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The severity of SARS-CoV-2 infection in K18-hACE2 mice is attenuated by a novel steroid-derivative in a gender-specific manner

British journal of pharmacology

2023 May 31

Gupte, SA;Bakshi, CS;Blackham, E;Duhamel, GE;Jordan, A;Salgame, P;D'silva, M;Khan, MY;Nadler, J;Gupte, R;
PMID: 37259182 | DOI: 10.1111/bph.16155

COVID-19 infections caused by SARS-CoV-2 disseminate through human-to-human transmission can evoke severe inflammation. Treatments to reduce the SARS-CoV-2-associated inflammation are needed and are the focus of much research. In this study, we investigated the effect of N-Ethyl-N'-[(3β,5α)-17-oxoandrostan-3-yl] urea (NEOU), a novel 17α-ketosteroid derivative, on the severity of COVID-19 infections.Studies were conducted in SARS-CoV-2-infected K18-hACE2 mice.SARS-CoV-2-infected K18-hACE2 mice developed severe inflammatory crises and immune responses along with up-regulation of genes in associated signaling pathways in male more than female mice. Notably, SARS-CoV-2 infection down-regulated genes encoding drug metabolizing cytochrome P450 enzymes in male but not female mice. Treatment with NEOU (1 mg/kg/day) 24 or 72 h post-viral infection alleviated lung injury by decreasing expression of genes encoding inflammatory cytokines and chemokines while increasing expression of genes encoding immunoglobins. In situ hybridization using RNA scope probes and immunohistochemical assays revealed that NEOU increased resident CD169+ immunoregulatory macrophages and IBA-1 immunoreactive macrophage-dendritic cells within alveolar spaces in the lungs of infected mice. Consequentially, NEOU reduced morbidity more prominently in male than female mice. However, NEOU increased median survival time and accelerated recovery from infection by 6 days in both males and females.These findings demonstrate that SARS-CoV-2 exhibits gender bias by differentially regulating genes encoding inflammatory cytokines, immunogenic factors, and drug-metabolizing enzymes, in male versus female mice. Most importantly, we identified a novel 17α-ketosteroid that reduces the severity of COVID-19 infection and could be beneficial for reducing impact of COVID-19.This article is protected by
Characterization and antiviral susceptibility of SARS-CoV-2 Omicron/BA.2

Research square

2022 Feb 24

Kawaoka, Y;Uraki, R;Kiso, M;Iida, S;Imai, M;Takashita, E;Kuroda, M;Halfmann, P;Loeber, S;Maemura, T;Yamayoshi, S;Fujisaki, S;Wang, Z;Ito, M;Ujie, M;Iwatsuki-Horimoto, K;Furusawa, Y;Wright, R;Chong, Z;Ozono, S;Yasuhara, A;Ueki, H;Sakai, Y;Li, R;Liu, Y;Larson, D;Koga, M;Tsutsumi, T;Adachi, E;Saito, M;Yamamoto, S;Matsubara, S;Hagihara, M;Mitamura, K;Sato, T;Hojo, M;Hattori, SI;Maeda, K;Okuda, M;Murakami, J;Duong, C;Godbole, S;Douek, D;Watanabe, S;Ohmagari, N;Yotsuyanagi, H;Diamond, M;Hasegawa, H;Mitsuya, H;Suzuki, T;
PMID: 35233565 | DOI: 10.21203/rs.3.rs-1375091/v1

The recent emergence of SARS-CoV-2 Omicron variants possessing large numbers of mutations has raised concerns of decreased effectiveness of current vaccines, therapeutic monoclonal antibodies, and antiviral drugs for COVID-19 against these variants1,2. While the original Omicron lineage, BA.1, has become dominant in many countries, BA.2 has been detected in at least 67 countries and has become dominant in the Philippines, India, and Denmark. Here, we evaluated the replicative ability and pathogenicity of an authentic infectious BA.2 isolate in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. In contrast to recent data with chimeric, recombinant SARS-CoV-2 strains expressing the spike proteins of BA.1 and BA.2 on an ancestral WK-521 backbone3, we observed similar infectivity and pathogenicity in mice and hamsters between BA.2 and BA.1, and less pathogenicity compared to early SARS-CoV-2 strains. We also observed a marked and significant reduction in the neutralizing activity of plasma from COVID-19 convalescent individuals and vaccine recipients against BA.2 compared to ancestral and Delta variant strains. In addition, we found that some therapeutic monoclonal antibodies (REGN10987/REGN10933, COV2-2196/COV2-2130, and S309) and antiviral drugs (molnupiravir, nirmatrelvir, and S-217622) can restrict viral infection in the respiratory organs of hamsters infected with BA.2. These findings suggest that the replication and pathogenicity of BA.2 is comparable to that of BA.1 in rodents and that several therapeutic monoclonal antibodies and antiviral compounds are effective against Omicron/BA.2 variants.
MVA vector expression of SARS-CoV-2 spike protein and protection of adult Syrian hamsters against SARS-CoV-2 challenge

