Noncoding RNA MaIL1 is an integral component of the TLR4-TRIF pathway.

RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin-proteasome system, our approach unveiled ncRNA MaIL1 as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification-mass spectrometry (RAP-MS) revealed MaIL1 binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. MaIL1 binding stabilized OPTN, and consequently, loss of MaIL1 blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. MaIL1 expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers MaIL1 as an integral RNA component of the TLR4-TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.

Diagnostic imaging in acute interstitial pneumonia in foals: High variability of interpretation of chest radiographs and good conformity between ultrasonographic and post-mortem findings.

Acute interstitial pneumonia (AIP) in foals is a rare but challenging syndrome. Diagnostic imaging is crucial for its diagnosis. While there are some reports on radiographic findings, ultrasound is sparsely described. Variability in the evaluation of radiographs in AIP has been well described in human literature. Part one of this study is a prospective, observational, observer agreement study investigating inter- and intraobserver agreement in the assessment of thoracic radiographs. Part two is a prospective, controlled, descriptive study on thoracic ultrasonography in foals with AIP. Eighteen foals with AIP were examined daily by thoracic ultrasound. Thoracic radiographs were taken on three successive occasions. Blinded radiographs were assessed by three observers based on two semi-quantitative scores (pattern recognition, subjective evaluation). Foals that died underwent postmortem examination (n = 9); postmortem findings were compared to ultrasound findings on the day of death. Ultrasonographic findings were consistent with postmortem findings. Comet tail scores in foals with AIP were significantly higher (p < 0.0001) than in control foals. Interrater agreement for the assessment of radiographs was none to moderate (κ = 0.07-0.65) for pattern recognition and weak to moderate (κ = 0.58-0.62) for subjective scoring. Intrarater agreement varied from minimal to strong (κ = 0.30-0.80) for pattern recognition but was strong (κ = 0.83) for subjective scoring. In conclusion, the diagnostic value of thoracic ultrasound in foals with AIP is high due to good conformity with postmortem findings. The evaluation of thoracic radiographs showed high variability in inter- and intra-agreement.

Cyclophilin inhibitors restrict Middle East respiratory syndrome coronavirus via interferon-λ in vitro and in mice.

While severe coronavirus infections, including Middle East respiratory syndrome coronavirus (MERS-CoV), cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use.We elucidated the molecular mechanisms by which the cyclophilin inhibitors cyclosporin A (CsA) and alisporivir (ALV) restrict MERS-CoV to validate their suitability as readily available therapy in MERS-CoV infection.Calu-3 cells and primary human alveolar epithelial cells (hAECs) were infected with MERS-CoV and treated with CsA or ALV or inhibitors targeting cyclophilin inhibitor-regulated molecules including calcineurin, nuclear factor of activated T-cells (NFATs) or mitogen-activated protein kinases. Novel CsA-induced pathways were identified by RNA sequencing and manipulated by gene knockdown or neutralising antibodies. Viral replication was quantified by quantitative real-time PCR and 50% tissue culture infective dose. Data were validated in a murine MERS-CoV infection model.Both CsA and ALV reduced MERS-CoV titres and viral RNA replication in Calu-3 cells and hAECs, improving epithelial integrity. While neither calcineurin nor NFAT inhibition reduced MERS-CoV propagation, blockade of c-Jun N-terminal kinase diminished infectious viral particle release but not RNA accumulation. Importantly, CsA induced interferon regulatory factor 1 (IRF1), a pronounced type III interferon (IFNλ) response and expression of antiviral genes. Downregulation of IRF1 or IFNλ increased MERS-CoV propagation in the presence of CsA. Importantly, oral application of CsA reduced MERS-CoV replication in vivo, correlating with elevated lung IFNλ levels and improved outcome.We provide evidence that cyclophilin inhibitors efficiently decrease MERS-CoV replication in vitro and in vivo via upregulation of inflammatory antiviral cell responses, in particular IFNλ. CsA might therefore represent a promising candidate for treating MERS-CoV infection.

Neutralizing Complement C5a Protects Mice with Pneumococcal Pulmonary Sepsis.

BACKGROUND: Community-acquired pneumonia and associated sepsis cause high mortality despite antibiotic treatment. Uncontrolled inflammatory host responses contribute to the unfavorable outcome by driving lung and extrapulmonary organ failure. The complement fragment C5a holds significant proinflammatory functions and is associated with tissue damage in various inflammatory conditions. The authors hypothesized that C5a concentrations are increased in pneumonia and C5a neutralization promotes barrier stabilization in the lung and is protective in pneumococcal pulmonary sepsis. METHODS: The authors investigated regulation of C5a in pneumonia in a prospective patient cohort and in experimental pneumonia. Two complementary models of murine pneumococcal pneumonia were applied. Female mice were treated with NOX-D19, a C5a-neutralizing L-RNA-aptamer. Lung, liver, and kidney injury and the inflammatory response were assessed by measuring pulmonary permeability (primary outcome), pulmonary and blood leukocytes, cytokine concentrations in lung and blood, and bacterial load in lung, spleen, and blood, and performing histologic analyses of tissue damage, apoptosis, and fibrin deposition (n = 5 to 13). RESULTS: In hospitalized patients with pneumonia (n = 395), higher serum C5a concentrations were observed compared to healthy subjects (n = 24; 6.3 nmol/l [3.9 to 10.0] vs. 4.5 nmol/l [3.8 to 6.6], median [25 to 75% interquartile range]; difference: 1.4 [95% CI, 0.1 to 2.9]; P = 0.029). Neutralization of C5a in mice resulted in lower pulmonary permeability in pneumococcal pneumonia (1.38 ± 0.89 vs. 3.29 ± 2.34, mean ± SD; difference: 1.90 [95% CI, 0.15 to 3.66]; P = 0.035; n = 10 or 11) or combined severe pneumonia and mechanical ventilation (2.56 ± 1.17 vs. 7.31 ± 5.22; difference: 4.76 [95% CI, 1.22 to 8.30]; P = 0.011; n = 9 or 10). Further, C5a neutralization led to lower blood granulocyte colony-stimulating factor concentrations and protected against sepsis-associated liver injury. CONCLUSIONS: Systemic C5a is elevated in pneumonia patients. Neutralizing C5a protected against lung and liver injury in pneumococcal pneumonia in mice. Early neutralization of C5a might be a promising adjunctive treatment strategy to improve outcome in community-acquired pneumonia.

