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Although type 1 diabetes (T1D) results from the autoimmune destruction of the insulin-producing β-cells, its treatment is largely restricted to exogenous insulin administration. Only few therapies targeting the autoaggressive immune system have been introduced into clinical practice or are considered in clinical trials. Here, we provide a gene expression profile of the islet microenvironment obtained by laser-dissection microscopy in an inducible mouse model. Thereby, we have identified novel targets for immune intervention. Increased gene expression of most inflammatory proteins was apparent at day 10 after T1D induction and largely paralleled the observed degree of insulitis. We further focused on genes involved in leukocyte migration, including chemokines and their receptors. Besides the critical chemokine CXCL10, we found several other chemokines upregulated locally in temporary or chronic manner. Localization of the chemokine ligand/receptor pairs to the islet microenvironment has been confirmed by RNAscope. Interference with the CXCL16-CXCR6 and CX 3 CL1-CX 3 CR1 axes, but not the CCL5-CCR1/3/5 axis, resulted in reduced insulitis and lower T1D incidence. Further, we found that the receptors for the differentially expressed chemokines CXCL10, CXCL16 and CX 3 CL1 are distributed unevenly among islet autoantigen-specific T cells, which explains why the interference with just one chemokine axis cannot completely abrogate insulitis and T1D. [Display omitted] • The autoaggressive immune response is still a neglected target in T1D. • By laser-dissecting microscopy of islet microenvironments we have identified novel targets for a therapeutic intervention. • Interference with the CXCL16-CXCR6 and CX 3 CL1-CX 3 CR1 axes resulted in reduced insulitis and lower T1D incidence. • There is uneven distribution of chemokine receptors on autoaggressive T cells. • Combination therapies targeting more than one chemokine axes might be more successful than monotherapies. [ABSTRACT FROM AUTHOR]

Abstract The expression of programmed cell death ligand 1 (PD‐L1) is a biomarker for immunotherapy, but approved detection method is absent in diffuse large B‐cell lymphoma (DLBCL). Here, we performed three methods including immunohistochemistry (IHC) (clone SP263 and SP142), RNAscope, and fluorescence in situ hybridization (FISH) to evaluate PD‐L1 status on a cohort of DLBCL including 94 of DLBCL‐NOS, 25 of primary mediastinal large B‐cell lymphoma (PMBCL) and 7 of double‐hit lymphoma (DHL). SP263 with 25% for immune cell (IC) or combined cell and SP142 with 10% for tumor cell (TC), 20% for both of IC and combined cell were proved to have corresponding survival prognostic. Combined+ showed comparable prognostic value with TC+ and IC+. SP263 and SP142 showed strong concordance (k = 0.788) with combined+ rates of 33.3% (42/126) and 34.9% (44/126), respectively. In DLBCL‐NOS, TC+ by SP263 preferred to non‐GCB and immunoblastic variant DLBCL‐NOS (P = 0.029 and P = 0.004). Combined+ (SP263 and SP142) were associated with more than one extranodal site involved (P = 0.006, P = 0.042), higher ECOG PS scores (P = 0.001, P

The prevalence of HIV-1 associated neurocognitive disorders (HAND) is significantly greater in older, relative to younger, HIV-1 seropositive individuals; the neural pathogenesis of HAND in older HIV-1 seropositive individuals, however, remains elusive. To address this knowledge gap, abnormal protein aggregates (i.e., β-amyloid) were investigated in the brains of aging (>12 months of age) HIV-1 transgenic (Tg) rats. In aging HIV-1 Tg rats, double immunohistochemistry staining revealed abnormal intraneuronal β-amyloid accumulation in the prefrontal cortex (PFC) and hippocampus, relative to F344/N control rats. Notably, in HIV-1 Tg animals, increased β-amyloid accumulation occurred in the absence of any genotypic changes in amyloid precursor protein (APP). Furthermore, no clear amyloid plaque deposition was observed in HIV-1 Tg animals. Critically, β-amyloid was co-localized with neurons in the cortex and hippocampus, supporting a potential mechanism underlying synaptic dysfunction in the HIV-1 Tg rat. Consistent with these neuropathological findings, HIV-1 Tg rats exhibited prominent alterations in the progression of temporal processing relative to control animals; temporal processing relies, at least in part, on the integrity of the PFC and hippocampus. In addition, in post-mortem HIV-1 seropositive individuals with HAND, intraneuronal β-amyloid accumulation was observed in the dorsolateral PFC and hippocampal dentate gyrus. Consistent with observations in the HIV-1 Tg rat, no amyloid plaques were found in these post-mortem HIV-1 seropositive individuals with HAND. Collectively, intraneuronal β-amyloid aggregation observed in the PFC and hippocampus of HIV-1 Tg rats supports a potential factor underlying HIV-1 associated synaptodendritic damage. Further, the HIV-1 Tg rat provides a biological system to model HAND in older HIV-1 seropositive individuals.

RNA is considered as an indicator of the dynamic genetic expression changes in a cell. RNAScope is a commercially available in situ hybridization assay for the detection of RNA in formalin-fixed paraffin-embedded tissue. In this work, we describe the use of RNAScope as a sensitive and specific method for the evaluation of c-KIT messenger RNA (mRNA) in canine mast cell tumor. We investigated the expression of c-KIT mRNA with RNAscope in 60 canine mast cell tumors (MCTs), comparing it with the histological grade and KIT immunohistochemical expression patterns. Our results showed an overall good expression of c-KIT mRNA in neoplastic cells if compared with control probes. We also observed a statistically significant correlation between histological grade and c-KIT mRNA expression. No correlations were found between KIT protein immunohistochemical distribution pattern and c-KIT mRNA expression or histological grade. Our results provide a reference basis to better understand c-KIT mRNA expression in canine MCTs and strongly encourage further studies that may provide useful information about its potential and significant role as a prognostic and predictive biological marker for canine MCTs clinical outcome.