NPJ vaccines

2021 Dec 03

Meseda, CA;Stauft, CB;Selvaraj, P;Lien, CZ;Pedro, C;Nuñez, IA;Woerner, AM;Wang, TT;Weir, JP;
PMID: 34862398 | DOI: 10.1038/s41541-021-00410-8

Numerous vaccine candidates against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are under development. The majority of vaccine candidates to date are designed to induce immune responses against the viral spike (S) protein, although different forms of S antigen have been incorporated. To evaluate the yield and immunogenicity of different forms of S, we constructed modified vaccinia virus Ankara (MVA) vectors expressing full-length S (MVA-S), the RBD, and soluble S ectodomain and tested their immunogenicity in dose-ranging studies in mice. All three MVA vectors induced spike-specific immunoglobulin G after one subcutaneous immunization and serum titers were boosted following a second immunization. The MVA-S and MVA-ssM elicited the strongest neutralizing antibody responses. In assessing protective efficacy, MVA-S-immunized adult Syrian hamsters were challenged with SARS-CoV-2 (USA/WA1/2020). MVA-S-vaccinated hamsters exhibited less severe manifestations of atypical pneumocyte hyperplasia, hemorrhage, vasculitis, and especially consolidation, compared to control animals. They also displayed significant reductions in gross pathology scores and weight loss, and a moderate reduction in virus shedding was observed post challenge in nasal washes. There was evidence of reduced viral replication by in situ hybridization, although the reduction in viral RNA levels in lungs and nasal turbinates did not reach significance. Taken together, the data indicate that immunization with two doses of an MVA vector expressing SARS-CoV-2 S provides protection against a stringent SARS-CoV-2 challenge of adult Syrian hamsters, reaffirm the utility of this animal model for evaluating candidate SARS-CoV-2 vaccines, and demonstrate the value of an MVA platform in facilitating vaccine development against SARS-CoV-2.
GLP-1 modulates the supramammillary nucleus-lateral hypothalamic neurocircuit to control ingestive and motivated behavior in a sex divergent manner.

Molecular Metabolism

2018 Nov 27

López-Ferreras L, Eerola K, Mishra D, Shevchouk OT, Richard JE, Nilsson FH, Hayes MR, Skibicka KP.
PMID: - | DOI: 10.1016/j.molmet.2018.11.005

Objective

The supramammillary nucleus (SuM) is nestled between the lateral hypothalamus (LH) and the ventral tegmental area (VTA). This neuroanatomical position is consistent with a potential role of this nucleus to regulate ingestive and motivated behavior. Here neuroanatomical, molecular, and behavior approaches are utilized to determine whether SuM contributes to ingestive and food-motivated behavior control.

Methods

Through the application of anterograde and retrograde neural tract tracing with novel designer viral vectors, the current findings show that SuM neurons densely innervate the LH in a sex dimorphic fashion. Glucagon-like peptide-1 (GLP-1) is a clinically targeted neuro-intestinal hormone with a well-established role in regulating energy balance and reward behaviors. Here we determine that GLP-1 receptors (GLP-1R) are expressed throughout the SuM of both sexes, and also directly on SuM LH-projecting neurons and investigate the role of SuM GLP-1R in the regulation of ingestive and motivated behavior in male and female rats.

