J Comp Pathol. 2015 Jul 16.
Palmer MV, Thacker TC, Waters WR.
PMID: 26189773 | DOI: 10.1016/j.jcpa.2015.06.004.
Mycobacterium bovis is the cause of tuberculosis in most animal species including cattle and is a serious zoonotic pathogen. In man, M. bovis infection can result in disease clinically indistinguishable from that caused by Mycobacterium tuberculosis, the cause of most human tuberculosis. Regardless of host, the typical lesion induced by M. bovis or M. tuberculosis is the tuberculoid granuloma. Tuberculoid granulomas are dynamic structures reflecting the interface between host and pathogen and, therefore, pass through various morphological stages (I to IV). Using a novel in-situ hybridization assay, transcription of various cytokine and chemokine genes was examined qualitatively and quantitatively using image analysis. In experimentally infected cattle, pulmonary granulomas of all stages were examined 150 days after aerosol exposure to M. bovis. Expression of mRNA encoding tumour necrosis factor (TNF)-α, transforming growth factor-β, interferon (IFN)-γ, interleukin (IL)-17A, IL-16, IL-10, CXCL9 and CXCL10 did not differ significantly between granulomas of different stages. However, relative expression of the various cytokines was characteristic of a Th1 response, with high TNF-α and IFN-γ expression and low IL-10 expression. Expression of IL-16 and the chemokines CXCL9 and CXCL10 was high, suggestive of granulomas actively involved in T-cell chemotaxis.
Schulz D, Zanotelli VRT, Fischer JR, Schapiro D, Engler S, Lun XK, Jackson HW, Bodenmiller B.
PMID: 29289569 | DOI: 10.1016/j.cels.2017.12.001
To build comprehensive models of cellular states and interactions in normal and diseased tissue, genetic and proteomic information must be extracted with single-cell and spatial resolution. Here, we extended imaging mass cytometry to enable multiplexed detection of mRNA and proteins in tissues. Three mRNA target species were detected by RNAscope-based metal in situ hybridization with simultaneous antibody detection of 16 proteins. Analysis of 70 breast cancer samples showed that HER2 and CK19 mRNA and protein levels are moderately correlated on the single-cell level, but that only HER2, and not CK19, has strong mRNA-to-protein correlation on the cell population level. The chemoattractant CXCL10 was expressed in stromal cell clusters, and the frequency of CXCL10-expressing cells correlated with T cell presence. Our flexible and expandable method will allow an increase in the information content retrieved from patient samples for biomedical purposes, enable detailed studies of tumor biology, and serve as a tool to bridge comprehensive genomic and proteomic tissue analysis.
The American journal of pathology
Ball, EE;Weiss, CM;Liu, H;Jackson, K;Keel, MK;Miller, CJ;Van Rompay, KKA;Coffey, LL;Pesavento, PA;
PMID: 36906263 | DOI: 10.1016/j.ajpath.2023.02.013
Clinical evidence of vascular dysfunction and hypercoagulability as well as pulmonary vascular damage and microthrombosis are frequently reported in severe cases of human coronavirus disease 2019 (COVID-19). Syrian golden hamsters recapitulate histopathologic pulmonary vascular lesions reported in patients with COVID-19. Here, special staining techniques and transmission electron microscopy further define vascular pathologies in a Syrian golden hamster model of human COVID-19. The results show that regions of active pulmonary inflammation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are characterized by ultrastructural evidence of endothelial damage with platelet marginalization and both perivascular and subendothelial macrophage infiltration. SARS-CoV-2 antigen/RNA was not detectable within affected blood vessels. Taken together, these findings suggest that the prominent microscopic vascular lesions in SARS-CoV-2-inoculated hamsters are likely due to endothelial damage followed by platelet and macrophage infiltration.