Introduction: Microglial TYROBP (DAP12) is a network hub and driver in sporadic late‐onset Alzheimer's disease (AD). TYROBP is a cytoplasmic adaptor for TREM2 and other receptors, but little is known about its roles and actions in AD. Herein, we demonstrate that endogenous Tyrobp transcription is specifically increased in recruited microglia. Methods: Using a novel transgenic mouse overexpressing TYROBP in microglia, we observed a decrease of the amyloid burden and an increase of TAU phosphorylation stoichiometry when crossed with APP/PSEN1 or MAPTP301S mice, respectively. Characterization of these mice revealed Tyrobp‐related modulation of apolipoprotein E (Apoe) transcription. We also showed that Tyrobp and Apoe mRNAs were increased in Trem2‐null microglia recruited around either amyloid beta deposits or a cortical stab injury. Conversely, microglial Apoe transcription was dramatically diminished when Tyrobp was absent. Conclusions: Our results provide evidence that TYROBP‐APOE signaling does not require TREM2 and could be an initiating step in establishment of the disease‐associated microglia (DAM) phenotype. [ABSTRACT FROM AUTHOR]

Background: Restaurant oil in poultry diets increases energy content, reduces production costs, and promotes sustainability within the food supply chain. However, variable oil composition and heating temperatures among restaurant oil sources can impact broiler chicken health due to heat-induced lipid modifications.Objectives: A 21-d experiment was conducted to evaluate ileal morphology, liver cytokine gene expression, and ileal immune cell populations in broilers fed control or peroxidized lipids with varying chain and saturation characteristics.Methods: Day-old broilers were housed in battery cages (5 birds per cage) and fed diets containing 5% control or peroxidized oils. Eight diets were randomly assigned in a 4 × 2 factorial arrangement consisting of oil source (palm, soybean, flaxseed, or fish) and peroxidation status (control or peroxidized). At day 21, samples were collected for ileal histomorphology [villus height (VH), crypt depth (CrD), and the VH:CrD ratio], and liver cytokine expression (qPCR). Ileum cytokine expression and T-cell markers were analyzed by RNAscope in situ hybridization (ISH). Data were analyzed as a mixed model (SAS 9.4) with fixed effects of lipid source, peroxidation, and lipid × peroxidation interaction.Results: CD3+ T-cells in the ileum decreased 16.2% due to peroxidation (P = 0.001) with 30.3% reductions observed in birds fed peroxidized flaxseed oil (P = 0.01). Peroxidation increased IL6+ and IL1B+ cells by 62.0% and 40.3%, respectively (P = 0.01). Soybean oil increased IFNG+ cells by 55.1% compared with palm oil, regardless of peroxidation status (P = 0.007). Lipid source and peroxidation did not alter ileal histomorphology or liver cytokine expression.Conclusions: Lipid peroxidation increased ileal IL1B and IL6 in broiler chickens, whereas soybean oil diets increased IFNG. Generally, peroxidation decreased overall CD3+ T-cell populations, suggesting impaired T-cell presence or recruitment. These results identify potential immunomodulatory lipid profiles in restaurant oil while supporting RNAscope-ISH as a method to describe avian tissue-level immune responses. [ABSTRACT FROM AUTHOR]

Background: The current diagnostic standard for detection of high‐risk human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma is via a two‐stage algorithm, namely p16 immunohistochemistry followed by HPV DNA in situ hybridization in p16 positive cases. This study evaluated the feasibility of automated RNA in situ hybridization on a clinical platform as a single‐step alternative to the two‐stage algorithm within a routine diagnostic histopathology setting. Methods: Thirty‐eight cases positive for both p16 and DNA in situ hybridization, 42 p16 negative cases and 20 cases positive for p16 but negative for DNA in situ hybridization were randomly selected. High‐risk HPV RNA in situ hybridization was undertaken on all cases on an automated clinical platform. Manufacturer‐recommended and on‐slide additional p16/HPV positive and negative controls were used. Test quality assurance and diagnostic RNA in situ hybridization were independently assessed by two observers. A consensus diagnosis was reached in the presence of a third observer on discordant cases. All RNA in situ hybridization results were then correlated against p16 and DNA ISH status. Results: Inter‐slide RNA in situ hybridization staining variation was observed in control sections. RNA in situ hybridization demonstrated a high inter‐observer agreement rate (κ =.897, P <.001). Following consensus review, there was full concordance between RNA in situ hybridization and the current standard. Conclusion: Human papillomavirus testing by standalone automated RNA in situ hybridization on a clinical diagnostic platform currently available in routine diagnostic histopathology laboratories is a feasible alternative to the two‐step algorithm of p16 and DNA in situ hybridization. Control tissue staining procedures need to be adapted to achieve the most accurate results. [ABSTRACT FROM AUTHOR]

Serotonin (5-hydroxytryptamine, 5HT) is a neurotransmitter and proinflammatory mediator found largely in the peripheral nervous system where it can initiate pain signaling. 5HT binds a variety of 5HT receptors on sensory nerve endings specialized in detecting noxious stimuli, termed nociceptors. A subset of sensory neurons involved in pain signaling express the transient receptor potential vanilloid 1 ion channel (TRPV1), a pain generator. 5HT can both directly activate sensory neurons and sensitize TRPV1 leading to enhanced nociceptor sensitivity (peripheral sensitization). Previous studies in male rats reported that the 5HT receptor subtype 3A (5HT3A) and TRPV1 are co-expressed on sensory neurons, but it is unknown if 5HT3A and TRPV1 are co-expressed on female sensory neurons. Given that craniofacial pain disorders occur at a 2-3x greater prevalence in women, examining pain mechanisms in female trigeminal sensory neurons that innervate the craniofacial region is critical to advancing craniofacial pain management in women. Here we examined whether (1) 5HT acting via the 5HT3A receptor produces sexually dimorphic orofacial pain behaviors in rats and (2) whether 5HT3A receptor mRNA is expressed in trigeminal sensory neurons, including the TRPV1-expressing subpopulation, and increase pain signaling. We report that 5HT evokes pain behaviors in male and female rats, which was not significantly reduced by antagonism of 5HT3A. We performed in situ hybridization to label 5HT3A and TRPV1 mRNA in trigeminal sensory neurons and found distinct cell populations with either 5HT3A mRNA or TRPV1 mRNA in males and females. Further, 5HT3A antagonism failed to reduce pain signaling in cultured trigeminal sensory neurons. These data suggest that the 5HT3A subtype on trigeminal sensory neurons innervating the orofacial soft tissues does not play a significant role in sexually dimorphic craniofacial pain disorders. As previous studies have reported that granisetron reduces masseter muscle pain, 5HT3 may play a role in sex differences in myofascial pain disorders but not in other craniofacial pain disorders. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) is a common cancer with poor prognosis. Transient Receptor Potential Ankyrin 1 (TRPA1) and Vanilloid 1 (TRPV1) receptors are non-selective cation channels expressed on primary sensory neurons and epithelial and immune cells. TRPV1 mRNA and immunopositivity, as well as TRPA1-like immunoreactivity upregulation, were demonstrated in OSCC, but selectivity problems with the antibodies still raise questions and their functional relevance is unclear. Therefore, here, we investigated TRPA1 and TRPV1 expressions in OSCC and analyzed their functions. TRPA1 and TRPV1 mRNA were determined by RNAscope in situ hybridization and qPCR. Radioactive 45Ca2+ uptake and ATP-based luminescence indicating cell viability were measured in PE/CA-PJ41 cells in response to the TRPA1 agonist allyl-isothiocyanate (AITC) and TRPV1 agonist capsaicin to determine receptor function. Both TRPA1 and TRPV1 mRNA are expressed in the squamous epithelium of the human oral mucosa and in PE/CA-PJ41 cells, and their expressions are significantly upregulated in OSCC compared to healthy mucosa. TRPA1 and TRPV1 activation (100 µM AITC, 100 nM capsaicin) induced 45Ca2+-influx into PE/CA-PJ41 cells. Both AITC (10 nM–5 µM) and capsaicin (100 nM–45 µM) reduced cell viability, reaching significant decrease at 100 nM AITC and 45 µM capsaicin. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the OSCC and confirm the expression of TRPV1 channel. These channels are functionally active and might regulate cancer cell viability.

