Wound healing, fibroblast heterogeneity, and fibrosis

Cell stem cell

2022 Aug 04

Talbott, HE;Mascharak, S;Griffin, M;Wan, DC;Longaker, MT;
PMID: 35931028 | DOI: 10.1016/j.stem.2022.07.006

Fibroblasts are highly dynamic cells that play a central role in tissue repair and fibrosis. However, the mechanisms by which they contribute to both physiologic and pathologic states of extracellular matrix deposition and remodeling are just starting to be understood. In this review article, we discuss the current state of knowledge in fibroblast biology and heterogeneity, with a primary focus on the role of fibroblasts in skin wound repair. We also consider emerging techniques in the field, which enable an increasingly nuanced and contextualized understanding of these complex systems, and evaluate limitations of existing methodologies and knowledge. Collectively, this review spotlights a diverse body of research examining an often-overlooked cell type-the fibroblast-and its critical functions in wound repair and beyond.

Midbrain Peptidergic Neurons Enable Maternal Nesting

SSRN Electronic Journal

2021 Jul 09

Topilko, T;Diaz, S;Pacheco, C;Verny, F;Deleuze, C;Gaspar, P;Renier, N;
| DOI: 10.2139/ssrn.3878409

Optimizing reproductive fitness in mammalian females requires behavioral adaptations during pregnancy. Maternal preparatory nesting is an essential behavior for the survival of the upcoming litter. Brain-wide immediate early gene mapping in mice evoked by nesting sequences revealed that phases of nest construction strongly activate peptidergic neurons of the Edinger-Westphal nucleus in pregnant mice. Genetic ablation, bidirectional neuromodulation, and in vitro and in vivo activity recordings demonstrated that these neurons are essential to modulate arousal before sleep to promote nesting specifically. We show that these neurons enable the behavioral effects of progesterone on preparatory nesting by modulating a broad network of downstream targets. Overall, our study deciphers the role of midbrain neurons in behavioral adaptations during pregnancy vital for reproductive fitness.

Top-down control of conditioned overconsumption is mediated by insular cortex Nos1 neurons

Cell metabolism

2021 Mar 23

Stern, SA;Azevedo, EP;Pomeranz, LE;Doerig, KR;Ivan, VJ;Friedman, JM;
PMID: 33761312 | DOI: 10.1016/j.cmet.2021.03.001

Associative learning allows animals to adapt their behavior in response to environmental cues. For example, sensory cues associated with food availability can trigger overconsumption even in sated animals. However, the neural mechanisms mediating cue-driven non-homeostatic feeding are poorly understood. To study this, we recently developed a behavioral task in which contextual cues increase feeding even in sated mice. Here, we show that an insular cortex to central amygdala circuit is necessary for conditioned overconsumption, but not for homeostatic feeding. This projection is marked by a population of glutamatergic nitric oxide synthase-1 (Nos1)-expressing neurons, which are specifically active during feeding bouts. Finally, we show that activation of insular cortex Nos1 neurons suppresses satiety signals in the central amygdala. The data, thus, indicate that the insular cortex provides top-down control of homeostatic circuits to promote overconsumption in response to learned cues.

Single-cell RNA-seq analysis reveals compartment-specific heterogeneity and plasticity of microglia

iScience

2021 Mar 01

Zheng, J;Ru, W;Adolacion, J;Spurgat, M;Liu, X;Yuan, S;Liang, R;Dong, J;Potter, A;Potter, S;Chen, K;Chen, R;Varadarajan, N;Tang, S;
| DOI: 10.1016/j.isci.2021.102186

Microglia are ubiquitous central nervous system (CNS)-resident macrophages that maintain homeostasis of neural tissues and protect them from pathogen attacks. Yet, their differentiation in different compartments remains elusive. We performed single-cell RNA-seq to compare microglial subtypes in the cortex and the spinal cord. A multi-way comparative analysis was carried out on samples from C57/BL and HIV gp120 transgenic mice at two, four, and eight months of age. The results revealed overlapping but distinct microglial populations in the cortex and the spinal cord. The differential heterogeneity of microglia in these CNS regions was further suggested by their disparity of plasticity in response to life span progression and HIV-1 pathogenic protein gp120. Our findings indicate that microglia in different CNS compartments are adapted to their local environments to fulfill region-specific biological functions.