Results

SuM microinjections of the GLP-1 analogue, exendin-4, reduced ad libitum intake of chow, fat, or sugar solution in both male and female rats, while food-motivated behaviors, measured using the sucrose motivated operant conditioning test, was only reduced in male rats. These data contrasted with the results obtained from a neighboring structure well known for its role in motivation and reward, the VTA, where females displayed a more potent response to GLP-1R activation by exendin-4. In order to determine the physiological role of SuM GLP-1R signaling regulation of energy balance, we utilized an adeno-associated viral vector to site-specifically deliver shRNA for the GLP-1R to the SuM. Surprisingly, and in contrast to previous results for the two SuM neighboring sites, LH and VTA, SuM GLP-1R knockdown increased food seeking and adiposity in obese male rats without altering food intake, body weight or food motivation in lean or obese, female or male rats.

Conclusion

Taken together, these results indicate that SuM potently contributes to ingestive and motivated behavior control; an effect contingent on sex, diet/homeostatic energy balance state and behavior of interest. These data also extend the map of brain sites directly responsive to GLP-1 agonists, and highlight key differences in the role that GLP-1R play in interconnected and neighboring nuclei.

Detection of SARS-CoV-2 RNA by In Situ Hybridization in Lung-Cancer Cells Metastatic to Brain and in Adjacent Brain Parenchyma

Pathogens (Basel, Switzerland)

2023 May 29

Valyi-Nagy, T;Fredericks, B;Wilson, J;Shukla, SD;Setty, S;Slavin, KV;Valyi-Nagy, K;
PMID: 37375462 | DOI: 10.3390/pathogens12060772

The mechanisms by which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may spread to the human brain are poorly understood, and the infection of cancer cells in the brain by SARS-CoV-2 in Coronavirus disease 2019 (COVID-19) patients has been the subject of only one previous case report. Here, we report the detection of SARS-CoV-2 RNA by in situ hybridization in lung-cancer cells metastatic to the brain and adjacent brain parenchyma in a 63-year-old male patient with COVID-19. These findings suggest that metastatic tumors may transport the virus from other parts of the body to the brain or may break down the blood-brain barrier to allow for the virus to spread to the brain. These findings confirm and extend previous observations that cancer cells in the brain can become infected by SARS-CoV-2 in patients with COVID-19 and raise the possibility that SARS-CoV-2 can have a direct effect on cancer growth and outcome.
Nrg1 is an injury-induced cardiomyocyte mitogen for the endogenous heart regeneration program in zebrafish.

Elife. 2015 Apr 1;4.

Gemberling M, Karra R, Dickson AL, Poss KD.
PMID: 25830562 | DOI: 10.7554/eLife.05871.

Heart regeneration is limited in adult mammals but occurs naturally in adult zebrafish through the activation of cardiomyocyte division. Several components of the cardiac injury microenvironment have been identified, yet no factor on its own is known to stimulate overt myocardial hyperplasia in a mature, uninjured animal. In this study, we find evidence that Neuregulin1 (Nrg1), previously shown to have mitogenic effects on mammalian cardiomyocytes, is sharply induced in perivascular cells after injury to the adult zebrafish heart. Inhibition of Erbb2, an Nrg1 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial Nrg1 overexpression enhances this proliferation. In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium. Our findings identify Nrg1 as a potent, induced mitogen for the endogenous adult heart regeneration program.
SARS-CoV-2 detection by digital polymerase chain reaction and immunohistochemistry in skin biopsies from 52 patients with different COVID-19-associated cutaneous phenotypes

Dermatology (Basel, Switzerland)

2023 Apr 19

Marzano, AV;Moltrasio, C;Genovese, G;De Andrea, M;Caneparo, V;Vezzoli, P;Morotti, D;Sena, P;Venturini, M;Battocchio, S;Caputo, V;Rizzo, N;Maronese, CA;Venegoni, L;Boggio, FL;Rongioletti, F;Calzavara-Pinton, P;Berti, E;
PMID: 37075721 | DOI: 10.1159/000530746