Trastuzumab deruxtecan (T-DXd) has been approved by the US Food and Drug Administration (FDA) to treat patients with metastatic HER2-positive and HER2-low breast cancer, and clinical trials are examining its efficacy against early-stage breast cancer. Current HER2 immunohistochemical (IHC) assays are suboptimal in evaluating HER2-low breast cancers and identifying which patients would benefit from T-DXd. HER2 expression in 526 breast cancer tissue microarray (TMA) cores was measured using the FDA-approved PATHWAY and HercepTest IHC assays, and the corresponding RNA levels were evaluated by RNAscope. HER2 protein levels by regression analysis using a quantitative immunofluorescence score against cell line arrays with known HER2 protein levels determined by mass spectrometry were available in 48 of the cores. RNAscope was also performed in 32 metastatic biopsies from 23 patients who were subsequently treated with T-DXd, and the results were correlated with response rate. HER2 RNA levels by RNAscope strongly correlated with HER2 protein levels (P < .0001) and with HER2 IHC H-scores from the PATHWAY and HercepTest assays (P < .0001). However, neither protein levels nor RNA levels significantly differed between cases scored 0, ultralow, and 1+ by PATHWAY and HercepTest. The RNA levels were significantly higher (P = .030) in responders (6.4 ± 8.2 dots/cell, n = 12) than those in nonresponders (2.6 ± 2.2, n = 20) to T-DXd. RNAscope is a simple assay that can be objectively quantified and is a promising alternative to current IHC assays in evaluating HER2 expression in breast cancers, especially HER2-low cases, and may identify patients who would benefit from T-DXd., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

This study was conducted to visualize the lobular distribution of enhanced mRNA expression levels of heat shock proteins (HSPs) in liver samples from carbon tetra chloride (CCl4)-treated mice using in-situ hybridization (ISH). Male BALB/c mice given a single oral administration of CCl4 were euthanized 6 hours or 1 day after the administration (6 h or 1 day). Paraffin-embedded liver samples were obtained, ISH for HSPs was conducted, as well as hematoxylin-eosin staining and immunohistochemistry (IHC). At 6 h, centrilobular hepatocellular vacuolization was observed, and increased signals for Hspa1a, Hspa1b , and Grp78 , which are HSPs, were noted in the centrilobular area using ISH. At 1 day, zonal hepatocellular necrosis was observed in the centrilobular area, but mRNA signal increases for HSPs were no longer observed there. Some discrepancies between ISH and IHC for HSPs were observed, and they might be partly caused by post-transcriptional gene regulation, including the ribosome quality control mechanisms. It is known that CCl4 damages centrilobular hepatocytes through metabolization by cytochrome P450, mainly located in the centrilobular region, and HSPs are induced under cellular stress. Therefore, our ISH results visualized increased mRNA expression levels of HSPs in the centrilobular hepatocytes of mice 6 hours after a single administration of CCl4 as a response to cellular stress, and it disappeared 1 day after the treatment when remarkable necrosis was observed there., Competing Interests: The authors declare no conflict of interest., (©2024 The Japanese Society of Toxicologic Pathology.)

Background: Joubert syndrome and related disorders (JSRD) and Jeune syndrome are multisystem ciliopathy disorders with overlapping phenotypes. There are a growing number of genetic causes for these rare syndromes, including the recently described genes ARL3 and CEP120. Methods: We sought to explore the developmental expression patterns of ARL3 and CEP120 in humans to gain additional understanding of these genetic conditions. We used an RNA in situ detection technique called RNAscope to characterise ARL3 and CEP120 expression patterns in human embryos and foetuses in collaboration with the MRC-Wellcome Trust Human Developmental Biology Resource. Results: Both ARL3 and CEP120 are expressed in early human brain development, including the cerebellum and in the developing retina and kidney, consistent with the clinical phenotypes seen with pathogenic variants in these genes. Conclusions: This study provides insights into the potential pathogenesis of JSRD by uncovering the spatial expression of two JSRD-causative genes during normal human development. [ABSTRACT FROM AUTHOR]

Besides classic tobacco and alcohol risk factors, human papillomavirus (HPV) plays a role in the development of a subset of head and neck squamous cell carcinomas (HNSCCs), and notably oropharynx squamous cell carcinomas (OPSCCs). HPV-induced OPSCCs have a different biological behavior and a better prognosis compared to non-HPV-induced OPSCCs and the eighth-edition TNM classification now separates these two entities. Therefore, determining the HPV status of patients with OPSCC is now essential for treatment, prognosis, and development of clinical trials. In this review, after reminding essential steps of HPV implication in the cell cycle, we describe the existing tools that are currently feasible in routine practice according to facilities available in health structures, with their benefits and drawbacks: HPV PCR, E6/E7 mRNA RT-PCR, E6/E7 mRNA in situ hybridization, HPV DNA in situ hybridization, and P16 immunochemistry. Besides these traditional HPV detection tools, novel diagnostic approaches are being evaluated for HPV-induced OPSCC “ultrastaging.” E6 humoral response and ddPCR-detecting HPVct DNA are two techniques performed on blood and are therefore non-invasive. Baseline E6 humoral levels could have a prognostic value, and HPVct DNA could be helpful for HPV OPSCC recurrence monitoring. At last, next-generation sequencing (NGS)-based “capture HPV” is a technique feasible on biopsies and circulating DNA material. It helps characterize HPV integration status and sites, and it could define prognostic subgroups in HPV-induced OPSCC. These novel precision detection tools could be further integrated in the care of patients with HPV-induced OPSCC.

Betaarterivirus suid 1 and 2 are the causative agents of porcine reproductive and respiratory syndrome (PRRS), which is one of the most significant diseases of the swine industry, causing significant economic losses in the main pig producing countries. Here, we report the development of a novel, RNA-based in situ hybridization technique (RNAscope) to detect PRRS virus (PRRSV) RNA in lung tissues of experimentally infected animals. The technique was applied to lung tissues of 20 piglets, which had been inoculated with a wild-type, highly pathogenic PRRSV-1 strain. To determine the RNAscope’s applicability as a semi-quantitative method, we analysed the association between the proportion of the virus-infected cells measured with an image analysis software (QuPath) and the outcome of the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) tests performed in parallel. The results of the quantitative approach of these two molecular biological methods show significant association (pseudo R2 = 0.3894, p = 0.004). This is the first time RNAscope assay has been implemented for the detection of PRRSV-1 in experimental animals.

