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Tissue factor upregulation is associated with SARS-CoV-2 in the lungs of COVID-19 patients
Journal of thrombosis and haemostasis : JTH
2021 Jul 08
Subrahmanian, S;Borczuk, A;Salvatore, S;Fung, KM;Merrill, JT;Laurence, J;Ahamed, J;
PMID: 34236752 | DOI: 10.1111/jth.15451
A substantial proportion of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe/critical coronavirus disease 2019 (COVID-19) characterized by acute respiratory distress syndrome (ARDS) with thrombosis.We tested the hypothesis that SARS-CoV-2--induced upregulation of tissue factor (TF) expression may be responsible for thrombus formation in COVID-19.We compared autopsy lung tissues from 11 patients with COVID-19--associated ARDS with samples from 6 patients with ARDS from other causes (non-COVID-19 ARDS) and 11 normal control lungs.Dual RNA in situ hybridization for SARS-CoV-2 and TF identified sporadic clustered SARS-CoV-2 with prominent co-localization of SARS-CoV-2 and TF RNA. TF expression was 2-fold higher in COVID-19 than in non-COVID-19 ARDS lungs (P = .017) and correlated with the intensity of SARS-CoV-2 staining (R2 = .36, P = .04). By immunofluorescence, TF protein expression was 2.1-fold higher in COVID-19 versus non-COVID-19 ARDS lungs (P = .0048) and 11-fold (P < .001) higher than control lungs. Fibrin thrombi and thrombi positive for platelet factor 4 (PF4) were found in close proximity to regions expressing TF in COVID-19 ARDS lung, and correlated with TF expression (fibrin, R2 = .52, P < .001; PF4, R2 = .59, P < .001).These data suggest that upregulation of TF expression is associated with thrombus formation in COVID-19 lungs and could be a key therapeutic target. Correlation of TF expression with SARS-CoV-2 in lungs of COVID-19 patients also raises the possibility of direct TF induction by the virus.
Brain Struct Funct.
2018 Jan 03
Howe WM, Brooks JL, Tierney PL, Pang J, Rossi A, Young D, Dlugolenski K, Guillmette E, Roy M, Hales K, Kozak R.
PMID: 29299690 | DOI: 10.1007/s00429-017-1601-1
A loss-of-function polymorphism in the α5 nicotinic acetylcholine receptor (nAChR) subunit gene has been linked to both drug abuse and schizophrenia. The α5 nAChR subunit is strategically positioned in the prefrontal cortex (PFC), where a loss-of-function in this subunit may contribute to cognitive disruptions in both disorders. However, the specific contribution of α5 to PFC-dependent cognitive functions has yet to be illustrated. In the present studies, we used RNA interference to knockdown the α5 nAChR subunit in the PFC of adult rats. We provide evidence that through its contribution to cholinergic modulation of cholinergic modulation of neurons in the PFC, the α5 nAChR plays a specific role in the recovery of attention task performance following distraction. Our combined data reveal the potent ability of this subunit to modulate the PFC and cognitive functions controlled by this brain region that are impaired in disease.
