Virchows Archiv : an international journal of pathology
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.
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.
Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood-brain barrier and knock down genes in the rodent CNS
Nagata, T;Dwyer, CA;Yoshida-Tanaka, K;Ihara, K;Ohyagi, M;Kaburagi, H;Miyata, H;Ebihara, S;Yoshioka, K;Ishii, T;Miyata, K;Miyata, K;Powers, B;Igari, T;Yamamoto, S;Arimura, N;Hirabayashi, H;Uchihara, T;Hara, RI;Wada, T;Bennett, CF;Seth, PP;Rigo, F;Yokota, T;
PMID: 34385691 | DOI: 10.1038/s41587-021-00972-x
Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5' end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood-brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration.
SARS-CoV-2 infection and transmission in the North American deer mouse
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.
Didiot MC, Ferguson CM, Ly S, Coles AH, Smith AO, Bicknell AA, Hall LM, Sapp E, Echeverria D, Pai AA, DiFiglia M, Moore MJ, Hayward LJ, Aronin N, Khvorova A.
PMID: 30184490 | DOI: 10.1016/j.celrep.2018.07.106
Huntington's disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ∼50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA subcellular localization might play a role in important neuronal regulatory mechanisms.
Sci Adv. 2018 Oct 17;4(10):eaat3386.
Ämmälä C, Drury WJ 3rd, Knerr L, Ahlstedt I, Stillemark-Billton P, Wennberg-Huldt C, Andersson EM, Valeur E, Jansson-Löfmark R, Janzén D, Sundström L, Meuller J, Claesson J, Andersson P, Johansson C, Lee RG, Prakash TP, Seth PP, Monia BP, Andersson S.
PMID: 30345352 | DOI: 10.1126/sciadv.aat3386
Antisense oligonucleotide (ASO) silencing of the expression of disease-associated genes is an attractive novel therapeutic approach, but treatments are limited by the ability to deliver ASOs to cells and tissues. Following systemic administration, ASOs preferentially accumulate in liver and kidney. Among the cell types refractory to ASO uptake is the pancreatic insulin-secreting β-cell. Here, we show that conjugation of ASOs to a ligand of the glucagon-like peptide-1 receptor (GLP1R) can productively deliver ASO cargo to pancreatic β-cells both in vitro and in vivo. Ligand-conjugated ASOs silenced target genes in pancreatic islets at doses that did not affect target gene expression in liver or other tissues, indicating enhanced tissue and cell type specificity. This finding has potential to broaden the use of ASO technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of ASOs to additional cell types.
Miao, H;Wu, F;Li, Y;Qin, C;Zhao, Y;Xie, M;Dai, H;Yao, H;Cai, H;Wang, Q;Song, X;Li, L;
PMID: 36563164 | DOI: 10.1126/sciadv.abq7289
Understanding how long noncoding RNAs (lncRNAs) cooperate with splicing factors (SFs) in alternative splicing (AS) control is fundamental to human biology and disease. We show that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a well-documented AS-implicated lncRNA, regulates AS via two SFs, polypyrimidine tract-binding protein 1 (PTBP1) and PTB-associated SF (PSF). MALAT1 stabilizes the interaction between PTBP1 and PSF, thereby forming a functional module that affects a network of AS events. The MALAT1-stabilized PTBP1/PSF interaction occurs in multiple cellular contexts; however, the functional module, relative to MALAT1 only, has more dominant pathological significance in hepatocellular carcinoma. MALAT1 also stabilizes the PSF interaction with several heterogeneous nuclear ribonucleoparticle proteins other than PTBP1, hinting a broad role in AS control. We present a model in which MALAT1 cooperates with distinct SFs for AS regulation and pose that, relative to analyses exclusively performed for lncRNAs, a comprehensive consideration of lncRNAs and their binding partners may provide more information about their biological functions.
