PLOS ONE: [sortOrder=DATE_NEWEST_FIRST, sort=Date, newest first, q=subject:"Histochemistry and cytochemistry techniques"]PLOShttps://journals.plos.org/plosone/webmaster@plos.orgaccelerating the publication of peer-reviewed sciencehttps://journals.plos.org/plosone/search/feed/atom?sortOrder=DATE_NEWEST_FIRST&unformattedQuery=subject:%22Histochemistry+and+cytochemistry+techniques%22&sort=Date,+newest+firstAll PLOS articles are Open Access.https://journals.plos.org/plosone/resource/img/favicon.icohttps://journals.plos.org/plosone/resource/img/favicon.ico2024-03-28T10:49:37ZEstablishment of a lethal mouse model of emerging tick-borne orthonairovirus infectionsTakuma AriizumiKoshiro TabataYukari ItakuraHiroko KobayashiWilliam W. HallMichihito SasakiHirofumi SawaKeita MatsunoYasuko Orba10.1371/journal.ppat.10121012024-03-19T14:00:00Z2024-03-19T14:00:00Z<p>by Takuma Ariizumi, Koshiro Tabata, Yukari Itakura, Hiroko Kobayashi, William W. Hall, Michihito Sasaki, Hirofumi Sawa, Keita Matsuno, Yasuko Orba</p>
Emerging and reemerging tick-borne orthonairovirus infections (family <i>Nairoviridae</i>), which are genetically distinct from Crimean-Congo hemorrhagic fever virus, have been recently reported in East Asia. Here, we have established a mouse infection model using type-I/II interferon receptor-knockout mice (AG129) both for a better understanding the pathogenesis of these infections and validation of antiviral agents using Yezo virus (YEZV), a novel virus causing febrile illnesses associated with tick bites in Japan and China. YEZV-inoculated AG129 mice developed hepatitis with body weight loss and died by 6 days post infection. Blood biochemistry tests showed elevated liver enzyme levels, similar to YEZV-infected human patients. AG129 mice treated with favipiravir survived lethal YEZV infection, demonstrating the anti-YEZV effect of this drug. The present mouse model will help us better understand the pathogenicity of the emerging tick-borne orthonairoviruses and the development of specific antiviral agents for their treatment.Higher densities of T-lymphocytes in the subsynovial connective tissue of people with carpal tunnel syndromeOliver Sandy-HindmarchMiguel Molina-AlvarezAkira WibergDominic FurnissAnnina B. Schmid10.1371/journal.pone.03000462024-03-07T14:00:00Z2024-03-07T14:00:00Z<p>by Oliver Sandy-Hindmarch, Miguel Molina-Alvarez, Akira Wiberg, Dominic Furniss, Annina B. Schmid</p>
Symptoms in people with carpal tunnel syndrome (CTS) are traditionally attributed to neural tissue, but recent studies suggest that the subsynovial connective tissue (SSCT) may also play a role in CTS. The SSCT undergoes fibrotic thickening which is generally described as “non-inflammatory” based on basic histology. This study uses immunohistochemistry to determine the presence of macrophages and T-cells within SSCT and their relationship with symptoms in people with CTS. SSCT was collected from twenty people with CTS and eight controls undergoing wrist fracture surgery. Immunohistochemical quantification of CD3+ T-cells and CD68+ macrophage densities as well as CD4+/CD8+ T-cell subpopulations were compared between groups using independent t-tests. Spearman correlations were used to identify associations between immune cell densities and CTS symptom scores. The density of CD3+ T-cells was significantly higher in SSCT of people with CTS compared to controls (CTS mean 26.7 (SD 13.7); controls 6.78 (6.3), p = 0.0005) while the density of CD68+ macrophages was lower (CTS mean 9.5 (SD 6.0); controls 17.7 (8.2), p = 0.0058). Neither CD68+ nor CD3+ cell densities correlated with symptom scores. In contrast to previous assumptions, our data show that the SSCT in the carpal tunnel in both people with CTS and controls is not devoid of immune cells. Whereas the higher density of CD68+ macrophages in control participants may be associated with their early recruitment after acute fracture, CD3+ cells within the SSCT may play a role in chronic CTS.A xenotransplantation mouse model to study physiology of the mammary gland from large mammalsJames L. MillerAlexandra ReddyRebecca M. HarmanGerlinde R. Van de Walle10.1371/journal.pone.02983902024-02-28T14:00:00Z2024-02-28T14:00:00Z<p>by James L. Miller, Alexandra Reddy, Rebecca M. Harman, Gerlinde R. Van de Walle</p>
