Next-Generation Digital Histopathology of the Tumor Microenvironment

Genes

2021 Apr 07

Mungenast, F;Fernando, A;Nica, R;Boghiu, B;Lungu, B;Batra, J;Ecker, RC;
PMID: 33917241 | DOI: 10.3390/genes12040538

Progress in cancer research is substantially dependent on innovative technologies that permit a concerted analysis of the tumor microenvironment and the cellular phenotypes resulting from somatic mutations and post-translational modifications. In view of a large number of genes, multiplied by differential splicing as well as post-translational protein modifications, the ability to identify and quantify the actual phenotypes of individual cell populations in situ, i.e., in their tissue environment, has become a prerequisite for understanding tumorigenesis and cancer progression. The need for quantitative analyses has led to a renaissance of optical instruments and imaging techniques. With the emergence of precision medicine, automated analysis of a constantly increasing number of cellular markers and their measurement in spatial context have become increasingly necessary to understand the molecular mechanisms that lead to different pathways of disease progression in individual patients. In this review, we summarize the joint effort that academia and industry have undertaken to establish methods and protocols for molecular profiling and immunophenotyping of cancer tissues for next-generation digital histopathology-which is characterized by the use of whole-slide imaging (brightfield, widefield fluorescence, confocal, multispectral, and/or multiplexing technologies) combined with state-of-the-art image cytometry and advanced methods for machine and deep learning.

Neuromodulation by the immune system: a focus on cytokines

Nature reviews. Immunology

2021 Mar 01

Salvador, AF;de Lima, KA;Kipnis, J;
PMID: 33649606 | DOI: 10.1038/s41577-021-00508-z

Interactions between the immune system and the nervous system have been described mostly in the context of diseases. More recent studies have begun to reveal how certain immune cell-derived soluble effectors, the cytokines, can influence host behaviour even in the absence of infection. In this Review, we contemplate how the immune system shapes nervous system function and how it controls the manifestation of host behaviour. Interactions between these two highly complex systems are discussed here also in the context of evolution, as both may have evolved to maximize an organism's ability to respond to environmental threats in order to survive. We describe how the immune system relays information to the nervous system and how cytokine signalling occurs in neurons. We also speculate on how the brain may be hardwired to receive and process information from the immune system. Finally, we propose a unified theory depicting a co-evolution of the immune system and host behaviour in response to the evolutionary pressure of pathogens.

Brain ethanol metabolism by astrocytic ALDH2 drives the behavioural effects of ethanol intoxication

Nature metabolism

2021 Mar 01

Jin, S;Cao, Q;Yang, F;Zhu, H;Xu, S;Chen, Q;Wang, Z;Lin, Y;Cinar, R;Pawlosky, RJ;Zhang, Y;Xiong, W;Gao, B;Koob, GF;Lovinger, DM;Zhang, L;
PMID: 33758417 | DOI: 10.1038/s42255-021-00357-z

Alcohol is among the most widely used psychoactive substances worldwide. Ethanol metabolites such as acetate, thought to be primarily the result of ethanol breakdown by hepatic aldehyde dehydrogenase 2 (ALDH2), contribute to alcohol's behavioural effects and alcoholism. Here, we show that ALDH2 is expressed in astrocytes in the mouse cerebellum and that ethanol metabolism by astrocytic ALDH2 mediates behavioural effects associated with ethanol intoxication. We show that ALDH2 is expressed in astrocytes in specific brain regions and that astrocytic, but not hepatocytic, ALDH2 is required to produce ethanol-derived acetate in the mouse cerebellum. Cerebellar astrocytic ALDH2 mediates low-dose ethanol-induced elevation of GABA levels, enhancement of tonic inhibition and impairment of balance and coordination skills. Thus, astrocytic ALDH2 controls the production, cellular and behavioural effects of alcohol metabolites in a brain-region-specific manner. Our data indicate that astrocytic ALDH2 is an important, but previously under-recognized, target in the brain to alter alcohol pharmacokinetics and potentially treat alcohol use disorder.

The impact of influenza pulmonary infection and inflammation on vagal bronchopulmonary sensory neurons

FASEB journal : official publication of the Federation of American Societies for Experimental Biology

2021 Mar 01

Verzele, NAJ;Chua, BY;Law, CW;Zhang, A;Ritchie, ME;Wightman, O;Edwards, IN;Hulme, KD;Bloxham, CJ;Bielefeldt-Ohmann, H;Trewella, MW;Moe, AAK;Chew, KY;Mazzone, SB;Short, KR;McGovern, AE;
PMID: 33660333 | DOI: 10.1096/fj.202001509R