medRxiv : the preprint server for health sciences
Gonzalez, V;Li, L;Buarpung, S;Prahl, M;Robinson, JF;Gaw, SL;
PMID: 36778281 | DOI: 10.1101/2023.02.01.23285349
Despite universal recommendations for COVID-19 mRNA vaccination in pregnancy, uptake has been lower than desired. There have been limited studies of the direct impact of COVID-19 mRNA vaccine exposure in human placental tissue. Using a primary human villous explant model, we investigated the uptake of two common mRNA vaccines (BNT162b2 Pfizer-BioNTech or mRNA-1273 Moderna), and whether exposure altered villous cytokine responses. Explants derived from second or third trimester chorionic villi were incubated with vaccines at supraphysiologic concentrations and analyzed at two time points. We observed minimal uptake of mRNA vaccines in placental explants by in situ hybridization and quantitative RT-PCR. No specific or global cytokine response was elicited by either of the mRNA vaccines in multiplexed immunoassays. Our results suggest that the human placenta does not readily absorb the COVID-19 mRNA vaccines nor generate a significant inflammatory response after exposure.
Nature biomedical engineering
Wang, Z;Popowski, KD;Zhu, D;de Juan Abad, BL;Wang, X;Liu, M;Lutz, H;De Naeyer, N;DeMarco, CT;Denny, TN;Dinh, PC;Li, Z;Cheng, K;
PMID: 35788687 | DOI: 10.1038/s41551-022-00902-5
The first two mRNA vaccines against infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that were approved by regulators require a cold chain and were designed to elicit systemic immunity via intramuscular injection. Here we report the design and preclinical testing of an inhalable virus-like-particle as a COVID-19 vaccine that, after lyophilisation, is stable at room temperature for over three months. The vaccine consists of a recombinant SARS-CoV-2 receptor-binding domain (RBD) conjugated to lung-derived exosomes which, with respect to liposomes, enhance the retention of the RBD in both the mucus-lined respiratory airway and in lung parenchyma. In mice, the vaccine elicited RBD-specific IgG antibodies, mucosal IgA responses and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile in the animals' lungs, and cleared them of SARS-CoV-2 pseudovirus after a challenge. In hamsters, two doses of the vaccine attenuated severe pneumonia and reduced inflammatory infiltrates after a challenge with live SARS-CoV-2. Inhalable and room-temperature-stable virus-like particles may become promising vaccine candidates.
Expert review of vaccines
Neil, JA;Griffith, M;Godfrey, DI;Purcell, DFJ;Deliyannis, G;Jackson, D;Rockman, S;Subbarao, K;Nolan, T;
PMID: 35652289 | DOI: 10.1080/14760584.2022.2071264
Evaluation of immunogenicity and efficacy in animal models provide critical data in vaccine development. Nonhuman primates (NHPs) have been used extensively in the evaluation of SARS-CoV-2 vaccines.A critical synthesis of SARS-CoV-2 vaccine development with a focus on challenge studies in NHPs is provided. The benefits and drawbacks of the NHP models are discussed. The citations were selected by the authors based on PubMed searches of the literature, summaries from national public health bodies, and press-release information provided by vaccine developers.We identify several aspects of NHP models that limit their usefulness for vaccine-challenge studies and numerous variables that constrain comparisons across vaccine platforms. We propose that studies conducted in NHPs for vaccine development should use a standardized protocol and, where possible, be substituted with smaller animal models. This will ensure continued rapid progression of vaccines to clinical trials without compromising assessments of safety or efficacy.
Pathology - Research and Practice
Schwab, C;Domke, L;Rose, F;Hausser, I;Schirmacher, P;Longerich, T;
| DOI: 10.1016/j.prp.2022.154000
Pulmonary capillary microthrombosis has been proposed as a major pathogenetic factor driving severe COVID-19. Autopsy studies reported endothelialitis but it is under debate if it is caused by SARS-CoV-2 infection of endothelial cells. In this study, RNA in situ hybridization was used to detect viral RNA and to identify the infected cell types in lung tissue of 40 patients with fatal COVID-19. SARS-CoV-2 Spike protein-coding RNA showed a steadily decreasing signal abundance over a period of three weeks. Besides the original virus strain the variants of concern Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529) could also be detected by the assay. Viral RNA was mainly detected in alveolar macrophages and pulmonary epithelial cells, while only single virus-positive endothelial cells were observed even in cases with high viral load suggesting that viral infection of endothelial cells is not a key factor for the development of pulmonary capillary microthrombosis.