Besides classic tobacco and alcohol risk factors, human papillomavirus (HPV) plays a role in the development of a subset of head and neck squamous cell carcinomas (HNSCCs), and notably oropharynx squamous cell carcinomas (OPSCCs). HPV-induced OPSCCs have a different biological behavior and a better prognosis compared to non-HPV-induced OPSCCs and the eighth-edition TNM classification now separates these two entities. Therefore, determining the HPV status of patients with OPSCC is now essential for treatment, prognosis, and development of clinical trials. In this review, after reminding essential steps of HPV implication in the cell cycle, we describe the existing tools that are currently feasible in routine practice according to facilities available in health structures, with their benefits and drawbacks: HPV PCR, E6/E7 mRNA RT-PCR, E6/E7 mRNA in situ hybridization, HPV DNA in situ hybridization, and P16 immunochemistry. Besides these traditional HPV detection tools, novel diagnostic approaches are being evaluated for HPV-induced OPSCC "ultrastaging." E6 humoral response and ddPCR-detecting HPVct DNA are two techniques performed on blood and are therefore non-invasive. Baseline E6 humoral levels could have a prognostic value, and HPVct DNA could be helpful for HPV OPSCC recurrence monitoring. At last, next-generation sequencing (NGS)-based "capture HPV" is a technique feasible on biopsies and circulating DNA material. It helps characterize HPV integration status and sites, and it could define prognostic subgroups in HPV-induced OPSCC. These novel precision detection tools could be further integrated in the care of patients with HPV-induced OPSCC. [ABSTRACT FROM AUTHOR]

Background: In situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatio-temporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-by-step method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the spatial distribution pattern of HvGAPDH and the low expressed disease resistance gene Rpg1 in leaf tissue sections of barley and discuss precautions that should be followed during image analysis. Results: We have shown the ubiquitous HvGAPH and predominantly stomatal guard cell associated subsidiary cell expressed Rpg1 expression pattern in barley leaf sections and described the improve RNAscope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues. Conclusions: RNAscope multiplex fluorescent RNA-ISH detection is well described and adapted for animal tissue samples, however due to morphological and structural differences in the plant tissues the standard protocol is deficient and required optimization. Utilizing barley specific HvGAPDH and Rpg1 RNA probes we report an optimized method which can be used for RNAscope detection to determine the spatial expression and semi-quantification of target RNAs. This optimized method will be immensely useful in other plant species such as the widely utilized Arabidopsis. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphomas (DLBCLs) are the most common types of Non-Hodgkin's lymphomas and are highly heterogeneous in terms of phenotype and treatment response. The natural course of DLBCLs tumor progression is featured by a flow of events leading to the enhancement of proliferative and invasive capabilities and, therefore, towards the establishment of a more aggressive phenotype. Angiogenesis is a constant hallmark of DLBCLs progression, has prognostic potential and promote DLBCLs dissemination. The study of DLBCLs angiogenesis mechanisms, and the tumor endothelium characterization, will allow us to identify new prognostic/predictive biomarkers to proper patient selection to antiangiogenic treatment. In our previous work, by RNAscope technology, we have demonstrated that Janus kinase (Jak) and signal transducer activator of transcription pathway (STAT) is one of the proangiogenic pathways activated in DLBCLs and it drives neoangiogenesis occurred by vasculogenesis mechanism. Here, we describe a detailed protocol to perform RNAscope technology alone and in combination with immunohistochemistry (called dual RNAscope ISH–IHC) in DLBCLs formalin-fixed, paraffin-embedded sections. We propose dual ISH–IHC as an extremely powerful method to study angiogenesis in DLBCLs, because it allows one to answer important biological questions that are difficult to address using other single methods. [ABSTRACT FROM AUTHOR]

Constitutively activated STAT3 is correlated with more advanced clinical stage and overall poor survival of diffuse large B-cell lymphoma (DLBCL). The aim of this study was to evaluate STAT3 and Ki67 tumor cell expression, inflammatory cell infiltration, microvascular density in DLBCL bioptic specimens. RNA-scope showed that activated B cell (ABC) tissue samples contained a significant higher number of STAT3+ cells as compared to germinal center B (GCB) tissue samples. Immunohistochemical analysis showed a significant increased levels of CD3, CD8, CD68, CD163, CD34, and Ki67 positive cells in ABC patients. A positive correlation between STAT3 and CD3, CD8, CD68, and CD163 was evidenced in ABC group. In ABC group, we found also a positive correlation between CD8 and CD34 and a positive correlation between Ki67 and, CD68, and CD163. These data indicate that in ABC—as compared to GCB-DLBCL, a higher STAT3 expression is associated with a higher CD163+ TAM and CD8+ cell infiltration which induces a strong angiogenic response. [ABSTRACT FROM AUTHOR]

Background: The diagnostic and prognostic values of glypican3 (GPC3) and glutamine synthetase (GS) proteins in hepatocellular carcinoma (HCC) have been reported, but their specificity and sensitivity remain low. Here, we applied RNAscope to improve HCC early pathological and differential diagnosis by estimating GPC3 and GS mRNAs. Methods: We performed RNAscope and immunohistochemistry (IHC) to detect GPC3 and GS biomarkers on the tissue sections of 194 cases, including high- and low-grade liver dysplastic nodules; highly, moderately, and poorly differentiated HCCs; intrahepatic cholangiocarcinomas (ICCs); metastatic HCC; and carcinomas from other organs. Results: The results showed that all the cases that were negative for GPC3 by RNAscope were also negative for this protein by IHC. The use of RNAscope assay improved the GPC3 and GS specificity and sensitivity by 20–30%. Hence, HCC shows early recognition and upgrades the metastatic HCC differentiation by 23% compared with IHC (p = 0.0001, 0.0064). Meanwhile, all liver cirrhosis, cholangiocytes and non-HCC samples were negative for GPC3 and GS except lymphocytes in lymphomas, and 2 (8.3%) out of the 24 ICC samples but not in the cancer cells. Conclusion: RNAscope for GPC3 and GS panel was highly specific and sensitive for the pathological identification of dysplastic nodules, early stages of HCCs, and would differentiate them from HCCs and metastatic tumors compared with IHC. [ABSTRACT FROM AUTHOR]

We report postmortem cardio-pulmonary findings including detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in formalin-fixed paraffin embedded tissue in 12 patients with COVID-19. The 5 women and 7 men (median age: 73 years; range 35–96) died 6–38 days after onset of symptoms (median: 14.5 days). Eight patients received mechanical ventilation. Ten patients showed diffuse alveolar damage (DAD), 7 as exudative and 3 as proliferative/organizing DAD. One case presented as acute fibrinous and organizing pneumonia. Seven patients (58%) had acute bronchopneumonia, 1/7 without associated DAD and 1/7 with aspergillosis and necrotic bronchitis. Microthrombi were present in 5 patients, only in exudative DAD. Reverse transcriptase quantitative PCR detected high virus amounts in 6 patients (50%) with exudative DAD and symptom-duration ≤14 days, supported by immunohistochemistry and in-situ RNA hybridization (RNAscope). The 6 patients with low viral copy levels were symptomatic for ≥15 days, comprising all cases with organizing DAD, the patient without DAD and one exudative DAD. We show the high prevalence of DAD as a reaction pattern in COVID-19, the high number of overlying acute bronchopneumonia, and high-level pulmonary virus detection limited to patients who died ≤2 weeks after onset of symptoms, correlating with exudative phase of DAD.