Influenza A virus (IAV) is rapidly detected in the airways by the immune system, with resident parenchymal cells and leukocytes orchestrating viral sensing and the induction of antiviral inflammatory responses. The airways are innervated by heterogeneous populations of vagal sensory neurons which also play an important role in pulmonary defense. How these neurons respond to IAV respiratory infection remains unclear. Here, we use a murine model to provide the first evidence that vagal sensory neurons undergo significant transcriptional changes following a respiratory IAV infection. RNA sequencing on vagal sensory ganglia showed that IAV infection induced the expression of many genes associated with an antiviral and pro-inflammatory response and this was accompanied by a significant increase in inflammatory cell recruitment into the vagal ganglia. Assessment of gene expression in single-vagal sensory neurons confirmed that IAV infection induced a neuronal inflammatory phenotype, which was most prominent in bronchopulmonary neurons, and also evident in some neurons innervating other organs. The altered transcriptome could be mimicked by intranasal treatment with cytokines and the lung homogenates of infected mice, in the absence of infectious virus. These data argue that IAV pulmonary infection and subsequent inflammation induces vagal sensory ganglia neuroinflammation and this may have important implications for IAV-induced morbidity.

Compromised counterselection by FAS creates an aggressive subtype of germinal center lymphoma

J Exp Med

2021 Mar 01

Razzaghi, R;Agarwal, S;Kotlov, N;Plotnikova, O;Nomie, K;Huang, DW;Wright, GW;Smith, GA;Li, M;Takata, K;Yamadi, M;Yao, C;O'Shea, JJ;Phelan, JD;Pittaluga, S;Scott, DW;Muppidi, JR;
PMID: 33237303 | DOI: 10.1084/jem.20201173

Fas is highly expressed on germinal center (GC) B cells, and mutations of FAS have been reported in diffuse large B cell lymphoma (DLBCL). Although GC-derived DLBCL has better overall outcomes than other DLBCL types, some cases are refractory, and the molecular basis for this is often unknown. We show that Fas is a strong cell-intrinsic regulator of GC B cells that promotes cell death in the light zone, likely via T follicular helper (Tfh) cell-derived Fas ligand. In the absence of Fas, GCs were more clonally diverse due to an accumulation of cells that did not demonstrably bind antigen. FAS alterations occurred most commonly in GC-derived DLBCL, were associated with inferior outcomes and an enrichment of Tfh cells, and co-occurred with deficiency in HVEM and PD-L1 that regulate the Tfh-B cell interaction. This work shows that Fas is critically required for GC homeostasis and suggests that loss of Tfh-mediated counterselection in the GC contributes to lethality in GC-derived lymphoma. This is a work of the U.S. Government and is not subject to

Basal forebrain mediates prosocial behavior via disinhibition of midbrain dopamine neurons

Proceedings of the National Academy of Sciences of the United States of America

2021 Feb 16

Wang, J;Li, J;Yang, Q;Xie, YK;Wen, YL;Xu, ZZ;Li, Y;Xu, T;Wu, ZY;Duan, S;Xu, H;
PMID: 33563763 | DOI: 10.1073/pnas.2019295118

Sociability is fundamental for our daily life and is compromised in major neuropsychiatric disorders. However, the neuronal circuit mechanisms underlying prosocial behavior are still elusive. Here we identify a causal role of the basal forebrain (BF) in the control of prosocial behavior via inhibitory projections that disinhibit the midbrain ventral tegmental area (VTA) dopamine (DA) neurons. Specifically, BF somatostatin-positive (SST) inhibitory neurons were robustly activated during social interaction. Optogenetic inhibition of these neurons in BF or their axon terminals in the VTA largely abolished social preference. Electrophysiological examinations further revealed that SST neurons predominantly targeted VTA GABA neurons rather than DA neurons. Consistently, optical inhibition of SST neuron axon terminals in the VTA decreased DA release in the nucleus accumbens during social interaction, confirming a disinhibitory action. These data reveal a previously unappreciated function of the BF in prosocial behavior through a disinhibitory circuitry connected to the brain's reward system.

Amylin antibodies frequently display cross-reactivity with CGRP: characterization of eight amylin antibodies

American journal of physiology. Regulatory, integrative and comparative physiology

2021 Feb 10

Rees, TA;Hay, DL;Walker, CS;
PMID: 33565362 | DOI: 10.1152/ajpregu.00338.2020

Amylin is a 37 amino acid endocrine hormone secreted from the pancreas in response to nutrient intake, acting centrally to promote meal ending satiation. With many studies linking amylin action to the nervous system, determining the distribution or expression of amylin in the nervous system is critical. However, amylin shares sequence identity and structural homology to the related neuropeptide calcitonin-gene related peptide (CGRP). This creates challenges in identifying selective amylin antibodies that do not cross-react with CGRP, especially in neural tissues, where CGRP is densely packed into secretory vesicles. Here, we characterized eight amylin antibodies to determine their ability to detect amylin and cross-react with rat or human αCGRP, using immunoblots and pre-absorption controls in rat pancreas. We observed that amylin antibodies frequently cross-reacted with αCGRP and are therefore not suitable for use in tissues which highly express CGRP. Prior work using these antibodies should be revisited in light of our findings.

