Comparative pathology of the nasal epithelium in K18-hACE2 Tg mice, hACE2 Tg mice, and hamsters infected with SARS-CoV-2

Veterinary pathology

2022 Jan 29

Yu, P;Deng, W;Bao, L;Qu, Y;Xu, Y;Zhao, W;Han, Y;Qin, C;
PMID: 35094625 | DOI: 10.1177/03009858211071016

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes severe viral pneumonia and is associated with a high fatality rate. A substantial proportion of patients infected by SARS-CoV-2 suffer from mild hyposmia to complete loss of olfactory function, resulting in anosmia. However, the pathogenesis of the olfactory dysfunction and comparative pathology of upper respiratory infections with SARS-CoV-2 are unknown. We describe the histopathological, immunohistochemical, and in situ hybridization findings from rodent models of SARS-CoV-2 infection. The main histopathological findings in the olfactory epithelia of K8-hACE2 Tg mice, hACE2 Tg mice, and hamsters were varying degrees of inflammatory lesions, including disordered arrangement, necrosis, exfoliation, and macrophage infiltration of the olfactory epithelia, and inflammatory exudation. On the basis of these observations, the nasal epithelia of these rodent models appeared to develop moderate, mild, and severe rhinitis, respectively. Correspondingly, SARS-CoV-2 viral RNA and antigen were mainly identified in the olfactory epithelia and lamina propria. Moreover, viral RNA was abundant in the cerebrum of K18-hACE2 Tg mice, including the olfactory bulb. The K8-hACE2 Tg mouse, hACE2 Tg mouse, and hamster models could be used to investigate the pathology of SARS-CoV-2 infection in the upper respiratory tract and central nervous system. These models could help to provide a better understanding of the pathogenic process of this virus and to develop effective medications and prophylactic treatments.

Matrix remodeling-associated protein 8 is a marker of a subset of cancer-associated fibroblasts in pancreatic cancer

Pathology international

2022 Jan 12

Ichihara, R;Shiraki, Y;Mizutani, Y;Iida, T;Miyai, Y;Esaki, N;Kato, A;Mii, S;Ando, R;Hayashi, M;Takami, H;Fujii, T;Takahashi, M;Enomoto, A;
PMID: 35020975 | DOI: 10.1111/pin.13198

Cancer-associated fibroblasts (CAFs), a compartment of the tumor microenvironment, were previously thought to be a uniform cell population that promotes cancer progression. However, recent studies have shown that CAFs are heterogeneous and that there are at least two types of CAFs, that is, cancer-promoting and -restraining CAFs. We previously identified Meflin as a candidate marker of cancer-restraining CAFs (rCAFs) in pancreatic ductal adenocarcinoma (PDAC). The precise nature of rCAFs, however, has remained elusive owing to a lack of understanding of their comprehensive gene signatures. Here, we screened genes whose expression correlated with Meflin in single-cell transcriptomic analyses of human cancers. Among the identified genes, we identified matrix remodeling-associated protein 8 (MXRA8), which encodes a type I transmembrane protein with unknown molecular function. Analysis of MXRA8 expression in human PDAC samples showed that MXRA8 was differentially co-expressed with other CAF markers. Moreover, in patients with PDAC or syngeneic tumors developed in MXRA8-knockout mice, MXRA8 expression did not affect the roles of CAFs in cancer progression, and the biological importance of MXRA8+ CAFs is still unclear. Overall, we identified MXRA8 as a new CAF marker; further studies are needed to determine the relevance of this marker.

Host parameters and mode of infection influence outcome in SARS-CoV-2 infected hamsters

iScience

2021 Nov 01

Griffin, B;Warner, B;Chan, M;Valcourt, E;Tailor, N;Banadyga, L;Leung, A;He, S;Boese, A;Audet, J;Cao, W;Moffat, E;Garnett, L;Tierney, K;Tran, K;Albietz, A;Manguiat, K;Soule, G;Bello, A;Vendramelli, R;Lin, J;Deschambault, Y;Zhu, W;Wood, H;Mubareka, S;Safronetz, D;Strong, J;Embury-Hyatt, C;Kobasa, D;
| DOI: 10.1016/j.isci.2021.103530

The golden hamster model of SARS-CoV-2 infection recapitulates key characteristics of COVID-19. In this work we examined the influence of the route of exposure, sex, and age on SARS-CoV-2 pathogenesis in hamsters. We report that delivery of SARS-CoV-2 by a low versus high volume intranasal or intragastric route results in comparable viral titers in the lung and viral shedding. However, low-volume intranasal exposure results in milder weight loss while intragastric exposure leads to a diminished capacity to regain body weight. Male hamsters, and particularly older male hamsters, display an impaired capacity to recover from illness and delayed viral clearance. These factors were found to influence the nature of the host inflammatory cytokine response, but had a minimal effect on the quality and durability of the humoral immune response and susceptibility to re-infection. These data further elucidate key factors that impact pre-clinical challenge studies carried out in the hamster model of COVID-19.