In situ cytokine gene expression in early stage of virulent Newcastle disease in chickens

Veterinary pathology

2021 Nov 18

Brown, C;Zhang, J;Pantin-Jackwood, M;Dimitrov, K;Ferreira, HL;Suarez, D;
PMID: 34794360 | DOI: 10.1177/03009858211045945

Selected lymphoid and reproductive tissues were examined from groups of 3-week-old chickens and 62-week-old hens that were inoculated choanally and conjunctivally with 106 EID50 of a virulent Newcastle disease virus (NDV) isolate from the California 2018-2020 outbreak, and euthanized at 1, 2, and 3 days postinfection. In the 3-week-old chickens, immunohistochemistry for NDV and for T and B cell lymphocytes, as well as in situ hybridization for IL-1β, IL-6, IFN-γ, and TNF-α revealed extensive expression of IL-1β and IL-6 in lymphoid tissues, often coinciding with NDV antigen. IFN-γ was only expressed infrequently in the same lymphoid tissues, and TNF-α was rarely expressed. T-cell populations initially expanded but by day 3 their numbers were below control levels. B cells underwent a similar expansion but remained elevated in some tissues, notably spleen, cecal tonsils, and cloacal bursa. Cytokine expression in the 62-week-old hens was overall lower than in the 3-week-old birds, and there was more prolonged infiltration of both T and B cells in the older birds. The strong pro-inflammatory cytokine response in young chickens is proposed as the reason for more severe disease.

Comprehensive BCMA Expression Profiling in Adult Normal Human Brain Suggests a Low Risk of On-target Neurotoxicity in BCMA-targeting Multiple Myeloma Therapy

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society

2022 Feb 22

Marella, M;Yao, X;Carreira, V;Bustamante, MF;Clark, HB;Jackson, CC;Zudaire, E;Schecter, JM;Glover, TD;Shenton, J;Cornax, I;
PMID: 35193424 | DOI: 10.1369/00221554221079579

B-cell maturation antigen (BCMA) is a target for the treatment of multiple myeloma with cytolytic therapies, such as chimeric antigen receptor T-cells or T-cell redirecting antibodies. To better understand the potential for "on-target/off-tumor" toxicity caused by BCMA-targeting cytolytic therapies in the brain, we investigated normal brain BCMA expression. An immunohistochemistry (IHC) assay using the E6D7B commercial monoclonal antibody was applied to 107 formalin-fixed, paraffin-embedded brain samples (cerebrum, basal ganglia, cerebellum, brainstem; 63 unique donors). Although immunoreactivity was observed in a small number of neurons in brain regions including the striatum, thalamus, midbrain, and medulla, this immunoreactivity was considered nonspecific and not reflective of BCMA expression because it was distinct from the membranous and Golgi-like pattern seen in positive control samples, was not replicated when a different IHC antibody (D6 clone) was used, and was not corroborated by in situ hybridization data. Analysis of RNA-sequencing data from 478 donors in the GTEx and Allen BrainSpan databases demonstrated low levels of BCMA RNA expression in the striatum of young donors with levels becoming negligible beyond 30 years of age. We concluded that BCMA protein is not present in normal adult human brain, and therefore on-target toxicity in the brain is unlikely.