Winter is coming-Temperature affects immune defenses and susceptibility to Batrachochytrium salamandrivorans

PLoS pathogens

2021 Feb 01

Carter, ED;Bletz, MC;Le Sage, M;LaBumbard, B;Rollins-Smith, LA;Woodhams, DC;Miller, DL;Gray, MJ;
PMID: 33600433 | DOI: 10.1371/journal.ppat.1009234

Environmental temperature is a key factor driving various biological processes, including immune defenses and host-pathogen interactions. Here, we evaluated the effects of environmental temperature on the pathogenicity of the emerging fungal pathogen, Batrachochytrium salamandrivorans (Bsal), using controlled laboratory experiments, and measured components of host immune defense to identify regulating mechanisms. We found that adult and juvenile Notophthalmus viridescens died faster due to Bsal chytridiomycosis at 14°C than at 6 and 22°C. Pathogen replication rates, total available proteins on the skin, and microbiome composition likely drove these relationships. Temperature-dependent skin microbiome composition in our laboratory experiments matched seasonal trends in wild N. viridescens, adding validity to these results. We also found that hydrophobic peptide production after two months post-exposure to Bsal was reduced in infected animals compared to controls, perhaps due to peptide release earlier in infection or impaired granular gland function in diseased animals. Using our temperature-dependent susceptibility results, we performed a geographic analysis that revealed N. viridescens populations in the northeastern United States and southeastern Canada are at greatest risk for Bsal invasion, which shifted risk north compared to previous assessments. Our results indicate that environmental temperature will play a key role in the epidemiology of Bsal and provide evidence that temperature manipulations may be a viable disease management strategy.

EMC3 Is Essential for Retinal Organization and Neurogenesis During Mouse Retinal Development

Investigative ophthalmology & visual science

2021 Feb 01

Cao, X;An, J;Cao, Y;Lv, J;Wang, J;Ding, Y;Lin, X;Zhou, X;
PMID: 33605987 | DOI: 10.1167/iovs.62.2.31

We used a mouse model to explore the role of the endoplasmic reticulum membrane protein complex subunit 3 (EMC3) in mammalian retinal development. The transcription pattern of Emc3 in C57BL/6 mice was analyzed by in situ hybridization. To explore the effects of EMC3 absence on retinal development, the Cre-loxP system was used to generate retina-specific Emc3 in knockout mice (Emc3flox/flox, Six3-cre+; CKO). Morphological changes in the retina of E13.5, E17.5, P0.5, and P7 mice were observed via hematoxylin and eosin staining. Immunofluorescence staining was used to assess protein distribution and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to assess apoptosis changes. Proteins were identified and quantified by Western blotting and proteomic analysis. Electroretinogram (ERG), fundus color photography, and optical coherence tomography were performed on 5-week-old mice to evaluate retinal function and structure. The Emc3 mRNA was widely distributed in the whole retina during development. Loss of retinal EMC3 led to retinal rosette degeneration with mislocalization of cell junction molecules (β-catenin, N-cadherin, and zonula occludens-1) and polarity molecules (Par3 and PKCζ). Endoplasmic reticulum stress and TUNEL apoptosis signals were present in retinal rosette-forming cells. Although the absence of EMC3 promoted the production of photoreceptor cells, 5-week-old mice lost all visual function and had severe retinal morphological degeneration. EMC3 regulates retinal structure by maintaining the polarity of retinal progenitor cells and regulating retinal cell apoptosis.

Intranasal Infection of Ferrets with SARS-CoV-2 as a Model for Asymptomatic Human Infection

Viruses

2021 Jan 15

Everett, HE;Lean, FZX;Byrne, AMP;van Diemen, PM;Rhodes, S;James, J;Mollett, B;Coward, VJ;Skinner, P;Warren, CJ;Bewley, KR;Watson, S;Hurley, S;Ryan, KA;Hall, Y;Simmons, H;Núñez, A;Carroll, MW;Brown, IH;Brookes, SM;
PMID: 33467732 | DOI: 10.3390/v13010113

Ferrets were experimentally inoculated with SARS-CoV-2 (severe acute respiratory syndrome (SARS)-related coronavirus 2) to assess infection dynamics and host response. During the resulting subclinical infection, viral RNA was monitored between 2 and 21 days post-inoculation (dpi), and reached a peak in the upper respiratory cavity between 4 and 6 dpi. Viral genomic sequence analysis in samples from three animals identified the Y453F nucleotide substitution relative to the inoculum. Viral RNA was also detected in environmental samples, specifically in swabs of ferret fur. Microscopy analysis revealed viral protein and RNA in upper respiratory tract tissues, notably in cells of the respiratory and olfactory mucosae of the nasal turbinates, including olfactory neuronal cells. Antibody responses to the spike and nucleoprotein were detected from 21 dpi, but virus-neutralizing activity was low. A second intranasal inoculation (re-exposure) of two ferrets after a 17-day interval did not produce re-initiation of viral RNA shedding, but did amplify the humoral response in one animal. Therefore, ferrets can be experimentally infected with SARS-CoV-2 to model human asymptomatic infection.