A single intranasal or intramuscular immunization with chimpanzee adenovirus vectored SARS-CoV-2 vaccine protects against pneumonia in hamsters

Cell Reports

2021 Jun 01

Bricker, T;Darling, T;Hassan, A;Harastani, H;Soung, A;Jiang, X;Dai, Y;Zhao, H;Adams, L;Holtzman, M;Bailey, A;Case, J;Fremont, D;Klein, R;Diamond, M;Boon, A;
| DOI: 10.1016/j.celrep.2021.109400

The development of an effective vaccine against SARS-CoV-2, the etiologic agent of COVID-19, is a global priority. Here, we compared the protective capacity of intranasal and intramuscular delivery of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in Golden Syrian hamsters. While immunization with ChAd-SARS-CoV-2-S induced robust spike protein specific antibodies capable of neutralizing the virus, antibody levels in serum were higher in hamsters vaccinated by an intranasal compared to intramuscular route. Accordingly, against challenge with SARS-CoV-2, ChAd-SARS-CoV-2-S immunized hamsters were protected against less weight loss and had reduced viral infection in nasal swabs and lungs, and reduced pathology and inflammatory gene expression in the lungs, compared to ChAd-Control immunized hamsters. Intranasal immunization with ChAd-SARS-CoV-2-S provided superior protection against SARS-CoV-2 infection and inflammation in the upper respiratory tract. These findings support intranasal administration of the ChAd-SARS-CoV-2-S candidate vaccine to prevent SARS-CoV-2 infection, disease, and possibly transmission.

Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-on-chip model

EMBO reports

2021 Apr 28

Thacker, VV;Sharma, K;Dhar, N;Mancini, GF;Sordet-Dessimoz, J;McKinney, JD;
PMID: 33908688 | DOI: 10.15252/embr.202152744

Severe cases of SARS-CoV-2 infection are characterized by hypercoagulopathies and systemic endotheliitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of an immune cell-mediated cytokine storm remain unknown. Using a vascularized lung-on-chip model, we find that infection of alveolar epithelial cells leads to limited apical release of virions, consistent with reports of monoculture infection. However, viral RNA and proteins are rapidly detected in underlying endothelial cells, which are themselves refractory to apical infection in monocultures. Although endothelial infection is unproductive, it leads to the formation of cell clusters with low CD31 expression, a progressive loss of barrier integrity and a pro-coagulatory microenvironment. Viral RNA persists in individual cells generating an inflammatory response, which is transient in epithelial cells but persistent in endothelial cells and typified by IL-6 secretion even in the absence of immune cells. Inhibition of IL-6 signalling with tocilizumab reduces but does not prevent loss of barrier integrity. SARS-CoV-2-mediated endothelial cell damage thus occurs independently of cytokine storm.

SARS-CoV-2 Infection Remodels the Phenotype and Promotes Angiogenesis of Primary Human Lung Endothelial Cells

Microorganisms

2021 Jul 03

Caccuri, F;Bugatti, A;Zani, A;De Palma, A;Di Silvestre, D;Manocha, E;Filippini, F;Messali, S;Chiodelli, P;Campisi, G;Fiorentini, S;Facchetti, F;Mauri, P;Caruso, A;
| DOI: 10.3390/microorganisms9071438

SARS-CoV-2-associated acute respiratory distress syndrome (ARDS) and acute lung injury are life-threatening manifestations of severe viral infection. The pathogenic mechanisms that lead to respiratory complications, such as endothelialitis, intussusceptive angiogenesis, and vascular leakage remain unclear. In this study, by using an immunofluorescence assay and in situ RNA-hybridization, we demonstrate the capability of SARS-CoV-2 to infect human primary lung microvascular endothelial cells (HL-mECs) in the absence of cytopathic effects and release of infectious particles. Preliminary data point to the role of integrins in SARS-CoV-2 entry into HL-mECs in the absence of detectable ACE2 expression. Following infection, HL-mECs were found to release a plethora of pro-inflammatory and pro-angiogenic molecules, as assessed by microarray analyses. This conditioned microenvironment stimulated HL-mECs to acquire an angiogenic phenotype. Proteome analysis confirmed a remodeling of SARS-CoV-2-infected HL-mECs to inflammatory and angiogenic responses and highlighted the expression of antiviral molecules as annexin A6 and MX1. These results support the hypothesis of a direct role of SARS-CoV-2-infected HL-mECs in sustaining vascular dysfunction during the early phases of infection. The construction of virus-host interactomes will be instrumental to identify potential therapeutic targets for COVID-19 aimed to inhibit HL-mEC-sustained inflammation and angiogenesis upon SARS-CoV-2 infection.