Although highly conserved in structure and function, many (patho)physiological processes of the mammary gland vary drastically between mammals, with mechanisms regulating these differences not well understood. Large mammals display variable lactation strategies and mammary cancer incidence, however, research into these variations is often limited to <i>in vitro</i> analysis due to logistical limitations. Validating a model with functional mammary xenografts from cryopreserved tissue fragments would allow for <i>in vivo</i> comparative analysis of mammary glands from large and/or rare mammals and would improve our understanding of postnatal development, lactation, and premalignancy across mammals. To this end, we generated functional mammary xenografts using mammary tissue fragments containing mammary stroma and parenchyma isolated via an antibody-independent approach from healthy, nulliparous equine and canine donor tissues to study these species <i>in vivo</i>. Cryopreserved mammary tissue fragments were xenotransplanted into de-epithelialized fat pads of immunodeficient mice and resulting xenografts were structurally and functionally assessed. Preimplantation of mammary stromal fibroblasts was performed to promote ductal morphogenesis. Xenografts recapitulated mammary lobule architecture and contained donor-derived stromal components. Mammatropic hormone stimulation resulted in (i) upregulation of lactation-associated genes, (ii) altered proliferation index, and (iii) morphological changes, indicating functionality. Preimplantation of mammary stromal fibroblasts did not promote ductal morphogenesis. This model presents the opportunity to study novel mechanisms regulating unique lactation strategies and mammary cancer induction <i>in vivo</i>. Due to the universal applicability of this approach, this model serves as proof-of-concept for developing mammary xenografts for <i>in vivo</i> analysis of virtually any mammals, including large and rare mammals.Smallpox lesion characterization in placebo-treated and tecovirimat-treated macaques using traditional and novel methodsTodd M. BellPaul FacemireJeremy J. BearssJo Lynne RaymondJennifer ChapmanXiankun ZengJoshua D. ShamblinJanice A. WilliamsDouglas W. GrosenbachDennis E. HrubyInger K. DamonArthur J. GoffEric M. Mucker10.1371/journal.ppat.10120072024-02-22T14:00:00Z2024-02-22T14:00:00Z<p>by Todd M. Bell, Paul Facemire, Jeremy J. Bearss, Jo Lynne Raymond, Jennifer Chapman, Xiankun Zeng, Joshua D. Shamblin, Janice A. Williams, Douglas W. Grosenbach, Dennis E. Hruby, Inger K. Damon, Arthur J. Goff, Eric M. Mucker</p>
Smallpox was the most rampant infectious disease killer of the 20<sup>th</sup> century, yet much remains unknown about the pathogenesis of the variola virus. Using archived tissue from a study conducted at the Centers for Disease Control and Prevention we characterized pathology in 18 cynomolgus macaques intravenously infected with the Harper strain of variola virus. Six macaques were placebo-treated controls, six were tecovirimat-treated beginning at 2 days post-infection, and six were tecovirimat-treated beginning at 4 days post-infection. All macaques were treated daily until day 17. Archived tissues were interrogated using immunohistochemistry, <i>in situ</i> hybridization, immunofluorescence, and electron microscopy. Gross lesions in three placebo-treated animals that succumbed to infection primarily consisted of cutaneous vesicles, pustules, or crusts with lymphadenopathy. The only gross lesions noted at the conclusion of the study in the three surviving placebo-treated and the Day 4 treated animals consisted of resolving cutaneous pox lesions. No gross lesions attributable to poxviral infection were present in the Day 2 treated macaques. Histologic lesions in three placebo-treated macaques that succumbed to infection consisted of proliferative and necrotizing dermatitis with intracytoplasmic inclusion bodies and lymphoid depletion. The only notable histologic lesion in the Day 4 treated macaques was resolving dermatitis; no notable lesions were seen in the Day 2 treated macaques. Variola virus was detected in all three placebo-treated animals that succumbed to infection prior to the study’s conclusion by all utilized methods (IHC, ISH, IFA, EM). None of the three placebo-treated animals that survived to the end of the study nor the animals in the two tecovirimat treatment groups showed evidence of variola virus by these methods. Our findings further characterize variola lesions in the macaque model and describe new molecular methods for variola detection.Quantitative detection and prognostic value of antibodies against M-type phospholipase A2 receptor and its cysteine-rich ricin domain and C-type lectin domains 1 and 6-7-8 in patients with idiopathic membranous nephropathyXiaobin LiuJing XueTing LiQingqing WuHuiming ShengXue YangBo LinXiumei ZhouYuan QinZijian HuangLeting ZhouLiang WangZhigang HuBiao Huang10.1371/journal.pone.02982692024-02-22T14:00:00Z2024-02-22T14:00:00Z<p>by Xiaobin Liu, Jing Xue, Ting Li, Qingqing Wu, Huiming Sheng, Xue Yang, Bo Lin, Xiumei Zhou, Yuan Qin, Zijian Huang, Leting Zhou, Liang Wang, Zhigang Hu, Biao Huang</p>
Background <p>M-type phospholipase A2 receptor (PLA2R) is the major autoantigen in adult idiopathic membranous nephropathy (IMN). Although reactive epitopes in the PLA2R domains have been identified, the clinical value of these domains recognized by anti-PLA2R antibodies remains controversial. Accordingly, this study aimed to quantitatively detect changes in the concentrations of different antibodies against epitopes of PLA2R in patients with IMN before and after treatment to evaluate the clinical value of epitope spreading.</p> Methods <p>Highly sensitive time-resolved fluorescence immunoassay was used to quantitatively analyze the concentrations of specific IgG and IgG4 antibodies against PLA2R and its epitopes (CysR, CTLD1, CTLD6-7-8) in a cohort of 25 patients with PLA2R-associated membranous nephropathy (13 and 12 in the remission and non-remission groups, respectively) before and after treatment, and the results were analyzed in conjunction with clinical biochemical indicators.</p> Results <p>The concentration of specific IgG (IgG4) antibodies against PLA2R and its epitopes (CysR, CTLD1 and CTLD6-7-8) in non-remission group was higher than that in remission group. The multipliers of elevation of IgG (IgG4) antibody were 5.6(6.2) fold, 3.0(24.3) fold, 1.6(9.0) fold, and 4.2(2.6) fold in the non-remission/remission group, respectively. However, the difference in antibody concentrations between the two groups at the end of follow-up was 5.6 (85.2), 1.7 (13.1), 1.0 (5.1), and 1.5 (22.3) times higher, respectively. When detecting concentrations of specific IgG antibodies against PLA2R and its different epitopes, the remission rate was 66.67% for only one epitope at M0 and 36.36% for three epitopes at M0. When detecting concentrations of specific IgG4 antibodies against PLA2R and its different epitopes, the remission rate was 100.00% for only one epitope at M0 and 50.00% for three epitopes at M0. A trivariate logistic regression model for the combined detection of eGFR, anti-CTLD678 IgG4, and urinary protein had an AUC of 100.00%.</p> Conclusion <p>Low concentrations of anti-CysR-IgG4, anti-CTLD1-IgG4, and anti-CTLD6-7-8-IgG4 at initial diagnosis predict rapid remission after treatment. The use of specific IgG4 against PLA2R and its different epitopes combined with eGFR and urinary protein provides a better assessment of the prognostic outcome of IMN.</p>Time-course analysis of liver and serum galectin-3 in acute liver injury after alpha-galactosylceramide injectionMikiko MatsuoAyumu KanbeKei NoguchiAyumi NiwaYuko ImaizumiTakahito KurodaKoki IchihashiTakafumi OkuboKosuke MoriTomohiro KanayamaHiroyuki TomitaAkira Hara10.1371/journal.pone.02982842024-02-08T14:00:00Z2024-02-08T14:00:00Z<p>by Mikiko Matsuo, Ayumu Kanbe, Kei Noguchi, Ayumi Niwa, Yuko Imaizumi, Takahito Kuroda, Koki Ichihashi, Takafumi Okubo, Kosuke Mori, Tomohiro Kanayama, Hiroyuki Tomita, Akira Hara</p>