G Protein-Biased Mu Opioid Receptor Agonist SR-17018 Has Low In Vivo Efficacy In Non-Human Primates

The Journal of Pain

2023 Apr 01

Ko, M;Ding, H;Kiguchi, N;Zhang, D;Zhang, Y;
| DOI: 10.1016/j.jpain.2023.02.116

SR-17018 was identified as a highly G protein-biased mu opioid peptide (MOP) receptor agonist and lacked MOP agonist-associated adverse effects in mice. The aim of this study was to determine the functional profile of spinal and systemic administration of SR-17018 in non-human primates. In vivo effects of SR-17018 were compared with those of MOP agonists in different intrinsic efficacies, DAMGO, morphine, heroin, and buprenorphine, in behavioral assays established in rhesus monkeys (Macaca mutatta). Nociceptive, itch-scratching, and operant behaviors were measured by experimenters blinded to the dosing conditions. Following intrathecal delivery, SR-17018 (30-300 ug), buprenorphine (3-10 ug), morphine (10-30 ug), and DAMGO (1-3 ug), dose-dependently attenuated capsaicin-induced thermal allodynia (p < 0.05). However, unlike DAMGO and morphine eliciting robust scratching activities, intrathecal SR-17018 and buprenorphine only elicited mild scratching responses, indicating that SR-17018 has low efficacy for activating spinal MOP receptors. In the intravenous drug self-administration assay, heroin (0.3-10 ug/kg/infusion) produced a higher reinforcing strength (abuse liability) as compared to lower reinforcing strengths by SR-17018 (3-30 ug/kg/infusion) and buprenorphine (1-10 ug/kg/infusion) in primates under the progressive-ratio schedule of reinforcement (p < 0.05). The intrathecal opioid-induced itch and intravenous drug self-administration have been documented to distinguish MOP receptor agonists with different intrinsic efficacies. Our findings reveal that in vivo apparent low efficacy of SR-17018 is similar to that of a MOP partial agonist buprenorphine measured by the primate assays with translation relevance. Such a low intrinsic efficacy explains its improved side-effect profile of a highly G protein-biased MOP agonist, S

Neuron-Keratinocyte Communication in the Epidermis in Painful Diabetic Neuropathy

The Journal of Pain

2022 May 01

George, D;Jayaraj, N;Belmadani, A;Ren, D;Miller, R;Menichella, D;
| DOI: 10.1016/j.jpain.2022.03.142

Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. PDN is characterized by small-fiber degeneration, which can progress to complete loss of cutaneous innervation and is accompanied by neuropathic pain. Uncovering the mechanisms underlying axonal degeneration in PDN remains a major challenge to finding effective and disease-modifying therapies. Sensory nerve afferents normally extend into the epidermis in close juxtaposition to keratinocytes but degenerate in diabetic skin. Our aim is to identify the changes in gene expression profiles and the interactions between dorsal root ganglion (DRG) neurons and keratinocytes to explore the mechanisms by which keratinocytes communicate with cutaneous afferents and how this communication impacts axonal degeneration underlying neuropathic pain in PDN. We used a mouse model of PDN where mice were fed a regular diet (RD, 11% fat) or a high-fat diet (HFD, 42% fat) for 10 weeks during which these mice develop glucose intolerance, mechanical allodynia, small fiber neuropathy. Using a single-cell RNA (scRNA-seq) sequencing approach we captured DRG and keratinocytes gene expression profiles and generated interactome maps. scRNA-seq identified both neuronal and non-neuronal clusters and several differentially expressed genes between RD and HFD from the DRG. We were able to identify several clusters of immune cells and keratinocytes at different stages of differentiation. scRNA-seq results were validated using RNAscope on DRG and skin frozen sections. Moreover, we generated interactome maps between DRG neurons and the peripheral cells to highlight ligand-receptor interactions and we looked to identify genes that were differentially expressed in these interactions. Taken together our data highlights the importance of studying neurons in conjunction with the cells in the tissues with which they interact to identify ligand-receptor interactions that may lead to the identification of neuron signaling in a chronic pain state such as PDN. Grant support from 1R01AR77691-01.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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