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.

Fibroblast Heterogeneity in Healthy and Wounded Skin

Cold Spring Harbor perspectives in biology

2022 Jun 06

Ganier, C;Rognoni, E;Goss, G;Lynch, M;Watt, FM;
PMID: 35667795 | DOI: 10.1101/cshperspect.a041238

Fibroblasts are the main cell type in the dermis. They are responsible for the synthesis and deposition of structural proteins such as collagen and elastin, which are integrated into the extracellular matrix (ECM). Mouse and human studies using flow cytometry, cell culture, skin reconstitution, and lineage tracing experiments have shown the existence of different subpopulations of fibroblasts, including papillary fibroblasts, reticular fibroblasts, and fibroblasts comprising the dermal papilla at the base of the hair follicle. In recent years, the technological advances in single-cell sequencing have allowed researchers to study the repertoire of cells present in full-thickness skin including the dermis. Multiple groups have confirmed that distinct fibroblast populations can be identified in mouse and human dermis on the basis of differences in the transcriptional profile. Here, we discuss the current state of knowledge regarding dermal fibroblast heterogeneity in healthy mouse and human skin, highlighting the similarities and differences between mouse and human fibroblast subpopulations. We also discuss how fibroblast heterogeneity may provide insights into physiological wound healing and its dysfunction in pathological states such as hypertrophic and keloid scars.

Hybridization Chain Reaction for mRNA Localization in Single Cells from Mouse and Human Cryosections

Current protocols

2022 May 01

May-Zhang, AA;Benthal, JT;Southard-Smith, EM;
PMID: 35612422 | DOI: 10.1002/cpz1.439

In situ hybridization has been a robust method for detection of mRNA expression in whole-mount samples or tissue sections for more than 50 years. Recent technical advances for in situ hybridization have incorporated oligo-based probes that attain greater tissue penetration and signal amplification steps with restricted localization for visualization of specific mRNAs within single cells. One such method is third-generation in situ hybridization chain reaction (V3HCR). Here, we report an optimized protocol for V3HCR detection of gene expression using sectioned frozen tissues from mouse and human on microscope slides. Our methods and modifications for cryosectioning, tissue fixation, and processing over a three-day V3HCR protocol are detailed along with recommendations for aliquoting and storing V3HCR single-stranded DNA probes and hairpin amplifiers. In addition, we describe a method for blocking background signal from lipofuscin, a highly autofluorescent material that is widespread in human neurons and often complicates imaging efforts. After testing multiple strategies for reduction of lipofuscin, we determined that application of a lipofuscin quencher dye is compatible with V3HCR, in contrast to other methods like cupric sulfate quenching or Sudan Black B blocking that cause V3HCR signal loss. This adaptation enables application of V3HCR for in situ detection of gene expression in human neuronal populations that are otherwise problematic due to lipofuscin autofluorescence.

An in situ hybridization study of syndecan family during the late stages of developing mouse molar tooth germ

Anatomical science international

2022 Feb 04

Fujikawa, K;Nonaka, N;Wang, X;Shibata, S;
PMID: 35119611 | DOI: 10.1007/s12565-022-00647-w

Expression of syndecan-1, 2, 3, and 4 mRNAs during the late stages of tooth germ formation was investigated by in situ hybridization, using [35S]-UTP-labeled cRNA probes. Syndecan-1 mRNA was mainly expressed in the stellate reticulum and stratum intermedium as well as at the cervical region of dental papilla/dental follicle during E18.5-P3.0. Expression in the dental epithelium was enhanced during the postnatal periods, which was supported by real-time RT-PCR analysis. These spatiotemporal expression patterns may suggest specific roles of syndecan-1 in tooth formation such as tooth eruption or root formation. Syndecan-3 mRNA expression became evident in odontoblasts at E18.5, but compared to collagen type I mRNA, which was strongly expressed at this stage, syndecan-3 expression in odontoblast was restricted in mature odontoblasts beneath the cusps during the postnatal periods. This result was also supported by real-time RT-PCR analysis, and indicated that syndecan-3 may be involved in the progress of dentinogenesis rather than in the initiation of it. Syndecan-4 mRNA roughly showed comparable expression patterns to those of syndecan-3. Syndecan-2 mRNA did not show significant expression during the experimental period, but real-time RT-PCR analysis suggested that syndecan-2 expression might be enhanced with hard tissue formation.