Immune checkpoints are a set of molecules dysregulated in several human and canine cancers and aberrations of the PD-1/PD-L1 axis are often correlated with a worse prognosis. To gain an insight into the role of immune checkpoints in canine diffuse large B-cell lymphoma (cDLBCL), we investigated PD-L1, PD-1 and CD8A expression by RNAscope. Results were correlated with several clinico-pathological features, including treatment, Ki67 index and outcome. A total of 33 dogs treated with chemotherapy (n = 12) or chemoimmunotherapy with APAVAC (n = 21) were included. PD-L1 signal was diffusely distributed among neoplastic cells, whereas PD-1 and CD8A were localized in tumor infiltrating lymphocytes. However, PD-1 mRNA was also retrieved in tumor cells. An association between PD-L1 and PD-1 scores was identified and a higher risk of relapse and lymphoma-related death was found in dogs treated with chemotherapy alone and dogs with higher PD-L1 and PD-1 scores. The correlation between PD-L1 and PD-1 is in line with the mechanism of immune checkpoints in cancers, where neoplastic cells overexpress PD-L1 that, in turn, binds PD-1 receptors in activated TIL. We also found that Ki67 index was significantly increased in dogs with the highest PD-L1 and PD-1 scores, indirectly suggesting a role in promoting tumor proliferation. Finally, even if the biological consequence of PD-1+ tumor cells is unknown, our findings suggest that PD-1 intrinsic expression in cDLBCL might contribute to tumor growth escaping adaptive immunity.

The persistence of HIV-1 viral reservoirs in the brain, despite treatment with combination antiretroviral therapy (cART), remains a critical roadblock for the development of a novel cure strategy for HIV-1. To enhance our understanding of viral reservoirs, two complementary studies were conducted to (1) evaluate the HIV-1 mRNA distribution pattern and major cell type expressing HIV-1 mRNA in the HIV-1 transgenic (Tg) rat, and (2) validate our findings by developing and critically testing a novel biological system to model active HIV-1 infection in the rat. First, a restricted, region-specific HIV-1 mRNA distribution pattern was observed in the HIV-1 Tg rat. Microglia were the predominant cell type expressing HIV-1 mRNA in the HIV-1 Tg rat. Second, we developed and critically tested a novel biological system to model key aspects of HIV-1 by infusing F344/N control rats with chimeric HIV (EcoHIV). In vitro, primary cultured microglia were treated with EcoHIV revealing prominent expression within 24 h of infection. In vivo, EcoHIV expression was observed seven days after stereotaxic injections. Following EcoHIV infection, microglia were the major cell type expressing HIV-1 mRNA, results that are consistent with observations in the HIV-1 Tg rat. Within eight weeks of infection, EcoHIV rats exhibited neurocognitive impairments and synaptic dysfunction, which may result from activation of the NogoA-NgR3/PirB-RhoA signaling pathway and/or neuroinflammation. Collectively, these studies enhance our understanding of HIV-1 viral reservoirs in the brain and offer a novel biological system to model HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse) in rats.

The locus coeruleus (LC)-norepinephrine (NE) system modulates a range of salient brain functions, including memory and response to stress. The LC-NE system is regulated by neurochemically diverse inputs, including a range of neuropeptides such as arginine-vasopressin (AVP). Whilst the origins of many of these LC inputs, their synaptic connectivity with LC neurons, and their contribution to LC-mediated brain functions, have been well characterized, this is not the case for the AVP-LC system. Therefore, our aims were to define the types of synapses formed by AVP+ fibers with LC neurons using immunohistochemistry together with confocal and transmission electron microscopy (TEM), the origins of such inputs, using retrograde tracers, and the plasticity of the LC AVP system in response to stress and spatial learning, using the maternal separation (MS) and Morris water maze (MWM) paradigms, respectively, in rat. Confocal microscopy revealed that AVP+ fibers contacting tyrosine hydroxylase (TH)+ LC neurons were also immunopositive for vesicular glutamate transporter 2, a marker of presynaptic glutamatergic axons. TEM confirmed that AVP+ axons formed Gray type I (asymmetric) synapses with TH+ dendrites thus confirming excitatory synaptic connections between these systems. Retrograde tracing revealed that these LC AVP+ fibers originate from hypothalamic vasopressinergic magnocellular neurosecretory neurons (AVPMNNs). MS induced a significant increase in the density of LC AVP+ fibers. Finally, AVPMNN circuit upregulation by water-deprivation improved MWM performance while increased Fos expression was found in LC and efferent regions such as hippocampus and prefrontal cortex, suggesting that AVPMMN projections to LC could integrate homeostatic responses modifying neuroplasticity.