COronaVIrus Disease 19 (COVID-19) is associated with a wide spectrum of skin manifestations, but SARS-CoV-2 RNA in lesional skin has been demonstrated only in few cases.To demonstrate SARS-CoV-2 presence in skin samples from patients with different COVID-19-related cutaneous phenotypes.Demographic and clinical data from 52 patients with COVID-19-associated cutaneous manifestations were collected. Immunohistochemistry and digital PCR (dPCR) were performed in all skin samples. RNA in situ hybridization (ISH) was used to confirm the presence of SARS-CoV-2 RNA.Twenty out of 52 (38%) patients presented SARS-CoV-2 positivity in the skin. Among these, 10/52 (19%) patients tested positive for spike protein on immunohistochemistry, five of whom had also positive testing on dPCR. Of the latter, one tested positive both for ISH and ACE-2 on immunohistochemistry while another one tested positive for nucleocapsid protein. Twelve patients showed positivity only for nucleocapsid protein on immunohistochemistry.SARS-CoV-2 was detected only in 38% of patients, without any association with a specific cutaneous phenotype, suggesting that the pathophysiology of cutaneous lesions mostly depends on the activation of the immune system. The combination of spike and nucleocapsid immunohistochemistry has higher diagnostic yield than dPCR. Skin persistence of SARS-CoV-2 may depend on timing of skin lesions, viral load and immune response.S. Karger AG, Basel.
Simultaneous detection and quantification of spike mRNA and protein in SARS-CoV-2 infected airway epithelium

MethodsX

2023 Feb 03

Jerome, K;Sattar, S;Mehedi, M;
PMID: 36779029 | DOI: 10.1016/j.mex.2023.102050

Visualizing and quantifying mRNA and its corresponding protein provides a unique perspective of gene expression at a single-molecule level. Here, we describe a method for differentiating primary cells for making airway epithelium and detecting SARS-CoV-2 Spike (S) mRNA and S protein in the paraformaldehyde-fixed paraffin-embedded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected airway epithelium. For simultaneous detection of mRNA and protein in the same cell, we combined two protocols: 1. RNA fluorescence-based in situ hybridization (RNA-FISH) based mRNA detection and 2. fluorescence-based immunohistochemistry (IHC) based protein detection. The detection of mRNA and proteins in the same cell also allows for quantifying them using the open-source software QuPath, which provides an accurate and more straightforward fluorescent-based quantification of mRNA and protein in the microscopic images of the infected cells. Additionally, we can achieve the subcellular distribution of both S mRNA and S protein. This method identifies SARS-CoV-2 S gene products' (mRNA and protein) degree of expression and their subcellular localization in the infected airway epithelium. Advantages of this method include: •Simultaneous detection and quantification of mRNA and protein in the same cell.•Universal use due to the ability to use mRNA-specific primer-probe and protein-specific antibodies.•An open-source software QuPath provides a straightforward fluorescent-based quantification.
Morphological changes without histological myocarditis in hearts of COVID-19 deceased patients

Scandinavian cardiovascular journal : SCJ

2022 Dec 01

Razaghi, A;Szakos, A;Al-Shakarji, R;Björnstedt, M;Szekely, L;
PMID: 35678649 | DOI: 10.1080/14017431.2022.2085320

Objective. Patients with underlying heart diseases have a higher risk of dying from Covid-19. It has also been suggested that Covid-19 affects the heart through myocarditis. Despite the rapidly growing research on the management of Covid-19 associated complications, most of the ongoing research is focused on the respiratory complications of Covid-19, and little is known about the prevalence of myocarditis. Design. This study aimed to characterize myocardial involvement by using a panel of antibodies to detect hypoxic and inflammatory changes and the presence of SARS-CoV-2 proteins in heart tissues obtained during the autopsy procedure of Covid-19 deceased patients. Thirty-seven fatal COVID-19 cases and 21 controls were included in this study. Results. Overall, the Covid-19 hearts had several histopathological changes like the waviness of myocytes, fibrosis, contract band necrosis, infiltration of polymorphonuclear neutrophils, vacuolization, and necrosis of myocytes. In addition, endothelial damage and activation were detected in heart tissue. However, viral replication was not detected using RNA in situ hybridization. Also, lymphocyte infiltration, as a hallmark of myocarditis, was not seen in this study. Conclusion. No histological sign of myocarditis was detected in any of our cases; our findings are thus most congruent with the hypothesis of the presence of a circulating endothelium activating factor such as VEGF, originating outside of the heart, probably from the hypoxic part of the Covid-19 lungs.
Adrenal tropism of SARS-CoV-2 and adrenal findings in a post-mortem case series of patients with severe fatal COVID-19