CircRPN2 inhibits aerobic glycolysis and metastasis in hepatocellular carcinoma

Cancer research

2022 Jan 19

Li, J;Hu, ZQ;Yu, S;Mao, L;Zhou, Z;Wang, P;Gong, Y;Su, S;Zhou, J;Fan, J;Zhou, SL;Huang, X;
PMID: 35045986 | DOI: 10.1158/0008-5472.CAN-21-1259

Although circular RNAs (circRNA) are known to modulate tumor initiation and progression, their role in hepatocellular carcinoma (HCC) metastasis remains poorly understood. Here, three metastasis-associated circRNAs identified in a previous circRNA-sequencing study were screened and validated in two HCC cohorts. CircRPN2 was downregulated in highly metastatic HCC cell lines and HCC tissues with metastasis. HCC patients with lower circRPN2 levels displayed shorter overall survival and higher rates of cumulative recurrence. Mechanistic studies in vitro and in vivo revealed that circRPN2 binds to enolase 1 (ENO1) and accelerates its degradation to promote glycolytic reprogramming through the AKT/mTOR pathway, thereby inhibiting HCC metastasis. CircRPN2 also acted as a competing endogenous RNA for miR-183-5p, which increases forkhead box protein O1 (FOXO1) expression to suppress glucose metabolism and tumor progression. In clinical samples, circRPN2 expression negatively correlated with ENO1 and positively correlated with FOXO1, and expression of circRPN2, either alone or in combination with ENO1 and FOXO1, was a novel indicator of HCC prognosis. These data support a model wherein circRPN2 inhibits HCC aerobic glycolysis and metastasis via acceleration of ENO1 degradation and regulation of the miR-183-5p/FOXO1 axis, suggesting that circRPN2 represents a possible therapeutic target in HCC.

A putative PCV3-associated disease in piglets from Southern Brazil

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]

2022 Jan 06

Molossi, FA;de Almeida, BA;de Cecco, BS;da Silva, MS;Mósena, ACS;Brandalise, L;Simão, GMR;Canal, CW;Vanucci, F;Pavarini, SP;Driemeier, D;
PMID: 34988935 | DOI: 10.1007/s42770-021-00644-7

Porcine circovirus type 3 (PCV3) is widely distributed worldwide, and its association with clinical disease in pigs has been studied in recent years. This study describes a novel PCV3-associated clinical disease in piglets from Brazil. Since September 2020, we received 48 piglets with large caudally rotated ears, weakness, and dyspnea. Most piglets were from gilts and died 1-5 days after birth. Two piglets that presented similar clinical signs and survived until 35-60 days had a marked decrease in growth rate. At post-mortem examination, the lungs did not collapse due to marked interlobular edema. Microscopically, the main feature was multisystemic vasculitis characterized by lymphocytes and plasma cells infiltrating and disrupting the wall of vessels, lymphohistiocytic interstitial pneumonia, myocarditis, and encephalitis. Viral replication was confirmed in these lesions through in situ hybridization (ISH-RNA). Seventeen cases were positive for PCV3 in PCR analysis, and all samples tested negative for porcine circovirus (PCV1, and PCV2); porcine parvovirus (PPV1, 2, 5, and 6); atypical porcine pestivirus (APPV); porcine reproductive and respiratory syndrome (PRRSV); and ovine herpesvirus-2 (OvHV-2). Phylogenetic analysis of the ORF2 sequence from five different pig farms showed that the PCV3a clade is circulating among Brazil's swineherds and causing neonatal piglet losses. This is the first report of PCV3a-associated disease in neonatal pigs from farms in Brazil.

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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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