Antemortem vs Postmortem Histopathologic and Ultrastructural Findings in Paired Transbronchial Biopsy Specimens and Lung Autopsy Samples From Three Patients With Confirmed SARS-CoV-2
American journal of clinical pathology
Gagiannis, D;Umathum, VG;Bloch, W;Rother, C;Stahl, M;Witte, HM;Djudjaj, S;Boor, P;Steinestel, K;
PMID: 34463314 | DOI: 10.1093/ajcp/aqab087
Respiratory failure is the major cause of death in coronavirus disease 2019 (COVID-19). Autopsy-based reports describe diffuse alveolar damage (DAD), organizing pneumonia, and fibrotic change, but data on early pathologic changes and during progression of the disease are rare.We prospectively enrolled three patients with COVID-19 and performed full clinical evaluation, including high-resolution computed tomography. We took transbronchial biopsy (TBB) specimens at different time points and autopsy tissue samples for histopathologic and ultrastructural evaluation after the patients' death.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed by reverse transcription polymerase chain reaction and/or fluorescence in situ hybridization in all TBBs. Lung histology showed reactive pneumocytes and capillary congestion in one patient who died shortly after hospital admission with detectable virus in one of two lung autopsy samples. SARS-CoV-2 was detected in two of two autopsy samples from another patient with a fulminant course and very short latency between biopsy and autopsy, showing widespread organizing DAD. In a third patient with a prolonged course, autopsy samples showed extensive fibrosis without detectable virus.We report the course of COVID-19 in paired biopsy specimens and autopsies, illustrating vascular, organizing, and fibrotic patterns of COVID-19-induced lung injury. Our results suggest an early spread of SARS-CoV-2 from the upper airways to the lung periphery with diminishing viral load during disease.
SARS-CoV-2 infection in the mouse olfactory system
Ye, Q;Zhou, J;He, Q;Li, RT;Yang, G;Zhang, Y;Wu, SJ;Chen, Q;Shi, JH;Zhang, RR;Zhu, HM;Qiu, HY;Zhang, T;Deng, YQ;Li, XF;Liu, JF;Xu, P;Yang, X;Qin, CF;
PMID: 34230457 | DOI: 10.1038/s41421-021-00290-1
SARS-CoV-2 infection causes a wide spectrum of clinical manifestations in humans, and olfactory dysfunction is one of the most predictive and common symptoms in COVID-19 patients. However, the underlying mechanism by which SARS-CoV-2 infection leads to olfactory disorders remains elusive. Herein, we demonstrate that intranasal inoculation with SARS-CoV-2 induces robust viral replication in the olfactory epithelium (OE), not the olfactory bulb (OB), resulting in transient olfactory dysfunction in humanized ACE2 (hACE2) mice. The sustentacular cells and Bowman's gland cells in the OE were identified as the major target cells of SARS-CoV-2 before invasion into olfactory sensory neurons (OSNs). Remarkably, SARS-CoV-2 infection triggers massive cell death and immune cell infiltration and directly impairs the uniformity of the OE structure. Combined transcriptomic and quantitative proteomic analyses revealed the induction of antiviral and inflammatory responses, as well as the downregulation of olfactory receptor (OR) genes in the OE from the infected animals. Overall, our mouse model recapitulates olfactory dysfunction in COVID-19 patients and provides critical clues for understanding the physiological basis for extrapulmonary manifestations of COVID-19.