CDK7 and MITF repress a transcription program involved in survival and drug tolerance in melanoma

EMBO reports

2021 Jul 23

Berico, P;Cigrang, M;Davidson, G;Braun, C;Sandoz, J;Legras, S;Vokshi, BH;Slovic, N;Peyresaubes, F;Gene Robles, CM;Egly, JM;Compe, E;Davidson, I;Coin, F;
PMID: 34296805 | DOI: 10.15252/embr.202051683

Melanoma cell phenotype switching between differentiated melanocytic and undifferentiated mesenchymal-like states drives metastasis and drug resistance. CDK7 is the serine/threonine kinase of the basal transcription factor TFIIH. We show that dedifferentiation of melanocytic-type melanoma cells into mesenchymal-like cells and acquisition of tolerance to targeted therapies is achieved through chronic inhibition of CDK7. In addition to emergence of a mesenchymal-type signature, we identify a GATA6-dependent gene expression program comprising genes such as AMIGO2 or ABCG2 involved in melanoma survival or targeted drug tolerance, respectively. Mechanistically, we show that CDK7 drives expression of the melanocyte lineage transcription factor MITF that in turn binds to an intronic region of GATA6 to repress its expression in melanocytic-type cells. We show that GATA6 expression is activated in MITF-low melanoma cells of patient-derived xenografts. Taken together, our data show how the poorly characterized repressive function of MITF in melanoma participates in a molecular cascade regulating activation of a transcriptional program involved in survival and drug resistance in melanoma.

Coinfection of porcine deltacoronavirus and porcine epidemic diarrhea virus altered viral tropism in gastrointestinal tract in a piglet model

Virology

2021 Mar 17

Jiao, Z;Liang, J;Yang, Y;Li, Y;Yan, Z;Hu, G;Gu, C;Hu, X;Cheng, G;Peng, G;Zhang, W;
PMID: 33756424 | DOI: 10.1016/j.virol.2021.03.006

Coinfection of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) is one of common findings in diarrheal piglets that cause massive economic losses to the pig industry globally. However, the mechanism of the co-infection is unclear. In this study, neonatal non-colostrum-fed piglets were exposed orally with a single infection of PDCoV or PEDV, or coinfection of PDCoV and PEDV. Clinically all viral infected piglets developed watery diarrhea and dehydration in 24 h post-exposure (hpe) and were succumbed to viral diarrhea disease and euthanized at 72 hpe. Histopathologically, acute gastroenteritis is evident in all viral infected piglet. Immunohistochemistry, RNAscope and RT-qPCR demonstrated that PEDV tropism changes from epithelial cells of small intestine to gastric epithelial cells and macrophages in Peyer's patches in the ileum. These findings suggest that coinfection of PDCoV and PEDV can alter PEDV tropism that may affect the outcome of viral disease in piglets. This animal model can be used for the pathogenesis and vaccination of viral coinfection in piglet in the future.

Murine astrovirus tropism for goblet cells and enterocytes facilitates an IFN-λ response in vivo and in enteroid cultures

Mucosal immunology

2021 Mar 05

Ingle, H;Hassan, E;Gawron, J;Mihi, B;Li, Y;Kennedy, EA;Kalugotla, G;Makimaa, H;Lee, S;Desai, P;McDonald, KG;Diamond, MS;Newberry, RD;Good, M;Baldridge, MT;
PMID: 33674763 | DOI: 10.1038/s41385-021-00387-6

Although they globally cause viral gastroenteritis in children, astroviruses are understudied due to the lack of well-defined animal models. While murine astroviruses (muAstVs) chronically infect immunodeficient mice, a culture system and understanding of their pathogenesis is lacking. Here, we describe a platform to cultivate muAstV using air-liquid interface (ALI) cultures derived from mouse enteroids, which support apical infection and release. Chronic muAstV infection occurs predominantly in the small intestine and correlates with higher interferon-lambda (IFN-λ) expression. MuAstV stimulates IFN-λ production in ALI, recapitulating our in vivo findings. We demonstrate that goblet cells and enterocytes are targets for chronic muAstV infection in vivo, and that infection is enhanced by parasite co-infection or type 2 cytokine signaling. Depletion of goblet cells from ALI limits muAstV infection in vitro. During chronic infection, muAstV stimulates IFN-λ production in infected cells and induces ISGs throughout the intestinal epithelium in an IFN-λ-receptor-dependent manner. Collectively, our study provides insights into the cellular tropism and innate immune responses to muAstV and establishes an enteroid-based culture system to propagate muAstV in vitro.