Nat Commun
2020 Apr 21
Tsukui T, Sun KH, Wetter JB, Wilson-Kanamori JR, Hazelwood LA, Henderson NC, Adams TS, Schupp JC, Poli SD, Rosas IO, Kaminski N, Matthay MA, Wolters PJ, Sheppard D
PMID: 32317643 | DOI: 10.1038/s41467-020-15647-5
Collagen-producing cells maintain the complex architecture of the lung and drive pathologic scarring in pulmonary fibrosis. Here we perform single-cell RNA-sequencing to identify all collagen-producing cells in normal and fibrotic lungs. We characterize multiple collagen-producing subpopulations with distinct anatomical localizations in different compartments of murine lungs. One subpopulation, characterized by expression of Cthrc1 (collagen triple helix repeat containing 1), emerges in fibrotic lungs and expresses the highest levels of collagens. Single-cell RNA-sequencing of human lungs, including those from idiopathic pulmonary fibrosis and scleroderma patients, demonstrate similar heterogeneity and CTHRC1-expressing fibroblasts present uniquely in fibrotic lungs. Immunostaining and in situ hybridization show that these cells are concentrated within fibroblastic foci. We purify collagen-producing subpopulations and find disease-relevant phenotypes of Cthrc1-expressing fibroblasts in in vitro and adoptive transfer experiments. Our atlas of collagen-producing cells provides a roadmap for studying the roles of these unique populations in homeostasis and pathologic fibrosis
Molecular Therapy - Methods & Clinical Development
2022 Dec 01
Dambra, R;Matter, A;Graca, K;Akhand, S;Mehta, S;Bell-Cohn, A;Swenson, J;Abid, S;Xin, D;Lewis, C;Coyle, L;Wang, M;Bunosso, K;Maugiri, M;Ruiz, R;Cirillo, C;Fogal, B;Grimaldi, C;Vigil, A;Wood, C;Ashour, J;
| DOI: 10.1016/j.omtm.2022.12.013
Viral replication places oncolytic viruses (OVs) in a unique niche in the field of drug pharmacokinetics (PK) as their self-amplification obscures exposure-response relationships. Moreover, standard bioanalytical techniques are unable to distinguish the input from replicated drug products. Here, we combine two novel approaches to characterize PK and biodistribution (BD) after systemic administration of vesicular stomatitis virus pseudotyped with lymphocytic choriomeningitis virus glycoprotein (VSV-GP) in healthy mice. First: to decouple input drug PK/BD versus replication PK/BD, we developed and fully characterized a replication-incompetent tool virus that retained all other critical attributes of the drug. We used this approach to quantify replication in blood and tissues and to determine its impact on PK and BD. Second: to discriminate the genomic and antigenomic viral RNA strands contributing to replication dynamics in tissues, we developed an in situ hybridization method using strand-specific probes and assessed their spatiotemporal distribution in tissues. This latter approach demonstrated that distribution, transcription, and replication localized to tissue-resident macrophages, indicating their role in PK and BD. Ultimately, our study results in a refined PK/BD profile for a replicating OV, new proposed PK parameters, and deeper understanding of OV PK/BD using unique approaches that could be applied to other replicating vectors.
Oncogene
2022 Apr 13
Kidger, AM;Saville, MK;Rushworth, LK;Davidson, J;Stellzig, J;Ono, M;Kuebelsbeck, LA;Janssen, KP;Holzmann, B;Morton, JP;Sansom, OJ;Caunt, CJ;Keyse, SM;
PMID: 35418690 | DOI: 10.1038/s41388-022-02302-0
The cytoplasmic phosphatase DUSP6 and its nuclear counterpart DUSP5 are negative regulators of RAS/ERK signalling. Here we use deletion of either Dusp5 or Dusp6 to explore the roles of these phosphatases in a murine model of KRASG12D-driven pancreatic cancer. By 56-days, loss of either DUSP5 or DUSP6 causes a significant increase in KRASG12D-driven pancreatic hyperplasia. This is accompanied by increased pancreatic acinar to ductal metaplasia (ADM) and the development of pre-neoplastic pancreatic intraepithelial neoplasia (PanINs). In contrast, by 100-days, pancreatic hyperplasia is reversed with significant atrophy of pancreatic tissue and weight loss observed in animals lacking either DUSP5 or DUSP6. On further ageing, Dusp6-/- mice display accelerated development of metastatic pancreatic ductal adenocarcinoma (PDAC), while in Dusp5-/- animals, although PDAC development is increased this process is attenuated by atrophy of pancreatic acinar tissue and severe weight loss in some animals before cancer could progress. Our data suggest that despite a common target in the ERK MAP kinase, DUSP5 and DUSP6 play partially non-redundant roles in suppressing oncogenic KRASG12D signalling, thus retarding both tumour initiation and progression. Our data suggest that loss of either DUSP5 or DUSP6, as observed in certain human tumours, including the pancreas, could promote carcinogenesis.