Gastrointestinal Pathology in Samples from Coronavirus Disease 2019 (COVID-19)-Positive Patients
Archives of pathology & laboratory medicine
Westerhoff, M;Jones, D;Hrycaj, SM;Chan, MP;Pantanowitz, L;Tu, H;Choi, K;Greenson, J;Lamps, L;
PMID: 33961007 | DOI: 10.5858/arpa.2021-0137-SA
-Although primarily considered a respiratory illness, coronavirus disease 2019 (COVID-19) can cause gastrointestinal manifestations. -To evaluate histopathology and in situ hybridization for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in gastrointestinal samples from patients with recent and remote COVID-19. -Patients with positive SARS-CoV-2 nasopharyngeal tests and a gastrointestinal tissue specimen were included. SARS-CoV-2 in situ hybridization (ISH) was performed on each sample. A subset had SARS-CoV-2 next generation sequencing (NGS) performed. -Twenty-five patients met inclusion criteria. Five had positive SARS-CoV-2 nasopharyngeal tests within 7 days of their gastrointestinal procedure. Two were ulcerative colitis patients on steroid therapy who lacked typical COVID-19 symptoms. Their colectomies showed severe ulcerative colitis; one demonstrated SARS-CoV-2 by NGS but a negative ISH. Another had an ischemic colon resected as a complication of the COVID-19 course; however, both ISH and NGS were negative. A fourth had a normal-appearing terminal ileum but positive ISH and NGS. The fifth patient had ileal ulcers with SARS-CoV-2 negativity by both modalities. The remaining 20 patients had positive nasopharyngeal tests an average of 53 days prior to procedure. None of their samples demonstrated SARS-CoV-2 ISH positivity, but one was positive on NGS despite a negative nasopharyngeal test. -Gastrointestinal findings from SARS-CoV-2-infected patients ranged from normal with virus detected by ISH and NGS, to bowel ischemia secondary to systemic viral effects, without evidence of virus in the tissue. No distinct histologic finding was identified in those with gastrointestinal tissue specimens demonstrating SARS-CoV-2 positivity in this cohort.
Yu Q, Hardin H, Chu YH, Rehrauer W, Lloyd RV.
PMID: 31119524 | DOI: 10.1007/s12022-019-9578-3
Parathyroid adenomas are slow growing benign neoplasms associated with hypercalcemia, while atypical parathyroid adenomas and parathyroid carcinomas are uncommon tumors and their histologic features may overlap with parathyroid adenomas. LncRNAs participate in transcription and in epigenetic or post-transcriptional regulation of gene expression, and probably contribute to carcinogenesis. We analyzed a group of normal, hyperplastic, and neoplastic parathyroid lesions to determine the best immunohistochemical markers to characterize these lesions and to determine the role of selected lncRNAs in tumor progression. A tissue microarray consisting of 111 cases of normal parathyroid (n = 14), primary hyperplasia (n = 15), secondary hyperplasia (n = 10), tertiary hyperplasia (n = 11), adenomas (n = 50), atypical adenomas (n = 7), and carcinomas (n = 4) was used. Immunohistochemical staining with antibodies against chromogranin A, synaptophysin, parathyroid hormone, and insulinoma-associated protein 1(INSM1) was used. Expression of lncRNAs including metastasis-associated lung adenocarcinoma transcript one (MALAT1), HOX transcript antisense intergenic RNA (HOTAIR), and long intergenic non-protein coding regulator of reprograming (Linc-ROR or ROR) was also analyzed by in situ hybridization and RT-PCR. All of the parathyroid tissues were positive for parathyroid hormone, while most cases were positive for chromogranin A (98%). Synaptophysin was expressed in only 12 cases (11%) and INMS1 was negative in all cases. ROR was significantly downregulated during progression from normal, hyperplastic, and adenomatous parathyroid to parathyroid carcinomas. These results show that parathyroid hormone and chromogranin A are useful markers for parathyroid neoplasms, while synaptophysin and INSM1 are not very sensitive broad-spectrum markers for these neoplasms. LincRNA ROR may function as a tumor suppressor during parathyroid tumor progression.
