High-multiplex tissue imaging in routine pathology-are we there yet?

Virchows Archiv : an international journal of pathology

2023 Feb 09

Einhaus, J;Rochwarger, A;Mattern, S;Gaudillière, B;Schürch, CM;
PMID: 36757500 | DOI: 10.1007/s00428-023-03509-6

High-multiplex tissue imaging (HMTI) approaches comprise several novel immunohistological methods that enable in-depth, spatial single-cell analysis. Over recent years, studies in tumor biology, infectious diseases, and autoimmune conditions have demonstrated the information gain accessible when mapping complex tissues with HMTI. Tumor biology has been a focus of innovative multiparametric approaches, as the tumor microenvironment (TME) contains great informative value for accurate diagnosis and targeted therapeutic approaches: unraveling the cellular composition and structural organization of the TME using sophisticated computational tools for spatial analysis has produced histopathologic biomarkers for outcomes in breast cancer, predictors of positive immunotherapy response in melanoma, and histological subgroups of colorectal carcinoma. Integration of HMTI technologies into existing clinical workflows such as molecular tumor boards will contribute to improve patient outcomes through personalized treatments tailored to the specific heterogeneous pathological fingerprint of cancer, autoimmunity, or infection. Here, we review the advantages and limitations of existing HMTI technologies and outline how spatial single-cell data can improve our understanding of pathological disease mechanisms and determinants of treatment success. We provide an overview of the analytic processing and interpretation and discuss how HMTI can improve future routine clinical diagnostic and therapeutic processes.

Advances in HIV Research Using Mass Cytometry

Current HIV/AIDS reports

2023 Jan 23

George, AF;Roan, NR;
PMID: 36689119 | DOI: 10.1007/s11904-023-00649-x

This review describes how advances in CyTOF and high-dimensional analysis methods have furthered our understanding of HIV transmission, pathogenesis, persistence, and immunity.CyTOF has generated important insight on several aspects of HIV biology: (1) the differences between cells permissive to productive vs. latent HIV infection, and the HIV-induced remodeling of infected cells; (2) factors that contribute to the persistence of the long-term HIV reservoir, in both blood and tissues; and (3) the impact of HIV on the immune system, in the context of both uncontrolled and controlled infection. CyTOF and high-dimensional analysis tools have enabled in-depth assessment of specific host antigens remodeled by HIV, and have revealed insights into the features of HIV-infected cells enabling them to survive and persist, and of the immune cells that can respond to and potentially control HIV replication. CyTOF and other related high-dimensional phenotyping approaches remain powerful tools for translational research, and applied HIV to cohort studies can inform on mechanisms of HIV pathogenesis and persistence, and potentially identify biomarkers for viral eradication or control.

Brain-to-BAT- and Back?: Crosstalk between the Central Nervous System and Thermogenic Adipose Tissue in Development and Therapy of Obesity

Brain sciences

2022 Dec 01

Till, A;Fries, C;Fenske, WK;
PMID: 36552107 | DOI: 10.3390/brainsci12121646

The body of mammals harbors two distinct types of adipose tissue: while cells within the white adipose tissue (WAT) store surplus energy as lipids, brown adipose tissue (BAT) is nowadays recognized as the main tissue for transforming chemical energy into heat. This process, referred to as 'non-shivering thermogenesis', is facilitated by the uncoupling of the electron transport across mitochondrial membranes from ATP production. BAT-dependent thermogenesis acts as a safeguarding mechanism under reduced ambient temperature but also plays a critical role in metabolic and energy homeostasis in health and disease. In this review, we summarize the evolutionary structure, function and regulation of the BAT organ under neuronal and hormonal control and discuss its mutual interaction with the central nervous system. We conclude by conceptualizing how better understanding the multifaceted communicative links between the brain and BAT opens avenues for novel therapeutic approaches to treat obesity and related metabolic disorders.