Galectin-3 is a beta-galactoside-binding lectin that plays important roles in diverse physiological functions, such as cell proliferation, apoptosis, and mRNA splicing. This protein is expressed on inflammatory cells and acts as a local inflammatory mediator. Recently, galectin-3 has been detected in several diseases, such as chronic liver, heart, and kidney diseases, diabetes, viral infection, autoimmune and neurodegenerative diseases, and tumors, and its role as a biomarker has attracted attention. Alpha-galactosylceramide is an artificially synthesized sphingolipid that can induce acute liver injury via the natural killer T pathway. However, the pathophysiological roles and kinetics of galectin-3 in acute liver injury are not fully understood. This study aimed to elucidate the expression and time course of galectin-3 in liver tissues during acute liver injury following alpha-galactosylceramide injection. Animals were histologically examined on days 1, 2, 4, and 7 after intraperitoneal injection of alpha-galactosylceramide, and the expressions of galectin-3 and ionized calcium-binding adaptor molecule 1 were analyzed. Notably, galectin-3 formed characteristic cluster foci, particularly on day 2 after injection. Cluster formation was not observed in chronic liver disease. Simultaneously, ionized calcium-binding adaptor molecule 1-positive cells were observed in the cluster foci. Serum galectin-3 levels increased on day 2 of treatment and correlated well with the number of galectin-3-positive cell clusters in the liver. Moreover, galectin-3 expression was an important mediator of the early phase of liver injury after alpha-galactosylceramide injection. These results suggest that serum galectin-3 may be a biomarker for the early diagnosis of acute liver injury and that clusters of galectin-3-positive cells may be a specific finding in acute liver injury.Detecting microsatellite instability in colorectal cancer using Transformer-based colonoscopy image classification and retrievalChung-Ming LoJeng-Kai JiangChun-Chi Lin10.1371/journal.pone.02922772024-01-25T14:00:00Z2024-01-25T14:00:00Z<p>by Chung-Ming Lo, Jeng-Kai Jiang, Chun-Chi Lin</p>
Colorectal cancer (CRC) is a major global health concern, with microsatellite instability-high (MSI-H) being a defining characteristic of hereditary nonpolyposis colorectal cancer syndrome and affecting 15% of sporadic CRCs. Tumors with MSI-H have unique features and better prognosis compared to MSI-L and microsatellite stable (MSS) tumors. This study proposed establishing a MSI prediction model using more available and low-cost colonoscopy images instead of histopathology. The experiment utilized a database of 427 MSI-H and 1590 MSS colonoscopy images and vision Transformer (ViT) with different feature training approaches to establish the MSI prediction model. The accuracy of combining pre-trained ViT features was 84% with an area under the receiver operating characteristic curve of 0.86, which was better than that of DenseNet201 (80%, 0.80) in the experiment with support vector machine. The content-based image retrieval (CBIR) approach showed that ViT features can obtain a mean average precision of 0.81 compared to 0.79 of DenseNet201. ViT reduced the issues that occur in convolutional neural networks, including limited receptive field and gradient disappearance, and may be better at interpreting diagnostic information around tumors and surrounding tissues. By using CBIR, the presentation of similar images with the same MSI status would provide more convincing deep learning suggestions for clinical use.Evaluating deep learning-based melanoma classification using immunohistochemistry and routine histology: A three center studyChristoph WiesLucas SchneiderSarah HaggenmüllerTabea-Clara BucherSarah HobelsbergerMarkus V. HepptGerardo FerraraEva I. Krieghoff-HenningTitus J. Brinker10.1371/journal.pone.02971462024-01-19T14:00:00Z2024-01-19T14:00:00Z<p>by Christoph Wies, Lucas Schneider, Sarah Haggenmüller, Tabea-Clara Bucher, Sarah Hobelsberger, Markus V. Heppt, Gerardo Ferrara, Eva I. Krieghoff-Henning, Titus J. Brinker</p>
