Cytokine expression by CD163+ monocytes in healthy and Actinobacillus pleuropneumoniae-infected pigs

Distinct monocyte subpopulations have been previously described in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae (APP). The CD163+ subpopulation of bone marrow (BM), peripheral blood (PB) and lung monocytes was found to play an important role in the inflammatory process. The inflammation is accompanied by elevation of inflammatory cytokines. The aim of the study was to evaluate the contribution of CD163+ monocytes and macrophages to cytokine production during APP-induced lung inflammation. Cytokine production was assessed by flow cytometry (FC) and quantitative PCR (qPCR) in CD163+ monocytes and by qPCR, immunohistochemistry/fluorescence in lungs and tracheobronchial lymph nodes (TBLN). Despite the systemic inflammatory response after APP infection, BM and PB CD163+ monocytes did not express elevated levels of a wide range of cytokines compared to control pigs. In contrast, significant amounts of IL-1β, IL-6, IL-8 and TNF-α were produced in lung lesions and IL-1β in the TBLN. At the protein level, TNF-α was expressed by both CD163+ monocytes and macrophages in lung lesions, whereas IL-1β, IL-6 and IL-8 expression was found only in CD163+ monocytes; no CD163+ macrophages were found to produce these cytokines. Furthermore, the quantification of CD163+ monocytes expressing the two cytokines IL-1β and IL-8 that were most elevated was performed. In lung lesions, 36.5% IL-1β positive CD163+ monocytes but only 18.3% IL-8 positive CD163+ monocytes were found. In conclusion, PB and BM CD163+ monocytes do not appear to contribute to the elevated cytokine levels in plasma. On the other hand, CD163+ monocytes contribute to inflammatory cytokine expression, especially IL-1β at the site of inflammation during the inflammatory process.Peer reviewe

N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

The interleukin-1α precursor suppresses hypersensitivity of <i>snf1</i>Δ strain to 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of the His3 imidazoleglycerol-phosphate dehydratase.

<p>This suppressive property of pre-IL-1alpha (pre) is more profound on SD media containing glycerol/ethanol as a carbon source (SDgly) in case of the <i>snf1-108</i> strain carrying incomplete <i>SNF1</i> deletion. Mature interleukin-1alpha (Mat) is not able to rescue the 3-AT hypersensitivity of both <i>snf1</i>Δ and <i>snf1-108</i> strains and was used as a control. We did not observe any differences in growth between strains producing pre-IL-1alpha and mature IL-1alpha on SD agar plates which did not contain and/or contain only a minute amount of 3-AT. This is an example of 3 independent biological experiments.</p

Modified Strategies for Invasive Management of Acute Coronary Syndrome during the COVID-19 Pandemic

The COVID-19 pandemic presents several challenges for managing patients with acute coronary syndrome (ACS). Modified treatment algorithms have been proposed for the pandemic. We assessed new algorithms proposed by The European Association of Percutaneous Cardiovascular Interventions (EAPCI) and the Acute Cardiovascular Care Association (ACCA) on patients with ACS admitted to the hospital during the COVID-19 pandemic. The COVID-19 period group (CPG) consisted of patients admitted into a high-volume centre in Prague between 1 February 2020 and 30 May 2020 (n = 181). The reference group (RG) included patients who had been admitted between 1 October 2018 and 31 January 2020 (n = 834). The proportions of patients with different types of ACS admitted before and during the pandemic did not differ significantly: in all ACS patients, KILLIP III-IV class was present in 13.9% in RG and in 9.4% of patients in CPG (p = 0.082). In NSTE-ACS patients, the ejection fraction was lower in the CPG than in the RG (44.7% vs. 50.7%, respectively; p &lt; 0.001). The time from symptom onset to first medical contact did not differ between CPG and RG patients in the respective NSTE-ACS and STEMI groups. The time to early invasive treatment in NSTE-ACS patients and the time to reperfusion in STEMI patients were not significantly different between the RG and the CPG. In-hospital mortality did not differ between the groups in NSTE-ACS patients (odds ratio in the CPG 0.853, 95% confidence interval (CI) 0.247 to 2.951; p = 0.960) nor in STEMI patients (odds ratio in CPG 1.248, 95% CI 0.566 to 2.749; p = 0.735). Modified treatment strategies for ACS during the COVID-19 pandemic did not cause treatment delays. Hospital mortality did not differ

The SAGA and ADA complex subunits co-purify as a part of the IL-1α precursor-binding complex.