Discrepancy of p16 immunohistochemical expression and HPV RNA in penile cancer. A multiplex in situ hybridization/immunohistochemistry approach study
Infectious agents and cancer
2021 Mar 31
Zito Marino, F;Sabetta, R;Pagliuca, F;Brunelli, M;Aquino, G;Perdonà, S;Botti, G;Facchini, G;Fiorentino, F;Di Lauro, G;De Sio, M;De Vita, F;Toni, G;Borges Dos Reis, R;Neder, L;Franco, R;
PMID: 33789689 | DOI: 10.1186/s13027-021-00361-8
The high-risk human papillomavirus (HPV) infection represents one of the main etiologic pathways of penile carcinogenesis in approximately 30-50 % of cases. Several techniques for the detection of HPV are currently available including Polymerase chain reaction-based techniques, DNA and RNA in situ hybridization (ISH), p16 immunohistochemistry (IHC). The multiplex HPV RNA ISH/p16 IHC is a novel technique for the simultaneous detection of HPV E6/E7 transcripts and p16INK4a overexpression on the same slide in a single assay. The main aim of this study was to evaluate the discrepancy of p16 IHC expression relatively to HPV RNA ISH in penile cancer tissue. We collected a series of 60 PCs. HPV has been analysed through the RNA ISH, p16 IHC and the multiplex HPV RNA ISH/p16 IHC. The multiplex HPV RNA ISH /p16 IHC results in the series were in complete agreement with the previous results obtained through the classic p16 IHC and HPV RNA scope carried out on two different slides. The multiplex HPV RNA ISH /p16 IHC showed that HPV positivity in our series is more frequently in usual squamous cell carcinoma than in special histotypes (19 out of 60 - 15 %- versus 6 out of 60 - 10 %-), in high-grade than in moderate/low grade carcinomas (6 out of 60 - 10 %- versus 4 out of 60 - 6.7 %-). In addition, our data revealed that in 5 out of 20 cases with p16 high intensity expression is not associated with HPV RNA ISH positivity. Our findings emphasize that the use of p16 as a surrogate of HPV positivity was unsuccessful in approximatively 8 % of cases analysed in our series. Indeed, p16 IHC showed a sensitivity of 100 % and a specificity of 71 %, with a positive predictive value (PPV) of 54 % and a negative predictive value of 100 %; when considering high intensity, p16 IHC showed a sensitivity of 100 %, a specificity of 89 %, with a PPV of 75 % and NPV of 100 %. Since HPV positivity could represent a relevant prognostic and predictive value, the correct characterization offered by this approach appears to be of paramount importance.
Human gene therapy
2021 Dec 21
Palazzi, X;Pardo, I;Sirivelu, M;Newman, L;Kumpf, S;Qian, J;Franks, T;Lopes, S;Liu, J;Monarski, L;Casinghino, S;Ritenour, C;Ritenour, H;Dubois, C;Olson, J;Graves, J;Alexander, K;Coskran, T;Lanz, TA;Brady, J;McCarty, D;Somanathan, S;Whiteley, L;
PMID: 34931542 | DOI: 10.1089/hum.2021.116
Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high dose intravenous (IV) administration. To characterize biodistribution, transduction and toxicity amongst common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron-1 (SMN-1) transgene (AAV-PHP.B-CBh-SMN1) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2x1013, 5x1013, or 1x1014 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5x1013 vg/kg was found dead on Day 4. Clinical pathology data on Days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization (ISH) demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while qPCR confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after one month in rats, and suggest adding the autonomic system in the watch-list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.
Veterinary pathology
2023 May 12
Tseng, AE;Carossino, M;Gertje, HP;O'Connell, AK;Gummuluru, S;Kolachalama, VB;Balasuriya, UBR;Connor, JH;Bennett, RS;Liu, DX;Hensley, LE;Crossland, NA;
PMID: 37170900 | DOI: 10.1177/03009858231171906
The liver is an early systemic target of Ebola virus (EBOV), but characterization beyond routine histopathology and viral antigen distribution is limited. We hypothesized Ebola virus disease (EVD) systemic proinflammatory responses would be reflected in temporally altered liver myeloid phenotypes. We utilized multiplex fluorescent immunohistochemistry (mfIHC), multispectral whole slide imaging, and image analysis to quantify molecular phenotypes of myeloid cells in the liver of rhesus macaques (Macaca mulatta; n = 21) infected with EBOV Kikwit. Liver samples included uninfected controls (n = 3), 3 days postinoculation (DPI; n = 3), 4 DPI (n = 3), 5 DPI (n = 3), 6 DPI (n = 3), and terminal disease (6-8 DPI; n = 6). Alterations in hepatic macrophages occurred at ≥ 5 DPI characterized by a 1.4-fold increase in CD68+ immunoreactivity and a transition from primarily CD14-CD16+ to CD14+CD16- macrophages, with a 2.1-fold decrease in CD163 expression in terminal animals compared with uninfected controls. An increase in the neutrophil chemoattractant and alarmin S100A9 occurred within hepatic myeloid cells at 5 DPI, followed by rapid neutrophil influx at ≥ 6 DPI. An acute rise in the antiviral myxovirus resistance protein 1 (MxA) occurred at ≥ 4 DPI, with a predilection for enhanced expression in uninfected cells. Distinctive expression of major histocompatibility complex (MHC) class II was observed in hepatocytes during terminal disease. Results illustrate that EBOV causes macrophage phenotype alterations as well as neutrophil influx and prominent activation of interferon host responses in the liver. Results offer insight into potential therapeutic strategies to prevent and/or modulate the host proinflammatory response to normalize hepatic myeloid functionality.