Pathologists routinely use immunohistochemical (IHC)-stained tissue slides against MelanA in addition to hematoxylin and eosin (H&E)-stained slides to improve their accuracy in diagnosing melanomas. The use of diagnostic Deep Learning (DL)-based support systems for automated examination of tissue morphology and cellular composition has been well studied in standard H&E-stained tissue slides. In contrast, there are few studies that analyze IHC slides using DL. Therefore, we investigated the separate and joint performance of ResNets trained on MelanA and corresponding H&E-stained slides. The MelanA classifier achieved an area under receiver operating characteristics curve (AUROC) of 0.82 and 0.74 on out of distribution (OOD)-datasets, similar to the H&E-based benchmark classification of 0.81 and 0.75, respectively. A combined classifier using MelanA and H&E achieved AUROCs of 0.85 and 0.81 on the OOD datasets. DL MelanA-based assistance systems show the same performance as the benchmark H&E classification and may be improved by multi stain classification to assist pathologists in their clinical routine.Propolis alleviates ulcerative colitis injury by inhibiting the protein kinase C ‐ transient receptor potential cation channel subfamily V member 1 ‐ calcitonin gene-related peptide/substance P (PKC-TRPV1-CGRP/SP) signaling axisZhen QianMengjie ZhangTaiyu LuJiayi YuSiyuan YinHaihua WangJing Wang10.1371/journal.pone.02941692024-01-11T14:00:00Z2024-01-11T14:00:00Z<p>by Zhen Qian, Mengjie Zhang, Taiyu Lu, Jiayi Yu, Siyuan Yin, Haihua Wang, Jing Wang</p>
This study investigated the protective effect of water-soluble propolis (WSP) on colonic tissues in ulcerative colitis (UC) and the role of the protein kinase C ‐ transient receptor potential cation channel subfamily V member 1 ‐ calcitonin gene-related peptide/substance P (PKC-TRPV1-CGRP/SP) signaling pathway. Male SD rats were divided into a control group, a UC model group, various WSP groups (Low-WSP, Medium-WSP, and High-WSP) with UC, and a salazosulfapyridine (SASP) positive control group with UC. After UC was established, the WSP and SASP groups were treated with WSP or SASP, respectively, for 7 d. Each day, body weight measurements were obtained, and the disease activity index (DAI) was recorded by observing fecal characteristics and blood in the stool. After the experiment, hematoxylin and eosin (HE) colonic tissue staining was performed to observe pathological changes, western blotting and immunohistochemistry were performed to detect PKC, TRPV1, CGRP, and SP expression in colonic tissues, and laser confocal microscopy was performed to observe the fluorescence colocalization of PKC/TRPV1, TRPV1/CGRP, and TRPV1/SP. HE staining showed significant colonic tissue structure disruption and inflammatory infiltration in the UC group. Western blotting and immunohistochemistry showed that the expression of PKC, TRPV1, CGRP, and SP in the colonic tissues of the UC group increased significantly compared with that of the control group. Compared with the UC group, the expression of PKC, TRPV1, CGRP, and SP in colonic tissues was significantly reduced in the High-WSP, Medium-WSP, and SASP groups. Immunofluorescence showed the colocalized expression of PKC/TRPV1, TRPV1/CGRP, and TRPV1/SP proteins in the colon tissue of the UC group was significantly reduced after WSP and SASP interventions compared with that of the control group. The results suggest that the mechanism of UC alleviation by propolis may inhibit the PKC-TRPV1-CGRP/SP signaling pathway and the release of inflammatory mediators, thus alleviating inflammation.FBXO3 stabilizes USP4 and Twist1 to promote PI3K-mediated breast cancer metastasisJing XuRongtian GuoNasi WenLuping LiYong YiJingzhen ChenZongyu HeJian YangZhi-Xiong Jim XiaoMengmeng Niu10.1371/journal.pbio.30024462023-12-22T14:00:00Z2023-12-22T14:00:00Z<p>by Jing Xu, Rongtian Guo, Nasi Wen, Luping Li, Yong Yi, Jingzhen Chen, Zongyu He, Jian Yang, Zhi-Xiong Jim Xiao, Mengmeng Niu</p>
Tumor metastasis is the major cause of breast cancer morbidity and mortality. It has been reported that the F-box protein FBXO3 functions as an E3 ubiquitin ligase in regulating various biological processes, including host autoimmune, antiviral innate immunity, and inflammatory response. However, the role of FBXO3 in tumor metastasis remains elusive. We have previously shown that ΔNp63α is a common inhibitory target in oncogene-induced cell motility and tumor metastasis. In this study, we show that FBXO3 plays a vital role in PI3K-mediated breast cancer metastasis independent of its E3 ligase activity and ΔNp63α in breast cancer cells and in mouse. FBXO3 can bind to and stabilize USP4, leading to Twist1 protein stabilization and increased breast cancer cell migration and tumor metastasis. Mechanistically, FBXO3 disrupts the interaction between USP4 and aspartyl