Patterns of Interferon γ Expression and C4d Deposition in Chronic Intervillositis of Unknown Etiology

Pediatric and developmental pathology : the official journal of the Society for Pediatric Pathology and the Paediatric Pathology Society

2022 Dec 26

Terry, J;
PMID: 36571293 | DOI: 10.1177/10935266221144083

The pathogenesis of chronic intervillositis of unknown etiology (CIUE) may involve IFNγ overexpression. This study assesses the extent of IFNγ expression in CIUE by immunohistochemistry and compares it to spontaneous pregnancy losses. C4d deposition is also assessed to see whether IFNγ and C4d might represent separate diagnostic categories. Placenta from first to early second trimester with high grade CIUE (CHG; 17 cases) and low grade CIUE (CLG; 12 cases) is compared to euploid (SPLN; 18 cases), aneuploid spontaneous pregnancy losses (SPLA, 17 cases), normal placenta (NP, 13 cases). Protein level expression of IFNγ and C4d is assessed on whole tissue sections by immunohistochemistry. 35% of CHG and 42% of CLG show some level of IFNγ expression localized to the luminal surface of syncytiotrophoblast. 12% of SPLA and no SPLN or NP cases are IFNγ positive. C4d deposition is seen in 100% of CIUE, 88% of SPLA, 83% of SPLN, and 46% of NP samples. IFNγ overexpression occurs in approximately 40% of CIUE-related pregnancy losses. IFNγ expression restricted to a subgroup of CIUE implies that IFNγ may define a distinct disease process. The non-discriminatory pattern of C4d deposition suggests it is a non-specific phenomenon possibly related to placental damage.

Analyses of Transcriptomics Cell Signalling for Pre-Screening Applications in the Integrated Approach for Testing and Assessment of Non-Genotoxic Carcinogens

International journal of molecular sciences

2022 Oct 22

Oku, Y;Madia, F;Lau, P;Paparella, M;McGovern, T;Luijten, M;Jacobs, MN;
PMID: 36361516 | DOI: 10.3390/ijms232112718

With recent rapid advancement of methodological tools, mechanistic understanding of biological processes leading to carcinogenesis is expanding. New approach methodologies such as transcriptomics can inform on non-genotoxic mechanisms of chemical carcinogens and can be developed for regulatory applications. The Organisation for the Economic Cooperation and Development (OECD) expert group developing an Integrated Approach to the Testing and Assessment (IATA) of Non-Genotoxic Carcinogens (NGTxC) is reviewing the possible assays to be integrated therein. In this context, we review the application of transcriptomics approaches suitable for pre-screening gene expression changes associated with phenotypic alterations that underlie the carcinogenic processes for subsequent prioritisation of downstream test methods appropriate to specific key events of non-genotoxic carcinogenesis. Using case studies, we evaluate the potential of gene expression analyses especially in relation to breast cancer, to identify the most relevant approaches that could be utilised as (pre-) screening tools, for example Gene Set Enrichment Analysis (GSEA). We also consider how to address the challenges to integrate gene panels and transcriptomic assays into the IATA, highlighting the pivotal omics markers identified for assay measurement in the IATA key events of inflammation, immune response, mitogenic signalling and cell injury.

A robust and tunable system for targeted cell ablation in developing embryos

Developmental cell

2022 Aug 22

Labbaf, Z;Petratou, K;Ermlich, L;Backer, W;Tarbashevich, K;Reichman-Fried, M;Luschnig, S;Schulte-Merker, S;Raz, E;
PMID: 35914525 | DOI: 10.1016/j.devcel.2022.07.008

Cell ablation is a key method in the research fields of developmental biology, tissue regeneration, and tissue homeostasis. Eliminating specific cell populations allows for characterizing interactions that control cell differentiation, death, behavior, and spatial organization of cells. Current methodologies for inducing cell death suffer from relatively slow kinetics, making them unsuitable for analyzing rapid events and following primary and immediate consequences of the ablation. To address this, we developed a cell-ablation system that is based on bacterial toxin/anti-toxin proteins and enables rapid and cell-autonomous elimination of specific cell types and organs in zebrafish embryos. A unique feature of this system is that it uses an anti-toxin, which allows for controlling the degree and timing of ablation and the resulting phenotypes. The transgenic zebrafish generated in this work represent a highly efficient tool for cell ablation, and this approach is applicable to other model organisms as demonstrated here for Drosophila.