ACS chemical neuroscience
Dagher, M;Perrotta, KA;Erwin, SA;Hachisuka, A;Iyer, R;Masmanidis, SC;Yang, H;Andrews, AM;
PMID: 35312275 | DOI: 10.1021/acschemneuro.1c00715
Targeting neurons with light-driven opsins is widely used to investigate cell-specific responses. We transfected midbrain dopamine neurons with the excitatory opsin Chrimson. Extracellular basal and stimulated neurotransmitter levels in the dorsal striatum were measured by microdialysis in awake mice. Optical activation of dopamine cell bodies evoked terminal dopamine release in the striatum. Multiplexed analysis of dialysate samples revealed that the evoked dopamine was accompanied by temporally coupled increases in striatal 3-methoxytyramine, an extracellular dopamine metabolite, and in serotonin. We investigated a mechanism for dopamine-serotonin interactions involving striatal dopamine receptors. However, the evoked serotonin associated with optical stimulation of dopamine neurons was not abolished by striatal D1- or D2-like receptor inhibition. Although the mechanisms underlying the coupling of striatal dopamine and serotonin remain unclear, these findings illustrate advantages of multiplexed measurements for uncovering functional interactions between neurotransmitter systems. Furthermore, they suggest that the output of optogenetic manipulations may extend beyond opsin-expressing neuronal populations.
Cellular & molecular immunology
Wang, Z;Lv, J;Yu, P;Qu, Y;Zhou, Y;Zhou, L;Zhu, Q;Li, S;Song, J;Deng, W;Gao, R;Liu, Y;Liu, J;Tong, WM;Qin, C;Huang, B;
PMID: 34983944 | DOI: 10.1038/s41423-021-00813-6
Exploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.
Han, Y;Yuan, K;Wang, Z;Liu, WJ;Lu, ZA;Liu, L;Shi, L;Yan, W;Yuan, JL;Li, JL;Shi, J;Liu, ZC;Wang, GH;Kosten, T;Bao, YP;Lu, L;
PMID: 34593760 | DOI: 10.1038/s41398-021-01629-8
The coronavirus disease 2019 (COVID-19) pandemic has caused large-scale economic and social losses and worldwide deaths. Although most COVID-19 patients have initially complained of respiratory insufficiency, the presence of neuropsychiatric manifestations is also reported frequently, ranging from headache, hyposmia/anosmia, and neuromuscular dysfunction to stroke, seizure, encephalopathy, altered mental status, and psychiatric disorders, both in the acute phase and in the long term. These neuropsychiatric complications have emerged as a potential indicator of worsened clinical outcomes and poor prognosis, thus contributing to mortality in COVID-19 patients. Their etiology remains largely unclear and probably involves multiple neuroinvasive pathways. Here, we summarize recent animal and human studies for neurotrophic properties of severe acute respiratory syndrome coronavirus (SARS-CoV-2) and elucidate potential neuropathogenic mechanisms involved in the viral invasion of the central nervous system as a cause for brain damage and neurological impairments. We then discuss the potential therapeutic strategy for intervening and preventing neuropsychiatric complications associated with SARS-CoV-2 infection. Time-series monitoring of clinical-neurochemical-radiological progress of neuropsychiatric and neuroimmune complications need implementation in individuals exposed to SARS-CoV-2. The development of a screening, intervention, and therapeutic framework to prevent and reduce neuropsychiatric sequela is urgently needed and crucial for the short- and long-term recovery of COVID-19 patients.