aminopeptidase (DNPEP), thereby protecting USP4 from DNPEP-mediated degradation. Furthermore, p110α<sup>H1047R</sup> facilitates the phosphorylation and stabilization of FBXO3 in an ERK1-dependent manner. Knockdown of either FBXO3 or USP4 leads to significant inhibition of PI3K-induced breast cancer metastasis. Clinically, elevated expression of p110α/FBXO3/USP4/Twist1 is associated with poor overall survival (OS) and recurrence-free survival (RFS) of breast cancer patients. Taken together, this study reveals that the FBXO3-USP4-Twist1 axis is pivotal in PI3K-mediated breast tumor metastasis and that FBXO3/USP4 may be potential therapeutic targets for breast cancer treatment.Evaluation of the osteogenic potential of demineralized and decellularized bovine bone granules following implantation in rat calvaria critical-size defect modelAli Al QabbaniK. G. Aghila RaniSausan AlKawasSuzina Sheikh Abdul HamidAbdullah Yap AbdullahA. R. SamsudinAhmad Azlina10.1371/journal.pone.02942912023-12-21T14:00:00Z2023-12-21T14:00:00Z<p>by Ali Al Qabbani, K. G. Aghila Rani, Sausan AlKawas, Suzina Sheikh Abdul Hamid, Abdullah Yap Abdullah, A. R. Samsudin, Ahmad Azlina</p>
The aim of this study was to compare the ability of demineralized (DMB) and decellularized (DCC) bovine bone granules to support bone regeneration in rat calvaria critical-size defects. DMB and DCC were prepared using a previously published method. The granule size used ranged between 500 and 750 μm. A total of forty-eight Sprague-Dawley rats were divided into two groups (n = 24). A pair of 5 mm diameter defects were created on the calvaria of the rats in the right and left parietal bone in both groups. Group A animals received DMB granules and Group B received DCC granules in the right parietal defect side while the left parietal untreated defect acted as sham surgery for both groups. Four animals per group were euthanized in a CO<sub>2</sub> chamber at day 7, 14 and 21 post-surgery and the calvaria implantation site biopsy harvested was subjected to osteogenic gene expression analysis. Another four animals per group were euthanized at days 15, 30 and 60 post surgery and the calvaria implantation site biopsy harvested was subjected to histological, immunohistochemistry, RAMAN spectroscopy and Micro-CT analysis at the mentioned time points. Statistical analysis was conducted using t-tests and ANOVA. Histomorphometry showed significantly higher new bone formation in the DCC sites (p<0.05) compared to DMB. Both DMB and DCC implantation sites showed distinct staining for osteocalcin and osteopontin proteins compared to their respective sham sites. By day 21 after implantation, DCC sites demonstrated significantly elevated mRNA levels of osteonectin (p<0.001), osteopontin (p<0.001), osteocalcin (p<0.0001), ALP (p<0.01), and BMP-2 (p<0.001) compared to DMB. However, VEGF expression showed no significant differences at this time point between the two groups. Micro-CT analysis also showed enhanced defect closure and higher bone density in DCC implanted sites while RAMAN spectra demonstrated increased abundance of collagen and bone minerals, especially, PO<sub>4</sub><sup>3-</sup> ions than DMB. In conclusion, both DMB and DCC granules demonstrated favorable osteogenic potential in critical-sized defects, with DCC exhibited superior osteoconductive, osteoinductive and osteogenesis properties.Gallium-68-labeled fibroblast activation protein inhibitor-46 PET in patients with resectable or borderline resectable pancreatic ductal adenocarcinoma: A phase 2, multicenter, single arm, open label non-randomized study protocolAashna KarbhariSherly MosessianKamaxi H. TrivediFrank Valla JrMark JacobsonMark J. TrutyNandakumar G. PatnamDiane M. SimeoneElcin ZanTracy BrennanHongli ChenPhillip H. KuoKen HerrmannAjit H. Goenka10.1371/journal.pone.02945642023-11-27T14:00:00Z2023-11-27T14:00:00Z<p>by Aashna Karbhari, Sherly Mosessian, Kamaxi H. Trivedi, Frank Valla Jr, Mark Jacobson, Mark J. Truty, Nandakumar G. Patnam, Diane M. Simeone, Elcin Zan, Tracy Brennan, Hongli Chen, Phillip H. Kuo, Ken Herrmann, Ajit H. Goenka</p>