<p>Co-immunoprecipitation experiments with an anti-Flag antibody using yeast strains from a TAP tag library transformed with IL-1α expression vectors revealed that both of the HAT complexes bound pre-IL-1α (pre) but not mature IL-1α (Mat). Control cells (ctrl) carry the empty plasmid pYX212. Western blotting was performed using an anti-CBP antibody that recognizes the TAP tag at the C-terminus of the respective HAT complex subunits. For each line of the IP experiment, 1.4 mL of the cell lysate prepared from 5.10⁸ yeast cells in average was used. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.</p

Disruption of the SAGA and ADA complexes confirmed binding of the IL-1α precursor to the HAT/Core module and suggested the mutually exclusive role of Spt7 and Ahc1 in SAGA complex assembly.

<p>Co-immunoprecipitation was performed using yeast lysates with an anti-Flag antibody that recognizes the Flag tag at the N-terminus of the IL-1α precursor. Western blotting was performed with an anti-CBP antibody which identifies the TAP tag at the C-terminus of the respective HAT complex subunits. (A) Gcn5 does not bind to pre-IL-1α, and because it is not required for SAGA or ADA complex integrity, its deletion has no effect on Ahc1 or Spt8 co-immunoprecipitation with the IL-1α precursor (pre). Deletion of the AHC2 gene doesn’t impair co-IP of pre-IL-1α with Gcn5, Spt8 and Spt7. (B) The disruption of the ADA HAT complex did not affect the co-immunoprecipitation of Gcn5 and Spt8 with IL-1α. However, the interaction between Spt7 and the IL-1α precursor was significantly weakened. In experiments with TAP/Spt7,<i>ahc1</i>Δ strain, we received either no or very low signal (the latter is depicted) of TAP-tagged Spt7, with a success rate 3∶1, respectively. (C) The disruption of the SAGA complex abolished the interaction between Spt8 and the IL-1α precursor but had no effect on Ahc1 binding to the IL-1α precursor. Control cells (ctrl) carry the empty plasmid pYX212. For each line of the IP experiment, 3.5 mL of cell lysate prepared from 20.10⁸ yeast cells in average was used, except TAP/Spt8,<i>gcn5</i>Δ, TAP/Spt8,<i>ahc1</i>Δ, TAP/Spt8,<i>spt7</i>Δ and TAP/Gcn5,<i>ahc1</i>Δ strains, where 1.3 mL of cell lysates from 9.10⁸ yeast cells each were applied. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.</p

Subcellular localization of pre-IL-1α and IL-1αMat in <i>Saccharomyces cerevisiae.</i>

<p>The IL-1α precursor (pre-IL-1α) is exclusively localized in the nucleus of yeast cells, which is in contrast to the observed cytoplasmic localization of mature IL-1α (IL-1αMat). Control cells (ctrl) carry the empty pUG36 vector. The cell nuclei are stained with DAPI.</p

Comparison of routine contrast‑enhanced computed tomography with late gadolinium enhancement cardiac magnetic resonance imaging in the detection of myocardial pathology

Bacground: Cardiac magnetic resonance imaging (MRI) represents the gold standard in noninvasive evaluation of myocardial tissue. However, some patients are unable to undergo cardiac MRI due to a variety of reasons. Aims: We sought to determine the diagnostic accuracy of routinely performed contrast‑enhanced computed tomography (CECT) compared with cardiac MRI in the evaluation of myocardial tissue. Methods: We retrospectively evaluated 96 consecutive patients (mean [SD] age, 51 [15] years; 41 women) who underwent both CECT and cardiac MRI within 30 days. All CECT scans that visualized the entire heart were analyzed, regardless of the indication for and protocol of the procedure. The presence of late gadolinium enhancement on cardiac MRI was compared with the finding of myocardial hypoattenuation on computed tomography scans. Results: With cardiac MRI as the gold standard, CECT revealed a per‑patient sensitivity of 66%, specificity of 89%, positive predictive value of 75%, negative predictive value of 84%, and accuracy of 81%. Per‑segment sensitivity was 54%; specificity, 98%; positive predictive value, 76%; negative predictive value, 94%; and accuracy, 92%. Conclusions: Our study suggests that routinely performed CECT has high specificity, but only moderate sensitivity, compared with cardiac MRI in the evaluation of myocardial tissue. This result supports the recommendation that all CECT scans that visualize the entire heart should be analyzed for myocardial tissue pathology

Nucleotide sequences of the primers used for the <i>loxP-kanMX-loxP</i> and <i>loxP-Leu2-loxP</i> gene disruption cassette amplification.

<p>Nucleotide sequences of the primers used for the <i>loxP-kanMX-loxP</i> and <i>loxP-Leu2-loxP</i> gene disruption cassette amplification.</p