-fold in photoreceptor outer segments of diabetic mice. Pharmacologic inhibition or genetic deletion of Capn1 significantly suppressed diabetes-induced oxidative stress and expression of pro-inflammatory proteins in retina. Proteomics identified a protein (WWOX; WW domain-containing oxidoreductase) whose expression was significantly increased in photoreceptors from mice diabetic for 2-months and was inhibited with CI. Knockdown of Wwox using specific siRNA in vitro inhibited increase in superoxide caused by the high glucose. These results suggest that reducing Ca2+ accumulation, suppressing calpain activation, and / or reducing Wwox upregulation are novel targets for treating early DR.
Bryce D. Warren, Soo Hyun Ahn, Kathryn Brittain, Manjunatha K. Nanjappa, Hao Wang, Jianrong Wang, Gustavo Blanco, Gladis Sanchez, Yong Fan, Brian K. Petroff, et al.
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.05.021

Abstract:
Male factors, including those of autoimmune origin, contribute to approximately 50% of infertility cases in humans. However, the mechanisms underlying autoimmune male infertility are poorly understood. Deficiency in autoimmune regulator (AIRE) impairs central immune tolerance because of diminished expression of self-antigens in the thymus. Humans with AIRE mutations and mice with engineered ablation of Aire develop multi-organ autoimmunity and infertility. To determine the immune targets contributing to infertility in male Aire-deficient (-/-) mice, we paired Aire-/- or wild-type (WT) males with WT females. Aire-/- males exhibited dramatically reduced mating frequency and fertility, hypogonadism, and reduced serum testosterone. Approximately 15% of mice exhibited lymphocytic infiltration into the testis, accompanied by atrophy, azoospermia, and reduced numbers of mitotically active germ cells; the remaining mice showed normal testicular morphology, sperm counts, and motility. However, spermatozoa from all Aire-/- mice were defective in their ability to fertilize wild-type oocytes in vitro. Lymphocytic infiltration into the epididymis, seminal vesicle, and prostate gland was evident. Aire-/- male mice generated autoreactive antibodies in an age-dependent manner against sperm, testis, epididymis, prostate gland and seminal vesicle. Finally, expression of Aire was evident in the seminiferous epithelium in an age-dependent manner, as well as the prostate gland. These findings suggest that Aire-dependent central tolerance plays a critical role in maintaining male fertility by stemming autoimmunity against multiple reproductive targets.
, Joost M. Meijer, , Gilles F.H. Diercks, , Nicolai Petkov
The American Journal of Pathology; doi:10.1016/j.ajpath.2021.05.024

Abstract:
The u-serrated immunodeposition pattern in direct immunofluorescence (DIF) microscopy is a recognizable feature and confirmative for diagnosis of epidermolysis bullosa acquisita (EBA). Due to unfamiliarity with serrated patterns, serration pattern recognition is still of limited use in routine DIF microscopy. The objective of this study is to investigate the feasibility of using convolutional neural networks (CNNs) for the recognition of u-serrated patterns that can assist in diagnosis of EBA. Nine most commonly used CNNs are trained and validated by using 220,800 manually delineated DIF image patches from 106 images of 46 different patients. The dataset was split into 10 subsets; 9 training subsets from 42 patients to train CNNs and the remaining subset from the remaining 4 patients for validation dataset of diagnostic accuracy. This process was repeated 10 times with a different subset used for validation. The best performing CNN achieved specificity of 89.3% and corresponding sensitivity of 89.3% in the classification of u-serrated DIF image patches, a diagnostic accuracy of expert level. Experiments and results demonstrate the effectiveness of convolutional neural networks approaches for u-serrated patterns recognition with a high accuracy. The proposed approach can assist clinicians and pathologists in recognition of u-serrated patterns in DIF images, and facilitate diagnosis of EBA.
Gun-Dong Kim, Hang Pong Ng, ,
The American Journal of Pathology, Volume 191, pp 1118-1134; doi:10.1016/j.ajpath.2021.03.008

Abstract:
Macrophages play crucial and diverse roles in the pathogenesis of inflammatory vascular diseases. Macrophages are the principal innate immune cells recruited to arterial walls to govern vascular homeostasis by modulating the proliferation of vascular smooth muscle cells, the reorganization of extracellular matrix components, the elimination of dead cells, and the restoration of normal blood flow. However, chronic sterile inflammation within the arterial walls draws inflammatory macrophages into intimal/neointimal regions that may contribute to disease pathogenesis. In this context, the accumulation and aberrant activation of macrophages in the neointimal regions govern the progression of inflammatory arterial wall diseases. Herein, we report that myeloid–hypoxia-inducible factor-1α (HIF1α) deficiency attenuates vascular smooth muscle cells and macrophage abundance in stenotic arteries and abrogates carotid neointima formation in vivo. The integrated transcriptomics, Gene Set Enrichment Analysis, metabolomics, and target gene evaluation showed that HIF1α represses oxidative phosphorylation, tricarboxylic acid cycle, fatty acid metabolism, and c-MYC signaling pathways while promoting inflammatory, glycolytic, hypoxia response gene expression in stenotic artery macrophages. At the molecular level, proinflammatory agents utilized STAT3 signaling pathways to elevate HIF1α expression in macrophages. Collectively, this study uncovers that macrophage-HIF1α deficiency restrains the pathogenesis of carotid artery stenosis by rewiring inflammatory and metabolic signaling pathways in macrophages.
, Saori Nishikawa, Junichi Hosoi, Satoshi Amano
The American Journal of Pathology, Volume 191, pp 1010-1019; doi:10.1016/j.ajpath.2021.03.007