Human alveolar progenitors generate dual lineage bronchioalveolar organoids.

Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280+/EpCAM+ population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3ß inhibitor, CHIR99021. By RNA-seq profiling of GSK-3ß knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction.

Subcutaneous seeding following surgical excision of an intracranial meningioma in a cat.

Traditionally patient owners express their concerns that surgical or diagnostic procedures on a tumor may induce metastasis. In pets, this has been documented in only very rare occasions, e. g. needle path metastases after diagnostic fine needle biopsies of urinary bladder or prostatic tumors. Here, we describe a case of subcutaneous seeding of a feline intracranial grade 1 meningioma 6 months after surgical resection. A 10-year-old male neutered domestic shorthaired cat with typical neurological signs was diagnosed with an extra-axial contrast enhancing mass in the dorsal frontotemporal lobes using magnetic resonance imaging (MRI). Transfronto-parietal bone craniotomy was performed and the 24 × 19 × 22 mm large tumor was largely removed. Tumor recurrence after 12 months resulted in a second surgical tumor removal. In addition, 2 subcutaneous masses of 10 × 4 × 4 mm in size were removed at the site of the original surgical site which were fully separated from the recurring meningeal tumor by the intact frontal bone. Histology and immunohistochemistry suggested the same tumor growth in all 4 masses. Most likely the tumor seeding had been caused during the first surgery. After all, the risk of surgical seeding of a benign tumor seems very low.

Acute interstitial pneumonia in foals: A severe, multifactorial syndrome with lung tissue recovery in surviving foals.

BACKGROUND: Acute interstitial pneumonia in foals has been sparsely described in literature, and the individual authors disagree on the underlying aetiology. Histopathological follow-up from surviving foals is not available. OBJECTIVES: Description of clinical and histopathological findings in the course of acute interstitial pneumonia and in recovery. Investigating the aetiology and possible triggering factors of acute interstitial pneumonia. STUDY DESIGN: Case series. METHODS: Post-mortem examination of nine affected foals; seven died during the acute phase, and two had recovered from acute interstitial pneumonia. Data from clinical examinations on the day of death were recorded for all foals. Complete necropsy, special histological staining, virological and microbiological examinations were performed. RESULTS: Seven foals died during the acute phase with severe respiratory distress, fever and increased numbers of comet tail artefacts in lung ultrasound. In post-mortem examination, a wide variety of possible triggering factors was identified. Microbiology revealed Escherichia coli, Rhodococcus equi and Klebsiella pneumoniae as the most common bacterial pathogens. Equine herpesvirus 2 was detected in all foals by PCR. Those with high viral loads also displayed histopathological changes suggestive of viral infections. Pneumocystis carinii was detected in all acutely affected foals. Histopathological changes in lung parenchyma clearly differed between the foals that had recovered from acute interstitial pneumonia and those dying in the acute phase. While lungs in acute phase featured marked parenchymal collapse and necrosis, the recovered foals revealed nearly normal ventilated lung parenchyma and alveolar structure. MAIN LIMITATIONS: Small number of cases. Because all foals are from the same breeding farm with endemic occurrence of pathogens, it is not certain whether the results of this study can be transferred to other foals without restrictions. CONCLUSIONS: Acute interstitial pneumonia seems to be based on a multifactorial aetiology. Lungs from foals that have survived acute interstitial pneumonia appear to be able to regenerate completely, leaving no permanent changes.

Fatal Neurotoxicosis in Dogs Associated with Tychoplanktic, Anatoxin-a Producing Tychonema sp. in Mesotrophic Lake Tegel, Berlin.

In May 2017, at least 12 dogs showed signs of acute neurotoxicosis after swimming in or drinking from Lake Tegel, a mesotrophic lake in Berlin, Germany, and several of the affected dogs died shortly afterwards despite intensive veterinary treatment. Cyanobacterial blooms were not visible at the water surface or the shorelines. However, detached and floating water moss (Fontinalis antipyretica) with high amounts of Tychonema sp., a potential anatoxin-a (ATX) producing cyanobacterium, was found near the beaches where the dogs had been swimming and playing. Necropsies of two of the dogs revealed no specific lesions beside the anamnestic neurotoxicosis. ATX was detected in concentrations up to 8700 µg L-1 in the stomach contents, while other (neuro)toxic substances were not found. In the aqueous fraction of Fontinalis/Tychonema clumps sampled after the casualties, ATX was found in concentrations up to 1870 µg L-1. This is the first report of a dense population of Tychonema sp. in stands of Fontinalis resulting in high ATX contents. This case emphasizes the need for further investigation of potentially toxic, non-bloom forming cyanobacteria in less eutrophic water bodies and underlines the novel challenge of developing appropriate surveillance schemes for respective bathing sites.