Cellular and molecular life sciences : CMLS
Zhu, A;Real, F;Capron, C;Rosenberg, AR;Silvin, A;Dunsmore, G;Zhu, J;Cottoignies-Callamarte, A;Massé, JM;Moine, P;Bessis, S;Godement, M;Geri, G;Chiche, JD;Valdebenito, S;Belouzard, S;Dubuisson, J;Lorin de la Grandmaison, G;Chevret, S;Ginhoux, F;Eugenin, EA;Annane, D;Bordé, EC;Bomsel, M;
PMID: 35708858 | DOI: 10.1007/s00018-022-04318-x
SARS-CoV-2, although not being a circulatory virus, spread from the respiratory tract resulting in multiorgan failures and thrombotic complications, the hallmarks of fatal COVID-19. A convergent contributor could be platelets that beyond hemostatic functions can carry infectious viruses. Here, we profiled 52 patients with severe COVID-19 and demonstrated that circulating platelets of 19 out 20 non-survivor patients contain SARS-CoV-2 in robust correlation with fatal outcome. Platelets containing SARS-CoV-2 might originate from bone marrow and lung megakaryocytes (MKs), the platelet precursors, which were found infected by SARS-CoV-2 in COVID-19 autopsies. Accordingly, MKs undergoing shortened differentiation and expressing anti-viral IFITM1 and IFITM3 RNA as a sign of viral sensing were enriched in the circulation of deadly COVID-19. Infected MKs reach the lung concomitant with a specific MK-related cytokine storm rich in VEGF, PDGF and inflammatory molecules, anticipating fatal outcome. Lung macrophages capture SARS-CoV-2-containing platelets in vivo. The virus contained by platelets is infectious as capture of platelets carrying SARS-CoV-2 propagates infection to macrophages in vitro, in a process blocked by an anti-GPIIbIIIa drug. Altogether, platelets containing infectious SARS-CoV-2 alter COVID-19 pathogenesis and provide a powerful fatality marker. Clinical targeting of platelets might prevent viral spread, thrombus formation and exacerbated inflammation at once and increase survival in COVID-19.
British journal of pharmacology
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
Polgár, E;Dickie, AC;Gutierrez-Mecinas, M;Bell, AM;Boyle, KA;Quillet, R;Rashid, EA;Clark, RA;German, MT;Watanabe, M;Riddell, JS;Todd, AJ;
PMID: 35543635 | DOI: 10.1097/j.pain.0000000000002677
Neurons in the superficial dorsal horn that express the gastrin-releasing peptide receptor (GRPR) are strongly implicated in spinal itch pathways. However, a recent study reported that many of these correspond to vertical cells, a population of interneurons that are thought to transmit nociceptive information. In this study, we have used a GRPRCreERT2 mouse line to identify and target cells that possess Grpr mRNA. We find that the GRPR cells are highly concentrated in lamina I and the outer part of lamina II, that they are all glutamatergic, and that they account for ∼15% of the excitatory neurons in the superficial dorsal horn. We had previously identified 6 neurochemically distinct excitatory interneuron populations in this region based on neuropeptide expression and the GRPR cells are largely separate from these, although they show some overlap with cells that express substance P. Anatomical analysis revealed that the GRPR neurons are indeed vertical cells, and that their axons target each other, as well as arborising in regions that contain projection neurons: lamina I, the lateral spinal nucleus and the lateral part of lamina V. Surprisingly, given the proposed role of GRPR cells in itch, we found that most of the cells received monosynaptic input from Trpv1-expressing (nociceptive) afferents, that the great majority responded to noxious and pruritic stimuli, and that chemogenetically activating them resulted in pain- and itch-related behaviours. Together, these findings suggest that the GRPR cells are involved in spinal cord circuits that underlie both pain and itch.