Abstract:
Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix of most mammalian tissues, including the epidermis. It is synthesized in epidermis, and mainly metabolized after transfer to the liver via lymphatic vessels in the dermis following its passage through the basement membrane (BM) at the dermal-epidermal junction. The aim of the present study was to investigate the influence of BM integrity on the level of HA in the epidermis. Epidermal HA content was decreased in sun-exposed skin of older subjects, whose BM structure was impaired, compared with sun-exposed young skin and sun-protected skin, in which BM integrity was well maintained. In an organotypic culture model of sun-exposed facial skin, epidermal HA was increased in the presence of inhibitors of BM-degrading matrix metalloproteinases and heparanase. In a skin equivalent model treated with these inhibitors, HA content was increased in the epidermis, but decreased in conditioned medium. These findings suggest that the BM at the dermal-epidermal junction plays an important role in maintaining epidermal HA levels.
Padmashree Rao, Xi Qiao, Winston Hua, , Mariah Tahan, Titi Chen, Hong Yu, Xiaojun Ren, Qi Cao, Yiping Wang, et al.
The American Journal of Pathology, Volume 191, pp 993-1009; doi:10.1016/j.ajpath.2021.03.005

Abstract:
Fibrosis is characterized by progressively excessive deposition of matrix components and may lead to organ failure. Transforming growth factor-β (TGF-β) is a key cytokine involved in tissue repair and fibrosis. TGF-β′s profibrotic signaling pathways converge at activation of β-catenin. β-Catenin is an important transcription cofactor whose function depends on its binding partner. Promoting β-catenin binding to forkhead box protein O (Foxo) via inhibition of its binding to T-cell factor (TCF) reduces kidney fibrosis in experimental murine models. Herein, we investigated whether β-catenin/Foxo diverts TGF-β signaling from profibrotic to physiological epithelial healing. In an in vitro model of wound healing (scratch assay), and in an in vivo model of kidney injury, unilateral renal ischemia reperfusion, TGF-β treatment in combination with either ICG-001 or iCRT3 (β-catenin/TCF inhibitors) increased β-catenin/Foxo interaction, increased scratch closure by increased cell proliferation and migration, reduced the TGF-β–induced mesenchymal differentiation, and healed the ischemia reperfusion injury with less fibrosis. In addition, administration of ICG-001 or iCRT3 reduced the contractile activity induced by TGF-β in C1.1 cells. Together, our results indicate that redirection of β-catenin binding from TCF to Foxo promotes β-catenin/Foxo–mediated epithelial repair. Targeting β-catenin/Foxo may rebuild normal structure of injured kidney.
Monica Sanchez-Ruiz, Ana-Maria Iorgu, Florian Küster, Martin Hellmich, Anna Brunn, Martina Deckert
The American Journal of Pathology, Volume 191, pp 1064-1076; doi:10.1016/j.ajpath.2021.02.021

Abstract:
In neuron-specific ovalbumin-transgenic CKTAC mice, antigen-specific OT-I CD8 T cells home to the enteric nervous system, where they attack and destroy neurons of the myenteric and submucosal plexus. Clinically, experimental autoimmune enteric ganglionitis (EAEG) manifests with gastrointestinal dysmotility and rapidly progresses to lethal ileus. Although interferon-γ has been identified as capable of damaging neurons in EAEG, the role of perforin, Fas/FasL, and tumor necrosis factor-α (TNF-α) in this disease is still a matter of debate. Thus, CKTAC mice were adoptively transferred with either perforin−/– or wild-type OT-I CD8 T cells. In addition, CKTAC mice that had received wild-type OT-I CD8 T cells were treated by either anti–TNF-α or anti-FasL. Furthermore, wild-type OT-I CD8 T cells were adoptively transferred into CKTAC mice with neuron-specific deletion of Fas. Although neither inactivation of enteric neuronal Fas nor anti-FasL treatment improved the disease, the absence of perforin from OT-I CD8 T cells and anti–TNF-α treatment significantly ameliorated EAEG and prevented lethal ileus by rescue of enteric neurons. Thus, these experiments identify perforin and TNF-α as important in the pathogenesis of EAEG.
The American Journal of Pathology, Volume 191, pp 967-967; doi:10.1016/j.ajpath.2021.04.002

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