Background <p>Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease prone to widespread metastatic dissemination and characterized by a desmoplastic stroma that contributes to poor outcomes. Fibroblast activation protein (FAP)-expressing Cancer-Associated Fibroblasts (CAFs) are crucial components of the tumor stroma, influencing carcinogenesis, fibrosis, tumor growth, metastases, and treatment resistance. Non-invasive tools to profile CAF identity and function are essential for overcoming CAF-mediated therapy resistance, developing innovative targeted therapies, and improved patient outcomes. We present the design of a multicenter phase 2 study (clinicaltrials.gov identifier NCT05262855) of [<sup>68</sup>Ga]FAPI-46 PET to image FAP-expressing CAFs in resectable or borderline resectable PDAC.</p> Methods <p>We will enroll up to 60 adult treatment-naïve patients with confirmed PDAC. These patients will be eligible for curative surgical resection, either without prior treatment (Cohort 1) or after neoadjuvant therapy (NAT) (Cohort 2). A baseline PET scan will be conducted from the vertex to mid-thighs approximately 15 minutes after administering 5 mCi (±2) of [<sup>68</sup>Ga]FAPI-46 intravenously. Cohort 2 patients will undergo an additional PET after completing NAT but before surgery. Histopathology and FAP immunohistochemistry (IHC) of initial diagnostic biopsy and resected tumor samples will serve as the truth standards. Primary objective is to assess the sensitivity, specificity, and accuracy of [<sup>68</sup>Ga]FAPI-46 PET for detecting FAP-expressing CAFs. Secondary objectives will assess predictive values and safety profile validation. Exploratory objectives are comparison of diagnostic performance of [<sup>68</sup>Ga]FAPI-46 PET to standard-of-care imaging, and comparison of pre- versus post-NAT [<sup>68</sup>Ga]FAPI-46 PET in Cohort 2.</p> Conclusion <p>To facilitate the clinical translation of [<sup>68</sup>Ga]FAPI-46 in PDAC, the current study seeks to implement a coherent strategy to mitigate risks and increase the probability of meeting FDA requirements and stakeholder expectations. The findings from this study could potentially serve as a foundation for a New Drug Application to the FDA.</p> Trial registration <p><sup>@</sup>ClinicalTrials.gov identifier NCT05262855.</p>Human dermal fibroblast subpopulations and epithelial mesenchymal transition signals in hidradenitis suppurativa tunnels are normalized by spleen tyrosine kinase antagonism in vivoAkshay FloraRebecca JepsenEmily K. KozeraJane A. WoodsGeoffrey D. CainsMichael RadzietaSlade O. JensenMatthew MaloneJohn W. Frew10.1371/journal.pone.02827632023-11-03T14:00:00Z2023-11-03T14:00:00Z<p>by Akshay Flora, Rebecca Jepsen, Emily K. Kozera, Jane A. Woods, Geoffrey D. Cains, Michael Radzieta, Slade O. Jensen, Matthew Malone, John W. Frew</p>
Hidradenitis Suppurativa is a chronic inflammatory disease of which the pathogenesis is incompletely understood. Dermal fibroblasts have been previously identified as a major source of inflammatory cytokines, however information pertaining to the characteristics of subpopulations of fibroblasts in HS remains unexplored. Using in silico-deconvolution of whole-tissue RNAseq, Nanostring gene expression panels and confirmatory immunohistochemistry we identified fibroblast subpopulations in HS tissue and their relationship to disease severity and lesion morphology. Gene signatures of SFRP2+ fibroblast subsets were increased in lesional tissue, with gene signatures of SFRP1+ fibroblast subsets decreased. SFRP2+ and CXCL12+ fibroblast numbers, measured by IHC, were increased in HS tissue, with greater numbers associated with epithelialized tunnels and Hurley Stage 3 disease. Pro-inflammatory CXCL12+ fibroblasts were also increased, with reductions in SFRP1+ fibroblasts compared to healthy controls. Evidence of Epithelial Mesenchymal Transition was seen via altered gene expression of SNAI2 and altered protein expression of ZEB1, TWIST1, Snail/Slug, E-Cadherin and N-Cadherin in HS lesional tissue. The greatest dysregulation of EMT associated proteins was seen in biopsies containing epithelialized tunnels. The use of the oral Spleen tyrosine Kinase inhibitor Fostamatinib significantly reduced expression of genes associated with chronic inflammation, fibroblast proliferation and migration suggesting a potential role for targeting fibroblast activity in HS.Apical-basal distribution of different subtypes of spiral ganglion neurons in the cochlea and the changes during agingMeijian WangShengyin LinRuili Xie10.1371/journal.pone.02926762023-10-26T14:00:00Z2023-10-26T14:00:00Z<p>by Meijian Wang, Shengyin Lin, Ruili Xie</p>
