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.
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.
Yoshida T, Takizawa N, Matsuda T, Yamada H, Kitada M, Tanaka S
PMID: 31949236 | DOI: 10.1038/s41598-019-57351-5
Adrenal cortex autotransplantation with ACTH stimulation may be an alternative therapy for patients with bilateral adrenalectomy to avoid adrenal crisis, but its underlying mechanism has not been elucidated. Previously, we detected Dhh upregulation in rat adrenocortical autografts after transplantation. Here, we investigated potential regulators such as Gata4, Gata6, Sry and Sox9 which affect Dhh transcription in adrenocortical autografts with or without ACTH stimulation. In ACTH-stimulated autografts, Gata4 and Gata6 were downregulated compared to control autografts. This response was linked to rDhh repression. A reporter assay using the upstream region of rDhh and a GATA binding motif revealed that rDhh promoters were significantly upregulated by co-transfection with Gata4 or Gata6 or both. Sry and Sox9 expression in autografts with or without ACTH stimulation were verified by PCR and RNAscope analyses. The ovarian differentiation factors Foxl2 and Rspo1 were also upregulated in the autografts. Gata4 and Gata6 were found to be significant factors in the regulation of rDhh expression and could be associated with adrenocortical autograft maintenance. Gonadal primordia with bipotential testicular and ovarian functions may also be present in these autografts.
Dermatology (Basel, Switzerland)
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.
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.
Scandinavian cardiovascular journal : SCJ
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.
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.
"Boldog E, Bakken TE, Hodge RD, Novotny M, Aevermann BD, Baka J, Bordé S, Close JL, Diez-Fuertes F, Ding SL, Faragó N, Kocsis AK, Kovács B, Maltzer Z, McCorrison JM, Miller JA, Molnár G, Oláh G, Ozsvár A, Rózsa M, Shehata SI, Smith KA, Sunkin SM, Tran D
PMID: 30150662 | DOI: 10.1038/s41593-018-0205-2
We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1-SST-CALB2-PVALB-) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.
American Journal of Transplantation
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.
Pulmonary stromal expansion and intra-alveolar coagulation are primary causes of COVID-19 death
Szekely, L;Bozoky, B;Bendek, M;Ostad, M;Lavignasse, P;Haag, L;Wu, J;Jing, X;Gupta, S;Saccon, E;Sönnerborg, A;Cao, Y;Björnstedt, M;Szakos, A;
PMID: 34056141 | DOI: 10.1016/j.heliyon.2021.e07134
Most COVID-19 victims are old and die from unrelated causes. Here we present twelve complete autopsies, including two rapid autopsies of young patients where the cause of death was COVID-19 ARDS. The main virus induced pathology was in the lung parenchyma and not in the airways. Most coagulation events occurred in the intra-alveolar and not in the intra-vascular space and the few thrombi were mainly composed of aggregated thrombocytes. The dominant inflammatory response was the massive accumulation of CD163 + macrophages and the disappearance of T killer, NK and B-cells. The virus was replicating in the pneumocytes and macrophages but not in bronchial epithelium, endothelium, pericytes or stromal cells. The lung consolidations were produced by a massive regenerative response, stromal and epithelial proliferation and neovascularization. We suggest that thrombocyte aggregation inhibition, angiogenesis inhibition and general proliferation inhibition may have a roll in the treatment of advanced COVID-19 ARDS.
Ruscitto A, Morel MM, Shawber CJ, Reeve G, Lecholop MK, Bonthius D, Yao H, Embree MC
PMID: 32030828 | DOI: 10.1096/fj.201902287R
Temporomandibular joint osteoarthritis (TMJ OA) leads to permanent cartilage destruction, jaw dysfunction, and compromises the quality of life. However, the pathological mechanisms governing TMJ OA are poorly understood. Unlike appendicular articular cartilage, the TMJ has two distinct functions as the synovial joint of the craniofacial complex and also as the site for endochondral jaw bone growth. The established dogma of endochondral bone ossification is that hypertrophic chondrocytes undergo apoptosis, while invading vasculature with osteoprogenitors replace cartilage with bone. However, contemporary murine genetic studies support the direct differentiation of chondrocytes into osteoblasts and osteocytes in the TMJ. Here we sought to characterize putative vasculature and cartilage to bone transdifferentiation using healthy and diseased TMJ tissues from miniature pigs and humans. During endochondral ossification, the presence of fully formed vasculature expressing CD31+ endothelial cells and ?-SMA+ vascular smooth muscle cells were detected within all cellular zones in growing miniature pigs. Arterial, endothelial, venous, angiogenic, and mural cell markers were significantly upregulated in miniature pig TMJ tissues relative to donor matched knee meniscus fibrocartilage tissue. Upon surgically creating TMJ OA in miniature pigs, we discovered increased vasculature and putative chondrocyte to osteoblast transformation dually marked by COL2 and BSP or RUNX2 within the vascular bundles. Pathological human TMJ tissues also exhibited increased vasculature, while isolated diseased human TMJ cells exhibited marked increased in vasculature markers relative to control 293T cells. Our study provides evidence to suggest that the TMJ in higher order species are in fact vascularized. There have been no reports of cartilage to bone transdifferentiation or vasculature in human-relevant TMJ OA large animal models or in human TMJ tissues and cells. Therefore, these findings may potentially alter the clinical management of TMJ OA by defining new drugs that target angiogenesis or block the cartilage to bone transformation
Roczkowsky, A;Limonta, D;Fernandes, JP;Branton, WG;Clarke, M;Hlavay, B;Noyce, RS;Joseph, JT;Ogando, NS;Das, SK;Elaish, M;Arbour, N;Evans, DH;Langdon, K;Hobman, TC;Power, C;
PMID: 37190821 | DOI: 10.1002/ana.26679
Peroxisome injury occurs in the central nervous system (CNS) during multiple virus infections that result in neurological disabilities. We investigated host neuroimmune responses and peroxisome biogenesis factors during SARS-CoV-2 infection using a multiplatform strategy.Brain tissues from COVID-19 (n=12) and other disease control (ODC) (n=12) patients, as well as primary human neural cells and Syrian hamsters, infected with a clinical variant of SARS-CoV-2, were investigated by ddPCR, RT-qPCR and immunodetection methods.SARS-CoV-2 RNA was detected in the CNS of four patients with COVID-19 with viral protein (NSP3 and spike) immunodetection in the brainstem. Olfactory bulb, brainstem, and cerebrum from patients with COVID-19 showed induction of pro-inflammatory transcripts (IL8, IL18, CXCL10, NOD2) and cytokines (GM-CSF and IL-18) compared to CNS tissues from ODC patients (p<0.05). Peroxisome biogenesis factor transcripts (PEX3, PEX5L, PEX11β and PEX14) and proteins (PEX3, PEX14, PMP70) were suppressed in the CNS of COVID-19 patients compared to ODCs (p<0.05). SARS-CoV-2 infection of hamsters revealed viral RNA detection in the olfactory bulb at days 4 and 7 post-infection while inflammatory gene expression was upregulated in the cerebrum of infected animals by day 14 post-infection (p<0.05). Pex3 transcript levels together with catalase and PMP70 immunoreactivity were suppressed in the cerebrum of SARS-CoV-2 infected animals (p<0.05).COVID-19 induced sustained neuroinflammatory responses with peroxisome biogenesis factor suppression despite limited brainstem SARS-CoV-2 neurotropism in humans. These observations offer insights into developing biomarkers and therapies, while also implicating persistent peroxisome dysfunction as a contributor to the neurological post-acute sequelae of COVID-19. This article is protected by