Neuroblastoma is a heterogeneous paediatric malignant tumour. Telomere maintenance mechanism (TMM) by telomerase activation or alternative lengthening of telomeres (ALT) is a hallmark of high-risk neuroblastoma. However, the prior assays for telomerase, such as TERT expression by RNA sequencing or microarrays, may not be easy to perform in many histopathology laboratories in hospitals. The aims of this study are to assess the utility of ultrasensitive single-cell RNA in situ hybridisation (RNAscope), immunohistochemistry, and RT-qPCR on formalin-fixed, paraffin-embedded tumour samples as diagnostic tools for detecting TERT expression in neuroblastoma. In this study, we detected MYCN amplification in 22 of 222 cases (10%), TERT rearrangements in 18 of 220 cases (8%), and ALT activation in 39 of 222 cases (18%) using fluorescence in situ hybridisation (FISH). By RNA in situ hybridisation, 36 of 210 (17%) pretreatment neuroblastomas were found to have TERT overexpression, which was significantly associated with the high-risk group (33/78, 42%), TERT rearrangements (16/18, 89%), and MYCN amplification (13/22, 59%). None of the tumours with ALT showed TERT staining. In our study, 19 of the 55 MYCN non-amplified high-risk neuroblastomas displayed TERT mRNA expression, including 13 of the 14 TERT rearrangements, none of the 30 ALT-positive cases, and a significant proportion (6/11, 55%) that did not have the aforementioned genomic anomalies. RT-qPCR results correlated well with RNAscope levels (Spearman's rho=0.621, p<0.001, n=94). In conclusion, TERT RNA in situ hybridisation and RT-qPCR are suitable methods to evaluate TERT expression in neuroblastoma. The combination of detection of the genomic alterations and TERT mRNA expression is a powerful strategy for TMM activation detection, which can categorise neuroblastomas into multiple clinical subgroups for risk stratification in routine histopathology practice., (Copyright © 2023 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Long non-coding RNA (lncRNA) plays an important role in the regulation of gene expression in normal and cancer cells. We previously discovered a novel tumor-suppressive lncRNA, DRAIC , in prostate cancer cells. Subsequent studies have demonstrated that DRAIC is dysregulated in various malignancies and exhibits a tumor-suppressive or pro-oncogenic function. However, details regarding its expression pattern in normal and cancerous tissues remain largely unknown. In this study, we performed chromogenic in situ hybridization (CISH) using RNAscope technology to assess DRAIC expression in formalin-fixed paraffin-embedded (FFPE) specimens. In the neuroendocrine-differentiated cancer cell line VMRC-LCD, CISH revealed a diffuse localization of DRAIC in the cytoplasm as well as specific accumulation in the nuclear compartment. DRAIC expression was comprehensively analyzed using tissue microarrays containing 89 normal and 155 tumor tissue samples. DRAIC was weakly expressed in normal epithelial cells of the colon, bronchiole, kidney, prostate, and testis. Conversely, DRAIC was moderately to highly expressed in some cancer tissues, including prostate adenocarcinoma, invasive ductal carcinoma of the breast, neuroendocrine carcinoma of the esophagus, lung adenocarcinoma, and small cell lung carcinoma. While DRAIC knockdown did not affect VMRC-LCD cellular viability and invasive ability, gene expression related to the neuroendocrine and cancer-related pathways was altered. Our expression analysis revealed the specific expression pattern of DRAIC in normal and cancerous FFPE tissues. The results presented here may lead to the elucidation of additional novel functions of DRAIC ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Identifying the source and dynamics of persistent HIV-1 at single-cell resolution during cART is crucial for the design of strategies to eliminate the latent HIV-1 reservoir. An assay to measure latent HIV-1 that can distinguish inducible from defective proviruses with high precision is essential to evaluate the efficacy of HIV-1 cure efforts but is presently lacking. The primary aim of this study was therefore to identify transcription and translation competent latently infected cells through detection of biomolecules that are dependent on transcriptional activation of the provirus. We investigated the applicability of two commercially available assays; PrimeFlowTM RNA Assay (RNAflow) and RNAscope® ISH (RNAscope) for evaluation of the efficacy of latency reversal agents (LRAs) to reactivate the HIV-1 latent reservoir. The J-Lat cell model (clones 6.3, 9.3, and 10.6) and four LRAs was used to evaluate the sensitivity, specificity, and lower detection limit of the RNAflow and RNAscope assays for the detection and description of the translation-competent HIV-1 reservoir. We also checked for HIV-1 subtype specificity of the RNAscope assay using patient-derived subtype A1, B, C, and CRF01_AE recombinant plasmids following transfection in 293T cells and the applicability of the method in patient-derived peripheral blood mononuclear cells (PBMCs). The lower detection limit of RNAflow was 575 HIV-1 infected cells/million and 45 cells/million for RNAscope. The RNAscope probes, designed for HIV-1B, also detected other subtypes (A1, B, C, and CRF01_AE). RNAscope was applicable for the detection of HIV-1 in patient-derived PBMCs following LRA activation. In conclusion, our study showed that RNAscope can be used to quantify the number of directly observed individual cells expressing HIV-1 mRNA following LRA activation. Therefore, it can be a useful tool for characterization of translation-competent HIV-1 in latently infected cell at single-cell resolution in the fields of HIV-1 pathogenesis and viral persistence.

Recent studies suggest that the ventral medial prefrontal cortex (vmPFC) encodes both operant drug self-administration and extinction memories. Here, we examined whether these opposing memories are encoded by distinct neuronal ensembles within the vmPFC with different outputs to the nucleus accumbens (NAc) in male and female rats. Using cocaine self-administration (3 h/d for 14 d) and extinction procedures, we demonstrated that vmPFC was similarly activated (indexed by Fos) during cocaine-seeking tests after 0 (no-extinction) or 7 extinction sessions. Selective Daun02 lesioning of the self-administration ensemble (no-extinction) decreased cocaine seeking, whereas Daun02 lesioning of the extinction ensemble increased cocaine seeking. Retrograde tracing with fluorescent cholera toxin subunit B injected into NAc combined with Fos colabeling in vmPFC indicated that vmPFC self-administration ensembles project to NAc core while extinction ensembles project to NAc shell. Functional disconnection experiments (Daun02 lesioning of vmPFC and acute dopamine D1-receptor blockade with SCH39166 in NAc core or shell) confirm that vmPFC ensembles interact with NAc core versus shell to play dissociable roles in cocaine self-administration versus extinction, respectively. Our results demonstrate that neuronal ensembles mediating cocaine self-administration and extinction comingle in vmPFC but have distinct outputs to the NAc core and shell that promote or inhibit cocaine seeking. [ABSTRACT FROM AUTHOR]

The long non-coding RNA (LncRNA) antisense RNA in the INK4 locus (ANRIL) is overexpressed in several cancers including breast cancer. To better understand the role of ANRIL in breast cancer this study investigated where ANRIL was expressed in breast tumors using in situ hybridization by RNAscope. Additional RNAscope assays for IL6, CCL2 , and POSTN were used to establish whether ANRIL correlated with increased tumor promoting cytokines. Breast tumors with ANRIL over expressed from real-time quantitative (RT-q) PCR assays were selected for analysis using RNAscope. All tumors showed ANRIL expression in malignant cells, but amongst tumors ANRIL showed different subcellular locations with 56% of tumors with ANRIL only in the nucleus, 16% with ANRIL only in the cytoplasm and 28% with ANRIL in both the nucleus and cytoplasm. Cases with nuclear ANRIL were positively correlated with POSTN expression in malignant cells (ρ = 0.57, P = 0.0086), and no correlation was found between ANRIL and IL6 or CCL2. Reduced POSTN was also found using siRNA to ANRIL in MDA-MB-231 and MCF7 breast cancer cells. These data indicate that ANRIL is expressed in malignant breast cells, and suggest its subcellular location may indicate its function in cancer progression. [ABSTRACT FROM AUTHOR]