Molecular metabolism
2022 Sep 29
Lewis, JE;Woodward, OR;Nuzzaci, D;Smith, CA;Adriaenssens, AE;Billing, L;Brighton, C;Phillips, BU;Tadross, JA;Kinston, SJ;Ciabatti, E;Göttgens, B;Tripodi, M;Hornigold, D;Baker, D;Gribble, FM;Reimann, F;
PMID: 36184065 | DOI: 10.1016/j.molmet.2022.101604
Insulin-like peptide 5 (INSL5) signalling, through its cognate receptor relaxin/insulin-like-family-peptide-receptor-4 (RXFP4), has been reported to be orexigenic, and the preference for high fat diet (HFD) observed in wildtype mice is altered in Rxfp4 knock-out mice. In this study, we used a new Rxfp4-Cre mouse model to investigate the mechanisms underlying these observations.We generated transgenic Rxfp4-Cre mice and investigated central expression of Rxfp4 by RT-qPCR, RNAscope and intraparenchymal infusion of INSL5. Rxfp4-expressing cells were chemogenetically manipulated in global Cre-reporter mice using designer receptors exclusively activated by designer drugs (DREADDs) or after stereotactic injection of Cre-dependent AAV-DIO-Dq-DREADD targeting a population located in the ventromedial hypothalamus (RXFP4VMH). Food intake and feeding motivation were assessed in the presence and absence of a DREADD agonist. Rxfp4-expressing cells in the hypothalamus were characterised by single-cell RNA-sequencing (scRNAseq) and the connectivity of RXFP4VMH cells was investigated using viral tracing.Rxfp4-Cre mice displayed Cre-reporter expression in the hypothalamus and active expression of Rxfp4 in the adult mouse brain was confirmed by RT-qPCR and RNAscope. Functional receptor expression was supported by cAMP-responses to INSL5 application in ex vivo brain slices and increased HFD and highly palatable liquid meal (HPM), but not chow, intake after intra-VMH INSL5 infusion. scRNAseq of hypothalamic RXFP4 neurons defined a cluster expressing VMH markers, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to nuclei implicated in hedonic feeding behaviour. Whole body chemogenetic inhibition (Di-DREADD) of Rxfp4-expressing cells, mimicking physiological INSL5-RXFP4 Gi-signalling, increased intake of HFD and HPM, but not chow, whilst activation (Dq-DREADD), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and motivation to work for HPM.These findings identify RXFP4VMH neurons as regulators of food intake and preference and reveal hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.
Molecular cancer therapeutics
2022 May 02
Martin, AL;Anadon, CM;Biswas, S;Mine, JA;Handley, KF;Payne, KK;Mandal, G;Chaurio, RA;Powers, JJ;Sprenger, KB;Rigolizzo, KE;Innamarato, P;Harro, CM;Mehta, S;Perez, BA;Wenham, RM;Conejo-Garcia, JR;
PMID: 35499393 | DOI: 10.1158/1535-7163.MCT-21-0872
Though chimeric antigen receptor (CAR) expressing T cells have proven success in hematologic malignancies, their effectiveness in solid tumors has been largely unsuccessful thus far. We found that some olfactory receptors are expressed in a variety of solid tumors of different histological subtypes, with a limited pattern of expression in normal tissues. Quantification of OR2H1 expression by RT-QPCR and western blot analysis of 17 normal tissues, 82 ovarian cancers of various histologies, 8 non-small cell lung cancers (NSCLC), and 17 breast cancers demonstrated widespread OR2H1 expression in solid epithelial tumors with expression in normal human tissues limited to the testis. CAR T cells recognizing the extracellular domain of the olfactory receptor OR2H1 were generated with a targeting motif identified through the screening of a phage display library and demonstrated OR2H1-specific cytotoxic killing in vitro and in vivo, using tumor cells with spontaneous expression of variable OR2H1 levels. Importantly, recombinant OR2H1 IgG generated with the VH/VL sequences of the CAR construct specifically detected OR2H1 protein signal in 60 human lung cancers, 40 ovarian carcinomas and 73 cholangiocarcinomas, at positivity rates comparable to mRNA expression and without OR2H1 staining in 58 normal tissues. CRISPR/Cas9-mediated ablation of OR2H1 confirmed targeting specificity of the CAR and the tumor-promoting role of OR2H1 in glucose metabolism. Therefore, T cells redirected against OR2H1-expressing tumor cells represent a promising therapy against a broad range of epithelial cancers, likely with an admissible toxicity profile.