Mitochondria-targeted nanozymes eliminate oxidative damage in retinal neovascularization disease

Journal of controlled release : official journal of the Controlled Release Society

2022 Aug 25

Xue, B;Ge, M;Fan, K;Huang, X;Yan, X;Jiang, W;Jiang, B;Yang, Z;
PMID: 35987352 | DOI: 10.1016/j.jconrel.2022.08.026

Retinal neovascularization is typically accompanied by hypoxia-induced oxidative injury in the vascular system. This study developed an ultrasmall (6-8 nm) platinum (Pt) nanozyme loaded mitochondria-targeted liposome (Pt@MitoLipo) to alleviate hypoxia and eliminate excess reactive oxygen species (ROS) for effective retinal neovascularization disease therapy. Pt nanozymes possess superoxide dismutase (SOD) and catalase (CAT) cascade enzyme-like activities, which convert cytotoxic O2•- and H2O2 into nontoxic H2O and O2. Triphenylphosphonium (TPP)-conjugated liposomes were coated on the surface of Pt nanozymes to increase their biocompatibility and help them penetrate the cell membrane, escape from the lysosomal barrier, and target mitochondria, thus achieving precise scavenging of mitochondrial O2•- and relief from hypoxia. Using an oxygen-induced retinopathy (OIR) mouse model, we demonstrated that Pt@MitoLipo nanozymes significantly suppressed hypoxia-induced abnormal neovascularization and facilitated avascular normalization of the retina in vivo without any noticeable toxicity. This study provides a promising way to break through cellular barriers and target scavenging mitochondrial O2•- and illustrates the potential of ROS-scavenging and hypoxia relief in retinal neovascularization disease therapy.

DLX1 and the NuRD complex cooperate in enhancer decommissioning and transcriptional repression

Development (Cambridge, England)

2022 Jun 01

Price, JD;Lindtner, S;Ypsilanti, A;Binyameen, F;Johnson, JR;Newton, BW;Krogan, NJ;Rubenstein, JLR;
PMID: 35695185 | DOI: 10.1242/dev.199508

In the developing subpallium, the fate decision between neurons and glia is driven by expression of Dlx1/2 or Olig1/2, respectively, two sets of transcription factors with a mutually repressive relationship. The mechanism by which Dlx1/2 repress progenitor and oligodendrocyte fate, while promoting transcription of genes needed for differentiation, is not fully understood. We identified a motif within DLX1 that binds RBBP4, a NuRD complex subunit. ChIP-seq studies of genomic occupancy of DLX1 and six different members of the NuRD complex show that DLX1 and NuRD colocalize to putative regulatory elements enriched near other transcription factor genes. Loss of Dlx1/2 leads to dysregulation of genome accessibility at putative regulatory elements near genes repressed by Dlx1/2, including Olig2. Consequently, heterozygosity of Dlx1/2 and Rbbp4 leads to an increase in the production of OLIG2+ cells. These findings highlight the importance of the interplay between transcription factors and chromatin remodelers in regulating cell-fate decisions.

In vivo imaging of mammary epithelial cell dynamics in response to lineage-biased Wnt/β-catenin activation

Cell reports

2022 Mar 08

Lloyd-Lewis, B;Gobbo, F;Perkins, M;Jacquemin, G;Huyghe, M;Faraldo, MM;Fre, S;
PMID: 35263603 | DOI: 10.1016/j.celrep.2022.110461

Real-time in vivo imaging provides an essential window into the spatiotemporal cellular events contributing to tissue development and pathology. By coupling longitudinal intravital imaging with genetic lineage tracing, here we capture the earliest cellular events arising in response to active Wnt/β-catenin signaling and the ensuing impact on the organization and differentiation of the mammary epithelium. This enables us to interrogate how Wnt/β-catenin regulates the dynamics of distinct subpopulations of mammary epithelial cells in vivo and in real time. We show that β-catenin stabilization, when targeted to either the mammary luminal or basal epithelial lineage, leads to cellular rearrangements that precipitate the formation of hyperplastic lesions that undergo squamous transdifferentiation. These results enhance our understanding of the earliest stages of hyperplastic lesion formation in vivo and reveal that, in mammary neoplastic development, β-catenin activation dictates a hair follicle/epidermal differentiation program independently of the targeted cell of origin.