Yaeger, JDW;Krupp, KT;Summers, TR;Summers, CH;
PMID: 35724928 | DOI: 10.1016/j.neuropharm.2022.109168
Fear-associated memories and behavior are often expressed in contexts/environments distinctively different from those in which they are created. This generalization process contributes to psychological disorders, particularly PTSD. Stress-related neurocircuits in the basolateral amygdala (BLA) receive inputs from hypothalamic orexin (Orx) neurons, which mediate neuronal activity by targeting orexin 1 (Orx1R) and orexin 2 (Orx2R) receptors via opposing functions. In BLA, inhibition of Orx1R or activation of Orx2R ameliorate stress responsiveness and behavior. We discovered that most Orx1R+ cells also express CamKIIα, while a majority of Orx2R+ cells are colocalized with GAD67. Further, Orx1R gene Hcrtr1 expression was positively correlated, and Orx2R gene Hcrtr2 expression was negatively correlated, with freezing in a phenotype-dependent fashion (Escape vs Stay) in the Stress Alternatives Model (SAM). The SAM consists of 4-days of social interaction between test mice and novel larger aggressors. Exits positioned at opposite ends of the SAM oval arena provide opportunities to actively avoid aggression. By Day 2, mice commit to behavioral phenotypes: Escape or Stay. Pharmacologically manipulating Orx receptor activity in the BLA, before Day 3 of the SAM, was followed with standard tests of anxiety: Open Field (OF) and Elevated Plus Maze (EPM). In Stay mice, freezing in response to social conflict and locomotion during SAM interaction (not home cage locomotion) were generalized to OF, and blocked by intra-BLA Orx1R antagonism, but not Orx2R antagonism. Moreover, patterns of social avoidance for Escape and Stay mice were recapitulated in OF, with generalization mediated by Orx1R and Orx2R antagonism, plus Orx2R stimulation.
Yaeger, J;Krupp, K;Jacobs, B;Onserio, B;Meyerink, B;Cain, J;Ronan, P;Renner, K;DiLeone, R;Summers, C;
| DOI: 10.1016/j.biopsych.2021.12.019
BACKGROUND Stress produces differential behavioral responses through select molecular modifications to specific neurocircuitry elements. The orexin system targets key components of this neurocircuitry in the basolateral amygdala (BLA). METHODS We assessed the contribution of intra-BLA Orexin 1 receptors (Orx1R) in the expression of stress-induced phenotypes of mice. Using the Stress Alternatives Model (SAM), a social stress paradigm that produces two behavioral phenotypes, we characterized the role of intra-BLA Orx1R using acute pharmacological inhibition (SB-674042) and genetic knockdown (AAV-U6-Orx1R-shRNA) strategies. RESULTS In the BLA, we observed that Orx1R (HCRTR1) mRNA is predominantly expressed in CamKIIα+ glutamatergic neurons and rarely in GABAergic cells. While there is a slight overlap in HCRTR1 and Orexin 2 receptor (Orx2R; HCRTR2) mRNA expression in the BLA, we find that these receptors are most often expressed in separate cells. Antagonism of intra-BLA Orx1R after phenotype formation shifted behavioral expression from stress sensitive (Stay) to resilient (Escape) responses, an effect that was mimicked by genetic knockdown. Acute inhibition of Orx1R in the BLA also reduced contextual and cued fear freezing responses in Stay animals. This phenotype-specific behavioral change was accompanied by biased molecular transcription favoring HCRTR2 over HCRTR1, and MAPK3 over PLCB1 cell signaling cascades and enhanced BDNF mRNA. CONCLUSIONS Functional reorganization of intra-BLA gene expression is produced by antagonism of Orx1R, which promotes elevated HCRTR2, greater MAPK3, and increased BDNF expression. Together, these results provide evidence for a receptor-driven mechanism that balances pro- and anti-stress responses within the BLA.
Hernández VS, Hernández OR, Perez de la Mora M, Gómora ML, Fuxe K, Eiden LE, Zhang L.