RNAScope in situ Hybridization as a Novel Technique for the Assessment of c-KIT mRNA Expression in Canine Mast Cell Tumor

Frontiers in veterinary science

2021 Feb 16

De Biase, D;Prisco, F;Piegari, G;Ilsami, A;d'Aquino, I;Baldassarre, V;Zito Marino, F;Franco, R;Papparella, S;Paciello, O;
PMID: 33665215 | DOI: 10.3389/fvets.2021.591961

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.

Genetic Depletion Of Acid-Sensing Ion Channel 3 In Macrophage/Monocytes Prevents Development Of Activity-Induced Muscle Pain

The Journal of Pain

2023 Apr 01

Hayashi, K;Lesnak, J;Plumb, A;Rasmussen, L;Sluka, K;
| DOI: 10.1016/j.jpain.2023.02.105

We developed an animal model of activity-induced muscle pain that combines muscle fatigue with a non-painful, low-dose muscle insult. We previously showed that pharmacological blockade of acid-sensing ion channel 3 (ASIC3) in muscle prevents the activity-induced muscle pain, however, genetic deletion of ASIC3 in primary afferents innervating muscle has no effect on pain. We hypothesized that genetic deletion of ASIC3 on macrophages would prevent activity-induced muscle pain. ASIC3fl/fl mice were crossed with Cx3cr1-Cre mice to generate macrophage/monocyte-specific deletion of ASIC3. To confirm the genetic deletion of ASIC3, expression of ASIC3 was assessed from the peritoneal macrophages using quantitative PCR. Muscle pain was induced by 2, 20μl pH 5.0 injections, 5 days apart, in the gastrocnemius muscle combined with fatiguing muscle contractions in male and female mice. To assess hyperalgesia, muscle withdrawal thresholds (MWT) of the gastrocnemius muscle were measured before and after induction of the model. There was a decrease in ASIC3 mRNA expression in peritoneal macrophages from Cx3cr1CreASIC3fl/fl mice. The decrease in MWT was prevented in Cx3cr1CreASIC3fl/fl mice in both male and female mice, when compared to genetic controls (Cx3cr1CreASIC3+/+)(p

A mast cell-thermoregulatory neuron circuit axis regulates hypothermia in anaphylaxis

Science immunology

2023 Mar 17

Bao, C;Chen, O;Sheng, H;Zhang, J;Luo, Y;Hayes, BW;Liang, H;Liedtke, W;Ji, RR;Abraham, SN;
PMID: 36930731 | DOI: 10.1126/sciimmunol.adc9417

IgE-mediated anaphylaxis is an acute life-threatening systemic reaction to allergens, including certain foods and venoms. Anaphylaxis is triggered when blood-borne allergens activate IgE-bound perivascular mast cells (MCs) throughout the body, causing an extensive systemic release of MC mediators. Through precipitating vasodilatation and vascular leakage, these mediators are believed to trigger a sharp drop in blood pressure in humans and in core body temperature in animals. We report that the IgE/MC-mediated drop in body temperature in mice associated with anaphylaxis also requires the body's thermoregulatory neural circuit. This circuit is activated when granule-borne chymase from MCs is deposited on proximal TRPV1+ sensory neurons and stimulates them via protease-activated receptor-1. This triggers the activation of the body's thermoregulatory neural network, which rapidly attenuates brown adipose tissue thermogenesis to cause hypothermia. Mice deficient in either chymase or TRPV1 exhibited limited IgE-mediated anaphylaxis, and, in wild-type mice, anaphylaxis could be recapitulated simply by systemically activating TRPV1+ sensory neurons. Thus, in addition to their well-known effects on the vasculature, MC products, especially chymase, promote IgE-mediated anaphylaxis by activating the thermoregulatory neural circuit.

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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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