Topographic connectivity and cellular profiling reveal detailed input pathways and functionally distinct cell types in the subthalamic nucleus

Cell reports

2022 Mar 01

Jeon, H;Lee, H;Kwon, DH;Kim, J;Tanaka-Yamamoto, K;Yook, JS;Feng, L;Park, HR;Lim, YH;Cho, ZH;Paek, SH;Kim, J;
PMID: 35235786 | DOI: 10.1016/j.celrep.2022.110439

The subthalamic nucleus (STN) controls psychomotor activity and is an efficient therapeutic deep brain stimulation target in individuals with Parkinson's disease. Despite evidence indicating position-dependent therapeutic effects and distinct functions within the STN, the input circuit and cellular profile in the STN remain largely unclear. Using neuroanatomical techniques, we construct a comprehensive connectivity map of the indirect and hyperdirect pathways in the mouse STN. Our circuit- and cellular-level connectivities reveal a topographically graded organization with three types of indirect and hyperdirect pathways (external globus pallidus only, STN only, and collateral). We confirm consistent pathways into the human STN by 7 T MRI-based tractography. We identify two functional types of topographically distinct glutamatergic STN neurons (parvalbumin [PV+/-]) with synaptic connectivity from indirect and hyperdirect pathways. Glutamatergic PV+ STN neurons contribute to burst firing. These data suggest a complex interplay of information integration within the basal ganglia underlying coordinated movement control and therapeutic effects.

Advances in Biomaterial‐Based Spinal Cord Injury Repair

Advanced Functional Materials

2021 Dec 01

Shen, H;Fan, C;You, Z;Xiao, Z;Zhao, Y;Dai, J;
| DOI: 10.1002/adfm.202110628

Spinal cord injury (SCI) often leads to the loss of motor and sensory functions and is a major challenge in neurological clinical practice. Understanding the pathophysiological changes and the inhibitory microenvironment is crucial to enable the identification of potential mechanisms for functional restoration and to provide guidance for the development of efficient treatment and repair strategies. To date, the implantation of specifically functionalized biomaterials in the lesion area has been shown to help promote axon regeneration and facilitate neuronal circuit generation by remolding SCI microenvironments. Moreover, structural and functional restoration of the spinal cord through the transplantation of naive spinal cord tissue grafts from adult donors, artificial spinal cord-like tissue developed from tissue engineering, and 3D printing will open up new avenues for SCI treatment. This review focuses on the dynamic pathophysiological changes in SCI microenvironments, biomaterials for SCI repairs, strategies for restoring spinal cord structure and function, experimental animal models, regenerative mechanisms, and clinical studies for SCI repair. The current status, recent advances, challenges, and prospects of scaffold-based SCI repair from basic to clinical settings are summarized and discussed, to provide a reference that will help to guide the future exploration and development of spinal cord regeneration strategies.

Glutamate in primary afferents is required for itch transmission

Neuron

2021 Dec 30

Cui, L;Guo, J;Cranfill, SL;Gautam, M;Bhattarai, J;Olson, W;Beattie, K;Challis, RC;Wu, Q;Song, X;Raabe, T;Gradinaru, V;Ma, M;Liu, Q;Luo, W;
PMID: 34986325 | DOI: 10.1016/j.neuron.2021.12.007

Whether glutamate or itch-selective neurotransmitters are used to confer itch specificity is still under debate. We focused on an itch-selective population of primary afferents expressing MRGPRA3, which highly expresses Vglut2 and the neuropeptide neuromedin B (Nmb), to investigate this question. Optogenetic stimulation of MRGPRA3+ afferents triggers scratching and other itch-related avoidance behaviors. Using a combination of optogenetics, spinal cord slice recordings, Vglut2 conditional knockout mice, and behavior assays, we showed that glutamate is essential for MRGPRA3+ afferents to transmit itch. We further demonstrated that MRGPRA3+ afferents form monosynaptic connections with both NMBR+ and NMBR- neurons and that NMB and glutamate together can enhance the activity of NMBR+ spinal DH neurons. Moreover, Nmb in MRGPRA3+ afferents and NMBR+ DH neurons are required for chloroquine-induced scratching. Together, our results establish a new model in which glutamate is an essential neurotransmitter in primary afferents for itch transmission, whereas NMB signaling enhances its activities.

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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
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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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