PMID: - | DOI: 10.3389/fncir.2016.00092
The arginine-vasopressin (AVP)-containing hypothalamic magnocellular neurosecretory neurons (VPMNNs) are known for their role in hydro-electrolytic balance control via their projections to the neurohypophysis. Recently, projections from these same neurons to hippocampus, habenula and other brain regions in which vasopressin infusion modulates contingent social and emotionally-affected behaviors, have been reported. Here, we present evidence that VPMNN collaterals also project to the amygdaloid complex, and establish synaptic connections with neurons in central amygdala (CeA). The density of AVP innervation in amygdala was substantially increased in adult rats that had experienced neonatal maternal separation (MS), consistent with our previous observations that MS enhances VPMNN number in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. In the CeA, V1a AVP receptor mRNA was only observed in GABAergic neurons, demonstrated by complete co-localization of V1a transcripts in neurons expressing Gad1 and Gad2 transcripts in CeA using the RNAscope method. V1b and V2 receptor mRNAs were not detected, using the same method. Water-deprivation (WD) for 24 h, which increased the metabolic activity of VPMNNs, also increased anxiety-like behavior measured using the elevated plus maze (EPM) test, and this effect was mimicked by bilateral microinfusion of AVP into the CeA. Anxious behavior induced by either WD or AVP infusion was reversed by CeA infusion of V1a antagonist. VPMNNs are thus a newly discovered source of CeA inhibitory circuit modulation, through which both early-life and adult stress coping signals are conveyed from the hypothalamus to the amygdala.
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.
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.
The Journal of neuroscience : the official journal of the Society for Neuroscience
Fudge, JL;Kelly, EA;Hackett, TA;
PMID: 36280261 | DOI: 10.1523/JNEUROSCI.1453-22.2022
The central extended amygdala (CEA) and ventral pallidum (VP) are involved in diverse motivated behaviors based on rodent models. These structures are conserved, but expanded, in higher primates including human. Corticotropin releasing factor (CRF), a canonical 'stress molecule' associated with the CEA and VP circuitry across species, is dynamically regulated by stress and drugs of abuse and misuse. CRF's effects on circuits critically depend on its colocation with primary 'fast' transmitters, making this crucial for understanding circuit effects. We surveyed the distribution and colocalization of CRF-, VGluT2- (vesicular glutamate transporter 2) and VGAT- (vesicular GABA transporter) mRNA in specific subregions of the CEA and VP in young male monkeys. Although CRF-containing neurons were clustered in the lateral central bed nucleus (BSTLcn), the majority were broadly dispersed throughout other CEA subregions, and the VP. CRF/VGAT-only neurons were highest in the BSTLcn, lateral central amygdala nucleus (CeLcn), and medial central amygdala nucleus (CeM) (74%, 73%, and 85%, respectively). In contrast, lower percentages of CRF/VGAT only neurons populated the sublenticular extended amygdala (SLEAc), ventrolateral bed nucleus (BSTLP), and VP (53%, 54%, 17%, respectively), which had higher complements of CRF/VGAT/VGluT2 labeled neurons (33%, 29%, 67%, respectively). Thus, the majority of CRF-neurons at the 'poles' (BSTLcn and CeLcn/CeM) of the CEA are inhibitory, while the 'extended' BSTLP and SLEAc subregions, and neighboring VP, have a more complex profile with admixtures of 'multiplexed' excitatory CRF neurons. CRF's colocalization with its various fast transmitters is likely circuit-specific, and relevant for understanding CRF actions on specific target sites.SIGNIFICANCE STATEMENT:The central extended amygdala (CEA) and ventral pallidum (VP) regulate multiple motivated behaviors through differential downstream projections. The stress neuropeptide corticotropin releasing factor (CRF) is enriched in the CEA, and is thought to 'set the gain' through modulatory effects on co-expressed primary transmitters. Using protein and transcript assays in monkey, we found that CRF neurons are broadly and diffusely distributed in CEA and VP. CRF mRNA+ neurons colocalize with VGAT (GABA) and VGluT2 (glutamate) mRNAs in different proportions depending on subregion. CRF mRNA was also co-expressed in a subpopulation of VGAT/VGluT2 mRNA ('multiplexed') cells which were most prominent in the VP and 'pallidal'-like parts of the CEA. Heterogeneous CRF and fast transmitter co-expression across CEA/VP subregions implies circuit-specific effects.
Pathogens (Basel, Switzerland)
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.