• PEDV strain BJ2011C can quickly colonize small intestine as early as 6 hpi. • The disease has a short incubation time of about 12 h. • The virus shedding starts much earlier than the appearance of clinical symptoms. • PEDV BJ2011C spreads within the gastrointestinal tract in an orderly manner. • PEDV BJ2011C mainly targets jejunum and ileum with a preference on the villous epithelial cells. The highly virulent porcine epidemic diarrhea virus (PEDV) variants cause the death of mainly neonatal piglets, but how the viruses spread within the gastro-intestinal tract in a temporal and spatial manner has remained poorly characterized but is critical to understand the viral pathogenesis. In this study, we used the Chinese PEDV epidemic strain BJ2011C as a model organism and took advantage of the newly developed RNAscope in situ hybridization technology to investigate the tempo-spatial infection dynamics in neonatal piglets. We found that the PEDV strain BJ2011C could quickly colonize the small intestine, which occurred in just 6 h post infection, with virus shedding starting at 6 hpi and peaking at 24 hpi. Jejunum was the first target tissue for infection and then ileum, followed by infrequent infection of duodenum. In these tissues, the virus nucleic acids were mainly present in the villous epithelial cells but not in crypt cells. Interestingly, the viral RNAs were not detectable by RNAscope in large intestines although tissue damages could be discerned by H & E staining. Overall, our results provide useful information about spread dynamics and tissue preference of PEDV epidemic strain BJ2011C. [ABSTRACT FROM AUTHOR]

The expression of programmed cell death ligand 1 (PD‐L1) is a biomarker for immunotherapy, but approved detection method is absent in diffuse large B‐cell lymphoma (DLBCL). Here, we performed three methods including immunohistochemistry (IHC) (clone SP263 and SP142), RNAscope, and fluorescence in situ hybridization (FISH) to evaluate PD‐L1 status on a cohort of DLBCL including 94 of DLBCL‐NOS, 25 of primary mediastinal large B‐cell lymphoma (PMBCL) and 7 of double‐hit lymphoma (DHL). SP263 with 25% for immune cell (IC) or combined cell and SP142 with 10% for tumor cell (TC), 20% for both of IC and combined cell were proved to have corresponding survival prognostic. Combined+ showed comparable prognostic value with TC+ and IC+. SP263 and SP142 showed strong concordance (k = 0.788) with combined+ rates of 33.3% (42/126) and 34.9% (44/126), respectively. In DLBCL‐NOS, TC+ by SP263 preferred to non‐GCB and immunoblastic variant DLBCL‐NOS (P = 0.029 and P = 0.004). Combined+ (SP263 and SP142) were associated with more than one extranodal site involved (P = 0.006, P = 0.042), higher ECOG PS scores (P = 0.001, P < 0.001), high IPI risk (P = 0.012, P = 0.005), and poor treatment response (P = 0.095, P = 0.002). IC+ by SP263 and SP142 were both independent risk factors (P = 0.027, P = 0.037). 9p24.1 locus amplification and gain were identified in 4.3% and 7.6% DLBCL‐NOS and indicated shorter overall survival (P = 0.004). Positive rate of PD‐L1 by RNAscope was 36.5%, while no clinical significance shown. PD‐L1 positive rates were all higher in PMBCL and DHL than in DLBCL‐NOS by SP263, SP142, RNAscope, and FISH (P = 0.001, P < 0.001, P = 0.005 and P < 0.001, respectively). In conclusion, combined PD‐L1 expression by IHC was potentially reliable and convenient as a predicting biomarker. SP263 staining was easier to evaluate and recognized more PD‐L1‐stained cells, but SP142 presented a better prognostic indicator. FISH and RNAscope could be used as supplementary assays. PMBCL itself was a sensitive cohort for immunotherapy. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis is a serious zoonotic pathogen and the cause of tuberculosis in many mammalian species, most notably, cattle. The hallmark lesion of tuberculosis is the granuloma. It is within the developing granuloma where host and pathogen interact; therefore, it is critical to understand host-pathogen interactions at the granuloma level. Cytokines and chemokines drive cell recruitment, activity, and function and ultimately determine the success or failure of the host to control infection. In calves, early lesions (ie, 15 and 30 days) after experimental aerosol infection were examined microscopically using in situ hybridization and immunohistochemistry to demonstrate early infiltrates of CD68+ macrophages within alveoli and alveolar interstitium, as well as the presence of CD4, CD8, and γδ T cells. Unlike lesions at 15 days, lesions at 30 days after infection contained small foci of necrosis among infiltrates of macrophages, lymphocytes, neutrophils, and multinucleated giant cells and extracellular acid-fast bacilli within necrotic areas. At both time points, there was abundant expression of the chemokines CXCL9, MCP-1/CCL2, and the cytokine transforming growth factor (TGF)–β. The proinflammatory cytokines tumor necrosis factor (TNF)–α and interleukin (IL)–1β, as well as the anti-inflammatory cytokine IL-10, were expressed at moderate levels at both time points, while expression of IFN-γ was limited. These findings document the early pulmonary lesions after M. bovis infection in calves and are in general agreement with the proposed pathogenesis of tuberculosis described in laboratory animal and nonhuman primate models of tuberculosis. [ABSTRACT FROM AUTHOR]

We present the first documented case of Trypanosoma cruzi‐induced orchitis in a rhesus macaque. Additionally, we describe an in situ hybridization–based assay to confirm T. cruzi infection in formalin‐fixed tissues. [ABSTRACT FROM AUTHOR]

The locus coeruleus (LC)-norepinephrine (NE) system modulates a range of salient brain functions, including memory and response to stress. The LC-NE system is regulated by neurochemically diverse inputs, including a range of neuropeptides such as arginine-vasopressin (AVP). Whilst the origins of many of these LC inputs, their synaptic connectivity with LC neurons, and their contribution to LC-mediated brain functions, have been well characterized, this is not the case for the AVP-LC system. Therefore, our aims were to define the types of synapses formed by AVP+ fibers with LC neurons using immunohistochemistry together with confocal and transmission electron microscopy (TEM), the origins of such inputs, using retrograde tracers, and the plasticity of the LC AVP system in response to stress and spatial learning, using the maternal separation (MS) and Morris water maze (MWM) paradigms, respectively, in rat. Confocal microscopy revealed that AVP+ fibers contacting tyrosine hydroxylase (TH)+ LC neurons were also immunopositive for vesicular glutamate transporter 2, a marker of presynaptic glutamatergic axons. TEM confirmed that AVP+ axons formed Gray type I (asymmetric) synapses with TH+ dendrites thus confirming excitatory synaptic connections between these systems. Retrograde tracing revealed that these LC AVP+ fibers originate from hypothalamic vasopressinergic magnocellular neurosecretory neurons (AVPMNNs). MS induced a significant increase in the density of LC AVP+ fibers. Finally, AVPMNN circuit upregulation by water-deprivation improved MWM performance while increased Fos expression was found in LC and efferent regions such as hippocampus and prefrontal cortex, suggesting that AVPMMN projections to LC could integrate homeostatic responses modifying neuroplasticity. [ABSTRACT FROM AUTHOR]