Journal of the American Society of Cytopathology
2022 Apr 01
Jager, L;Felicelli, C;Alexiev, B;Samant, S;Johnson, D;
| DOI: 10.1016/j.jasc.2022.03.004
Introduction The presence of tumor cell anaplasia and multinucleation (A/M) in oropharyngeal squamous cell carcinoma (OPSCC) has recently been found to be associated with increased disease recurrence and poorer disease-specific survival, regardless of HPV status. We aim to study the detection of A/M in cytology specimens. Materials and Methods A comprehensive data search for all patients with OPSCC diagnosed and treated at Northwestern Memorial Hospital between January 2013 and April 2020. All cytology and histopathologic slides were reviewed for the presence of A/M in patients with both surgical resection or biopsy specimens and fine needle aspiration cytology of a metastatic site. Results 87 patients were identified with both surgical and cytology specimens available for review. A/M was identified in 21 cytology specimens and 14 surgical specimens. Cytologic A/M was seen in 11 of the 14 patients (78.5%) with corresponding histologic A/M and in 10 of the 73 patients (13.7%) without histologic A/M. Disease-specific survival was significantly worse in patients with cytologic A/M regardless of the presence of histologic A/M (P = 0.0064) and in patients with cytologic A/M only (P = 0.0271). In patients with p16 positive/HPV-associated carcinoma, disease-specific survival was significantly worse in patients with both histologic and cytologic A/M (P = 0.0305). Conclusions A/M can be reliably identified in cytology specimens among all the various stains and preparations irrespective of primary tumor histology. Identification of A/M on cytology specimens may indicate more aggressive clinical behavior and help guide patient management.
Skin Health and Disease
2023 Jan 29
Mattsson, J;Israelsson, E;Björhall, K;Yrlid, L;Thörn, K;Thorén, A;Toledo, E;Jinton, L;Öberg, L;Wingren, C;Tapani, S;Jackson, S;Skogberg, G;Lundqvist, A;Hendrickx, R;Cavallin, A;Österlund, T;Grimster, N;Nilsson, M;Åstrand, A;
| DOI: 10.1002/ski2.209
Background Janus Kinase (JAK) inhibition has recently demonstrated therapeutic efficacy in both restoring hair growth and resolving inflammation in Alopecia Areata (AA). These effects are dose dependent and mainly efficacious at ranges close to a questionable risk profile. Objectives We explored the possibility to separate the beneficial and adverse effects of JAK inhibition by selectively inhibiting JAK1 and thereby avoiding side effects associated with JAK2 blockade. Methods The C3H/HeJ mouse model of AA was used to demonstrate therapeutic efficacy in vivo with different regimens of a selection of JAK inhibitors in regards to systemic versus local drug exposure. Human peripheral blood lymphocytes were stimulated in vitro to demonstrate translation to the human situation. Results We demonstrate that selective inhibition of JAK1 produces fast resolution of inflammation and complete restoration of hair growth in the C3H/HeJ mouse model of AA. Furthermore, we show that topical treatment does not restore hair growth and that treatment needs to be extended well beyond that of restored hair growth in order to reach treatment-free remission. For translatability to human disease, we show that cytokines involved in AA pathogenesis are similarly inhibited by selective JAK1 and pan-JAK inhibition in stimulated human peripheral lymphocytes and specifically in CD8+ T cells. Conclusion This study demonstrates that systemic exposure is required for efficacy in AA and we propose that a selective JAK1 inhibitor will offer a treatment option with a superior safety profile to pan-JAK inhibitors for these patients.
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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 | |
| EnEm | Probe targets exons n and m | |
| En-Em | Probe 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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