UCP1 governs liver extracellular succinate and inflammatory pathogenesis

Nature metabolism

2021 May 01

Mills, EL;Harmon, C;Jedrychowski, MP;Xiao, H;Garrity, R;Tran, NV;Bradshaw, GA;Fu, A;Szpyt, J;Reddy, A;Prendeville, H;Danial, NN;Gygi, SP;Lynch, L;Chouchani, ET;
PMID: 34002097 | DOI: 10.1038/s42255-021-00389-5

Non-alcoholic fatty liver disease (NAFLD), the most prevalent liver pathology worldwide, is intimately linked with obesity and type 2 diabetes. Liver inflammation is a hallmark of NAFLD and is thought to contribute to tissue fibrosis and disease pathogenesis. Uncoupling protein 1 (UCP1) is exclusively expressed in brown and beige adipocytes, and has been extensively studied for its capacity to elevate thermogenesis and reverse obesity. Here we identify an endocrine pathway regulated by UCP1 that antagonizes liver inflammation and pathology, independent of effects on obesity. We show that, without UCP1, brown and beige fat exhibit a diminished capacity to clear succinate from the circulation. Moreover, UCP1KO mice exhibit elevated extracellular succinate in liver tissue that drives inflammation through ligation of its cognate receptor succinate receptor 1 (SUCNR1) in liver-resident stellate cell and macrophage populations. Conversely, increasing brown and beige adipocyte content in mice antagonizes SUCNR1-dependent inflammatory signalling in the liver. We show that this UCP1-succinate-SUCNR1 axis is necessary to regulate liver immune cell infiltration and pathology, and systemic glucose intolerance in an obesogenic environment. As such, the therapeutic use of brown and beige adipocytes and UCP1 extends beyond thermogenesis and may be leveraged to antagonize NAFLD and SUCNR1-dependent liver inflammation.

A point mutation R122C in RUNX3 promotes the expansion of isthmus stem cells and inhibits their differentiation in the stomach

Cellular and molecular gastroenterology and hepatology

2022 Jan 21

Douchi, D;Yamamura, A;Matsuo, J;Lee, JW;Nuttonmanit, N;Melissa Lim, YH;Suda, K;Shimura, M;Chen, S;Pang, S;Kohu, K;Kaneko, M;Kiyonari, H;Kaneda, A;Yoshida, H;Taniuchi, I;Osato, M;Yang, H;Unno, M;Bok-Yan So, J;Yeoh, KG;Huey Chuang, LS;Bae, SC;Ito, Y;
PMID: 35074568 | DOI: 10.1016/j.jcmgh.2022.01.010

RUNX transcription factors play pivotal roles in embryonic development and neoplasia. We previously identified the single missense mutation R122C in RUNX3 from human gastric cancer. However, how RUNX3R122C mutation disrupts stem cell homeostasis and promotes gastric carcinogenesis remained unclear.To understand the oncogenic nature of this mutation in vivo, we generated the RUNX3R122C knock-in mice. Stomach tissues were harvested, followed by histological and immunofluorescence staining, organoid culture, flow cytometry to isolate gastric corpus isthmus and non-isthmus epithelial cells, and RNA extraction for transcriptomic analysis.The corpus tissue of RUNX3R122C/R122C homozygous mice exhibited a precancerous phenotype such as spasmolytic polypeptide-expressing metaplasia (SPEM). We observed mucous neck cell hyperplasia, massive reduction of pit, parietal, and chief cell populations, as well as a dramatic increase in the number of rapidly proliferating isthmus stem/progenitor cells in the corpus of RUNX3R122C/R122C mice. Transcriptomic analyses of the isolated epithelial cells showed that the cell cycle-related MYC target gene signature was enriched in the corpus epithelial cells of RUNX3R122C/R122C mice compared with the wild-type corpus. Mechanistically, RUNX3R122C mutant protein disrupted the regulation of the restriction point where cells decide to enter either proliferative or quiescent state, thereby driving stem cell expansion and limiting the ability of cells to terminally differentiate.RUNX3R122C missense mutation is associated with the continuous cycling of isthmus stem/progenitor cells, maturation arrest and development of a precancerous state. This work highlights the importance of RUNX3 in prevention of metaplasia and gastric cancer.