Signal transduction and targeted therapy
Song, Z;Bao, L;Deng, W;Liu, J;Ren, E;Lv, Q;Liu, M;Qi, F;Chen, T;Deng, R;Li, F;Liu, Y;Wei, Q;Gao, H;Yu, P;Han, Y;Zhao, W;Zheng, J;Liang, X;Yang, F;Qin, C;
PMID: 35091528 | DOI: 10.1038/s41392-022-00891-6
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted on mink farms between minks and humans in many countries. However, the systemic pathological features of SARS-CoV-2-infected minks are mostly unknown. Here, we demonstrated that minks were largely permissive to SARS-CoV-2, characterized by severe and diffuse alveolar damage, and lasted at least 14 days post inoculation (dpi). We first reported that infected minks displayed multiple organ-system lesions accompanied by an increased inflammatory response and widespread viral distribution in the cardiovascular, hepatobiliary, urinary, endocrine, digestive, and immune systems. The viral protein partially co-localized with activated Mac-2+ macrophages throughout the body. Moreover, we first found that the alterations in lipids and metabolites were correlated with the histological lesions in infected minks, especially at 6 dpi, and were similar to that of patients with severe and fatal COVID-19. Particularly, altered metabolic pathways, abnormal digestion, and absorption of vitamins, lipids, cholesterol, steroids, amino acids, and proteins, consistent with hepatic dysfunction, highlight metabolic and immune dysregulation. Enriched kynurenine in infected minks contributed to significant activation of the kynurenine pathway and was related to macrophage activation. Melatonin, which has significant anti-inflammatory and immunomodulating effects, was significantly downregulated at 6 dpi and displayed potential as a targeted medicine. Our data first illustrate systematic analyses of infected minks to recapitulate those observations in severe and fetal COVID-19 patients, delineating a useful animal model to mimic SARS-CoV-2-induced systematic and severe pathophysiological features and provide a reliable tool for the development of effective and targeted treatment strategies, vaccine research, and potential biomarkers.
Vanderheiden, A;Thomas, J;Soung, AL;Davis-Gardner, ME;Floyd, K;Jin, F;Cowan, DA;Pellegrini, K;Shi, PY;Grakoui, A;Klein, RS;Bosinger, SE;Kohlmeier, JE;Menachery, VD;Suthar, MS;
PMID: 34749524 | DOI: 10.1128/mBio.02749-21
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a historic pandemic of respiratory disease (coronavirus disease 2019 [COVID-19]), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2 signaling restricts the viral burden in the lung. We find that a recently developed mouse-adapted SARS-CoV-2 (MA-SARS-CoV-2) strain as well as the emerging B.1.351 variant trigger an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Single-cell RNA sequencing (scRNA-Seq) analysis of lung homogenates identified a hyperinflammatory monocyte profile. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a potential CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts the viral burden in the lung. We find that SARS-CoV-2 triggers an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using RNA sequencing and flow cytometry approaches, we demonstrate that SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Mechanistically, CCR2 signaling promoted the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified that the CCR2 pathway is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.
Röltgen, K;Nielsen, S;Silva, O;Younes, S;Maxim Zaslavsky, ;Costales, C;Yang, F;Wirz, O;Solis, D;Hoh, R;Wang, A;Arunachalam, P;Colburg, D;Zhao, S;Haraguchi, E;Lee, A;Shah, M;Manohar, M;Chang, I;Gao, F;Mallajosyula, V;Li, C;Liu, J;Shoura, M;Sindher, S;Parsons, E;Dashdorj, N;Dashdorj, N;Monroe, R;Serrano, G;Beach, T;Chinthrajah, R;Charville, G;Wilbur, J;Wohlstadter, J;Davis, M;Pulendran, B;Troxell, M;Sigal, G;Natkunam, Y;Pinsky, B;Nadeau, K;Boyd, S;
| DOI: 10.1016/j.cell.2022.01.018
During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including 3rd dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is less after infection compared to all vaccines evaluated, but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks post-vaccination in some cases. SARS-CoV-2 antibody specificity, breadth and maturation are affected by imprinting from exposure history, and distinct histological and antigenic contexts in infection compared to vaccination.