Nature communications

2022 Mar 24

Paul, T;Ledderose, S;Bartsch, H;Sun, N;Soliman, S;Märkl, B;Ruf, V;Herms, J;Stern, M;Keppler, OT;Delbridge, C;Müller, S;Piontek, G;Kimoto, YS;Schreiber, F;Williams, TA;Neumann, J;Knösel, T;Schulz, H;Spallek, R;Graw, M;Kirchner, T;Walch, A;Rudelius, M;
PMID: 35332140 | DOI: 10.1038/s41467-022-29145-3

Progressive respiratory failure and hyperinflammatory response is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. Despite mounting evidence of disruption of the hypothalamus-pituitary-adrenal axis in COVID-19, relatively little is known about the tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to adrenal glands and associated changes. Here we demonstrate adrenal viral tropism and replication in COVID-19 patients. Adrenal glands showed inflammation accompanied by inflammatory cell death. Histopathologic analysis revealed widespread microthrombosis and severe adrenal injury. In addition, activation of the glycerophospholipid metabolism and reduction of cortisone intensities were characteristic for COVID-19 specimens. In conclusion, our autopsy series suggests that SARS-CoV-2 facilitates the induction of adrenalitis. Given the central role of adrenal glands in immunoregulation and taking into account the significant adrenal injury observed, monitoring of developing adrenal insufficiency might be essential in acute SARS-CoV-2 infection and during recovery.
NPY mediates the rapid feeding and glucose metabolism regulatory functions of AgRP neurons

Nat Commun

2020 Jan 23

Engstr�m Ruud L Pereira MMA, de Solis AJ, Fenselau H Br�ning JC
PMID: 31974377 | DOI: 10.1038/s41467-020-14291-3

Activation of Agouti-Related Peptide (AgRP)-expressing neurons promotes feeding and insulin resistance. Here, we examine the contribution of neuropeptide Y (NPY)-dependent signaling to the diverse physiological consequences of activating AgRP neurons. NPY-deficient mice fail to rapidly increase food intake during the first hour of either chemo- or optogenetic activation of AgRP neurons, while the delayed increase in feeding is comparable between control and NPY-deficient mice. Acutely stimulating AgRP neurons fails to induce systemic insulin resistance in NPY-deficient mice, while increased locomotor activity upon AgRP neuron stimulation in the absence of food remains unaffected in these animals. Selective re-expression of NPY in AgRP neurons attenuates the reduced feeding response and reverses the protection from insulin resistance upon optogenetic activation of AgRP neurons in NPY-deficient mice. Collectively, these experiments reveal a pivotal role of NPY-dependent signaling in mediating the rapid feeding inducing effect and the acute glucose regulatory function governed by AgRP neurons
Successful lung transplantation using an allograft from a COVID-19-recovered donor: a potential role for subgenomic RNA to guide organ utilization

American Journal of Transplantation

2022 Jan 01

Saharia, KK;Ramelli, SC;Stein, SR;Roder, AE;
| DOI: 10.1016/j.ajt.2022.09.001

Although the risk of SARS-CoV-2 transmission through lung transplantation from acutely infected donors is high, the risks of virus transmission and long-term lung allograft outcomes are not as well described when using pulmonary organs from COVID-19-recovered donors. We describe successful lung transplantation for a COVID-19-related lung injury using lungs from a COVID-19-recovered donor who was retrospectively found to have detectable genomic SARS-CoV-2 RNA in the lung tissue by multiple highly sensitive assays. However, SARS-CoV-2 subgenomic RNA (sgRNA), a marker of viral replication, was not detectable in the donor respiratory tissues. One year after lung transplantation, the recipient has a good functional status, walking 1 mile several times per week without the need for supplemental oxygen and without any evidence of donor-derived SARS-CoV-2 transmission. Our findings highlight the limitations of current clinical laboratory diagnostic assays in detecting the persistence of SARS-CoV-2 RNA in the lung tissue. The persistence of SARS-CoV-2 RNA in the donor tissue did not appear to represent active viral replication via sgRNA testing and, most importantly, did not negatively impact the allograft outcome in the first year after lung transplantation. sgRNA is easily performed and may be a useful assay for assessing viral infectivity in organs from donors with a recent infection.

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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
EnEmProbe targets exons n and m
En-EmProbe 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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