Sound information is transmitted from the cochlea to the brain mainly by type I spiral ganglion neurons (SGNs), which consist of different subtypes with distinct physiological properties and selective expression of molecular markers. It remains unclear how these SGN subtypes distribute along the tonotopic axis, and whether the distribution pattern changes during aging that might underlie age-related hearing loss (ARHL). We investigated these questions using immunohistochemistry in three age groups of CBA/CaJ mice of either sex, including 2–5 months (young), 17–19 months (middle-age), and 28–32 months (old). Mouse cochleae were cryo-sectioned and triple-stained using antibodies against Tuj1, calretinin (CR) and calbindin (CB), which are reportedly expressed in all type I, subtype I<sub>a</sub>, and subtype I<sub>b</sub> SGNs, respectively. Labeled SGNs were classified into four groups based on the expression pattern of stained markers, including CR<sup>+</sup> (subtype I<sub>a</sub>), CB<sup>+</sup> (subtype I<sub>b</sub>), CR<sup>+</sup>CB<sup>+</sup> (dual-labeled I<sub>a</sub>/I<sub>b</sub>), and CR<sup>-</sup>CB<sup>-</sup> (subtype I<sub>c</sub>) neurons. The distribution of these SGN groups was analyzed in the apex, middle, and base regions of the cochleae. It showed that the prevalence of subtype I<sub>a</sub>, I<sub>b</sub> and dual-labeled I<sub>a</sub>/I<sub>b</sub> SGNs are high in the apex and low in the base. In contrast, the distribution pattern is reversed in I<sub>c</sub> SGNs. Such frequency-dependent distribution is largely maintained during aging except for a preferential reduction of I<sub>c</sub> SGNs, especially in the base. These findings corroborate the prior study based on RNAscope that SGN subtypes show differential vulnerability during aging. It suggests that sound processing of different frequencies involves distinct combinations of SGN subtypes, and the age-dependent loss of I<sub>c</sub> SGNs in the base may especially impact high-frequency hearing during ARHL.A modified method to analyse cell proliferation using EdU labelling in large insect brainsAmaia Alcalde AntonMax S. FarnworthLaura HebberechtC. Jill HarrisonStephen H. Montgomery10.1371/journal.pone.02920092023-10-05T14:00:00Z2023-10-05T14:00:00Z<p>by Amaia Alcalde Anton, Max S. Farnworth, Laura Hebberecht, C. Jill Harrison, Stephen H. Montgomery</p>
The study of neurogenesis is critical to understanding of the evolution of nervous systems. Within invertebrates, this process has been extensively studied in <i>Drosophila melanogaster</i>, which is the predominant model thanks to the availability of advanced genetic tools. However, insect nervous systems are extremely diverse, and by studying a range of taxa we can gain additional information about how nervous systems and their development evolve. One example of the high diversity of insect nervous system diversity is provided by the mushroom bodies. Mushroom bodies have critical roles in learning and memory and vary dramatically across species in relative size and the type(s) of sensory information they process. Heliconiini butterflies provide a useful snapshot of this diversity within a closely related clade. Within Heliconiini, the genus <i>Heliconius</i> contains species where mushroom bodies are 3–4 times larger than other closely related genera, relative to the rest of the brain. This variation in size is largely explained by increases in the number of Kenyon cells, the intrinsic neurons which form the mushroom body. Hence, variation in mushroom body size is the product of changes in cell proliferation during Kenyon cell neurogenesis. Studying this variation requires adapting labelling techniques for use in less commonly studied organisms, as methods developed for common laboratory insects often do not work. Here, we present a modified protocol for EdU staining to examine neurogenesis in large-brained insects, using Heliconiini butterflies as our primary case, but also demonstrating applicability to cockroaches, another large-brained insect.