LB740 SARS-CoV-2-associated ‘covid toes:’ multiplex immunofluorescent characterization of pathophysiology
Journal of Investigative Dermatology
Moon, J;Costa da Silva, A;Tran, J;Kim, C;Sharma, R;Hinshaw, M;Shields, B;Brooks, E;Cowen, E;Singh, A;Drolet, B;Mays, J;Arkin, L;
| DOI: 10.1016/j.jid.2021.07.093
Coincident with the start of the COVID-19 pandemic, dermatologists worldwide have reported an uncharacteristic increase in pernio or chilblains (aka ‘COVID toes’). However, the lack of systemic illness, low PCR positivity and lack of consistent seroconversion have led some authors to postulate an epiphenomenon. SARS-CoV-2 spike protein has been identified in a limited number of skin biopsies in few publications, yet there remain conflicting reports regarding other SARS-CoV-2 associated proteins, the presence or absence of viral RNA, and a unifying pathophysiology. In cooperation with the COVID Human Genome Effort, our “COVID toes” biobank was established to identify both the genetic and immunologic basis and provide clinically relevant insights into targeted therapeutics. As of March 2021, we have enrolled 96 patients, creating a prospective biorepository with clinical data, saliva, serial blood collection, and skin biopsies. Here we aim to comprehensively investigate the conflicting findings, detail the inflammatory response, and identify the source of interferon signaling with multiplex immunofluorescence (IFA) and the RNAscope fluorescent assay to detect viral mRNA. Median patient age was 17 (range 2 e 72) and 44/96 (46%) were male. Preliminary IFA results demonstrate detection of SARS-CoV-2 components, robust MxA detection and plasmacytoid dendritic cell (pDC) colocalization, identifying PDCs as the likely primary source of IFN-I production and implicates an excessive localized IFN-I response in affected patients.
Claypool, SM;Behdin, S;Applebey, SV;Orihuel, J;Ma, Z;Reiner, DJ;
PMID: 35768212 | DOI: 10.1523/ENEURO.0496-21.2022
The orbitofrontal cortex (OFC) and piriform cortex (Pir) play a role in fentanyl relapse after food choice-induced voluntary abstinence, a procedure mimicking abstinence because of availability of alternative nondrug rewards. We used in situ hybridization and pharmacology to determine the role of OFC and Pir cannabinoid and dopamine receptors in fentanyl relapse. We trained male and female rats to self-administer food pellets for 6 d (6 h/d) and intravenous fentanyl (2.5 µg/kg/infusion) for 12 d (6 h/d). We assessed fentanyl relapse after 12 discrete choice sessions between fentanyl and food (20 trials/d), in which rats voluntarily reduced fentanyl self-administration. We used RNAscope to determine whether fentanyl relapse is associated with activity (indicated by Fos) in OFC and Pir cells expressing Cnr1 [which encodes cannabinoid 1 (CB1) receptors] or Drd1 and Drd2 (which encode dopamine D1 and D2 receptors). We injected a CB1 receptor antagonist or agonist (0.3 or 1.0 µg AM251 or WIN55,212-2/hemisphere) into OFC or a dopamine D1 receptor antagonist (1.0 or 3.0 µg SCH39166/hemisphere) into Pir to determine the effect on fentanyl relapse. Fentanyl relapse was associated with OFC cells co-expressing Fos and Cnr1 and Pir cells co-expressing Fos and Drd1 However, injections of the CB1 receptor antagonist AM251 or agonist WIN55,212-2 into OFC or the dopamine D1 receptor antagonist SCH39166 into Pir had no effect on fentanyl relapse. Fentanyl relapse is associated with activation of Cnr1-expressing OFC cells and Drd1-expressing Pir cells, but pharmacological manipulations do not support causal roles of OFC CB1 receptors or Pir dopamine D1 receptors in fentanyl relapse.