RNAscope® technology provided by Advanced Cell Diagnostics (ACD) allows the detection and evaluation of coinciding mRNA expression profiles in the same or adjacent cells in unprecedented quantitative detail using multicolor fluorescent in situ hybridization (FISH). While already extensively used in thinly sectioned material of various pathological tissues and, to a lesser extent, in some whole mounts, we provide here a detailed approach to use the fluorescent RNAscope method in the mouse inner ear and thick brain sections by modifying and adapting existing techniques of whole mount fluorescent in situ hybridization (WH-FISH). We show that RNAscope WH-FISH can be used to quantify local variation in overlaying mRNA expression intensity, such as neurotrophin receptors along the length of the mouse cochlea. We also show how RNAscope WH-FISH can be combined with immunofluorescence (IF) of some epitopes that remain after proteinase digestion and, to some extent, with fluorescent protein markers such as tdTomato. Our WH-FISH technique provides an approach to detect cell-specific quantitative differences in developing and mature adjacent cells, an emerging issue revealed by improved cellular expression profiling. Further, the presented technique may be useful in validating single-cell RNAseq data on expression profiles in a range of tissue known or suspected to have locally variable mRNA expression levels. [ABSTRACT FROM AUTHOR]

Behaviors associated with distress can affect the anxiety-like states in observers and this social transfer of affect shapes social interactions among stressed individuals. We hypothesized that social reactions to stressed individuals engage the serotonergic dorsal raphe nucleus (DRN) which promotes anxiety-like behavior via postsynaptic action of serotonin at serotonin 2C (5-HT 2C) receptors in the forebrain. First, we inhibited the DRN by administering an agonist (8-OH-DPAT, 1 μg in 0.5 μL) for the inhibitory 5-HT 1A autoreceptors which silences 5-HT neuronal activity. 8-OH-DPAT prevented the approach and avoidance, respectively, of stressed juvenile (PN30) or stressed adult (PN60) conspecifics in the social affective preference (SAP) test in rats. Similarly, systemic administration of a 5-HT 2C receptor antagonist (SB242084, 1 mg/kg, i.p.) prevented approach and avoidance of stressed juvenile or adult conspecifics, respectively. Seeking a locus of 5-HT 2C action, we considered the posterior insular cortex which is critical for social affective behaviors and rich with 5-HT 2C receptors. SB242084 administered directly into the insular cortex (5 μM in 0.5 μL bilaterally) interfered with the typical approach and avoidance behaviors observed in the SAP test. Finally, using fluorescent in situ hybridization, we found that 5-HT 2C receptor mRNA (htr2c) is primarily colocalized with mRNA associated with excitatory glutamatergic neurons (vglut1) in the posterior insula. Importantly, the results of these treatments were the same in male and female rats. These data suggest that interactions with stressed others require the serotonergic DRN and that serotonin modulates social affective decision-making via action at insular 5-HT 2C receptors. [Display omitted] [ABSTRACT FROM AUTHOR]

A primary obstacle to an HIV-1 cure is long-lived viral reservoirs, which must be eliminated or greatly reduced. Cure strategies have largely focused on monitoring changes in T cell reservoirs in peripheral blood (PB), even though the lymphoid tissues (LT) are primary sites for viral persistence. To track and discriminate viral reservoirs within tissue compartments we developed a specific and sensitive next-generation in situ hybridization approach to detect vRNA, including vRNA+ cells and viral particles (“RNAscope”), vDNA+ cells (“DNAscope”) and combined vRNA and vDNA with immunohistochemistry to detect and phenotype active and latently infected cells in the same tissue section. RNAscope is highly sensitive with greater speed of analysis compared to traditional in situ hybridization. Highly sensitive and specific DNAscope detected SIV/HIV vDNA+ cells, including duplexed detection of vDNA and vRNA or immunophenotypic markers in the same section. Analysis of LT samples from macaques prior to and during combination antiretroviral therapy demonstrated that B cell follicles are an important anatomical compartment for both latent and active viral persistence during treatment. These new tools should allow new insights into viral reservoir biology and evaluation of cure strategies.

Whether HPV is causative of pregnancy complications is uncertain. E6 and E7 affect functions underling preeclampsia (PET) in cultured trophoblasts, but whether E6 and E7 is produced in the placenta is uncertain. Here, we investigated whether E6/E7 was expressed in the placentae from pregnancies with PET, other pregnancy complications (fetal growth restriction (FGR) and diabetes mellitus), and uncomplicated pregnancies. Placental tissues collected from two geographical locations were subjected to RNAscope analyses of high- and low- risk E6/E7, and individual HPV types identified using an HPV array. High-risk E6/E7 expression was increased in both PET cohorts, (81% and 86% of patients positive for high-risk HPV DNA compared to 13% of control patients). Various HPV types were identified. Although HPV 18 was the most frequent in all cohorts, the majority of individuals had multiple HPV types (55% of the PET compared to 25% of the control cohort). Further evidence that E6 and E7 is present early when placental pathology underlying preeclampsia is established, is provided with the finding of high-risk E6/E7 in the first-trimester placenta anchoring trophoblast. In conclusion, E6/E7 expression and multiple HPV types were frequent in placentae from preeclampsia-complicated pregnancies.

Estrogens play an essential role in multiple physiological functions in the brain, including reproductive neuroendocrine, learning and memory, and anxiety-related behaviors. To determine these estrogen functions, many studies have tried to characterize neurons expressing estrogen receptors known as ERα and ERβ. However, the characteristics of ERβ-expressing neurons in the rat brain still remain poorly understood compared to that of ERα-expressing neurons. The main aim of this study is to determine the neurochemical characteristics of ERβ-expressing neurons in the rat hypothalamus using RNAscope in situ hybridization (ISH) combined with immunofluorescence. Strong Esr2 signals were observed especially in the anteroventral periventricular nucleus (AVPV), bed nucleus of stria terminalis, hypothalamic paraventricular nucleus (PVN), supraoptic nucleus, and medial amygdala, as previously reported. RNAscope ISH with immunofluorescence revealed that more than half of kisspeptin neurons in female AVPV expressed Esr2, whereas few kisspeptin neurons were found to co-express Esr2 in the arcuate nucleus. In the PVN, we observed a high ratio of Esr2 co-expression in arginine-vasopressin neurons and a low ratio in oxytocin and corticotropin-releasing factor neurons. The detailed neurochemical characteristics of ERβ-expressing neurons identified in the current study can be very essential to understand the estrogen signaling via ERβ.