Variable levels of spike and ORF1ab RNA in post-mortem lung samples of SARS-CoV-2-positive subjects: comparison between ISH and RT-PCR

Virchows Archiv : an international journal of pathology

2022 Feb 01

Zito Marino, F;De Cristofaro, T;Varriale, M;Zannini, G;Ronchi, A;La Mantia, E;Campobasso, CP;De Micco, F;Mascolo, P;Municinò, M;Municinò, E;Vestini, F;Pinto, O;Moccia, M;De Stefano, N;Nappi, O;Sementa, C;Zotti, G;Pianese, L;Giordano, C;Franco, R;
PMID: 35103846 | DOI: 10.1007/s00428-021-03262-8

Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.

Development of a Hamster Natural Transmission Model of SARS-CoV-2 Infection

Viruses

2021 Nov 09

Dowall, S;Salguero, FJ;Wiblin, N;Fotheringham, S;Hatch, G;Parks, S;Gowan, K;Harris, D;Carnell, O;Fell, R;Watson, R;Graham, V;Gooch, K;Hall, Y;Mizen, S;Hewson, R;
PMID: 34835057 | DOI: 10.3390/v13112251

The global pandemic of coronavirus disease (COVID-19) caused by infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to an international thrust to study pathogenesis and evaluate interventions. Experimental infection of hamsters and the resulting respiratory disease is one of the preferred animal models since clinical signs of disease and virus shedding are similar to more severe cases of human COVID-19. The main route of challenge has been direct inoculation of the virus via the intranasal route. To resemble the natural infection, we designed a bespoke natural transmission cage system to assess whether recipient animals housed in physically separate adjacent cages could become infected from a challenged donor animal in a central cage, with equal airflow across the two side cages. To optimise viral shedding in the donor animals, a low and moderate challenge dose were compared after direct intranasal challenge, but similar viral shedding responses were observed and no discernible difference in kinetics. The results from our natural transmission set-up demonstrate that most recipient hamsters are infected within the system developed, with variation in the kinetics and levels of disease between individual animals. Common clinical outputs used for the assessment in directly-challenged hamsters, such as weight loss, are less obvious in hamsters who become infected from naturally acquiring the infection. The results demonstrate the utility of a natural transmission model for further work on assessing the differences between virus strains and evaluating interventions using a challenge system which more closely resembles human infection.

Ovarian Cancer Cells in Ascites Form Aggregates That Display a Hybrid Epithelial-Mesenchymal Phenotype and Allows Survival and Proliferation of Metastasizing Cells

International journal of molecular sciences

2022 Jan 13

Capellero, S;Erriquez, J;Battistini, C;Porporato, R;Scotto, G;Borella, F;Di Renzo, MF;Valabrega, G;Olivero, M;
PMID: 35055018 | DOI: 10.3390/ijms23020833

Peritoneal metastases are the leading cause of morbidity and mortality in ovarian cancer. Cancer cells float in peritoneal fluid, named ascites, together with a definitely higher number of non neo-neoplastic cells, as single cells or multicellular aggregates. The aim of this work is to uncover the features that make these aggregates the metastasizing units. Immunofluorescence revealed that aggregates are made almost exclusively of ovarian cancer cells expressing the specific nuclear PAX8 protein. The same cells expressed epithelial and mesenchymal markers, such as EPCAM and αSMA, respectively. Expression of fibronectin further supported a hybrid epithelia-mesenchymal phenotype, that is maintained when aggregates are cultivated and proliferate. Hematopoietic cells as well as macrophages are negligible in the aggregates, while abundant in the ascitic fluid confirming their prominent role in establishing an eco-system necessary for the survival of ovarian cancer cells. Using ovarian cancer cell lines, we show that cells forming 3D structures neo-expressed thoroughly fibronectin and αSMA. Functional assays showed that αSMA and fibronectin are necessary for the compaction and survival of 3D structures. Altogether these data show that metastasizing units display a hybrid phenotype that allows maintenance of the 3D structures and the plasticity necessary for implant and seeding into peritoneal lining.

SARS-CoV-2 infection and transmission in the North American deer mouse

Nature communications

2021 Jun 14

Griffin, BD;Chan, M;Tailor, N;Mendoza, EJ;Leung, A;Warner, BM;Duggan, AT;Moffat, E;He, S;Garnett, L;Tran, KN;Banadyga, L;Albietz, A;Tierney, K;Audet, J;Bello, A;Vendramelli, R;Boese, AS;Fernando, L;Lindsay, LR;Jardine, CM;Wood, H;Poliquin, G;Strong, JE;Drebot, M;Safronetz, D;Embury-Hyatt, C;Kobasa, D;
PMID: 34127676 | DOI: 10.1038/s41467-021-23848-9

Widespread circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive nondomesticated animals. Here we report that North American deer mice (Peromyscus maniculatus) are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naïve deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment of Peromyscus rodents as a North American reservoir for SARS-CoV-2 remains unknown.

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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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