Background Heterologous vaccine regimens have been widely discussed as a way to mitigate intermittent supply shortages and to improve immunogenicity and safety of COVID-19 vaccines. We aimed to assess the reactogenicity and immunogenicity of heterologous immunisations with ChAdOx1 nCov-19 (AstraZeneca, Cambridge, UK) and BNT162b2 (Pfizer-BioNtech, Mainz, Germany) compared with homologous BNT162b2 and ChAdOx1 nCov-19 immunisation. Methods This is an interim analysis of a prospective observational cohort study enrolling health-care workers in Berlin (Germany) who received either homologous ChAdOx1 nCov-19 or heterologous ChAdOx1 nCov-19-BNT162b2 vaccination with a 10-12-week vaccine interval or homologous BNT162b2 vaccination with a 3-week vaccine interval. We assessed reactogenicity after the first and second vaccination by use of electronic questionnaires on days 1, 3, 5, and 7. Immunogenicity was measured by the presence of SARS-CoV-2-specific antibodies (full spike-IgG, S1-IgG, and RBD-IgG), by an RBD-ACE2 binding inhibition assay (surrogate SARS-CoV-2 virus neutralisation test), a pseudovirus neutralisation assay against two variants of concerns (alpha [B.1.1.7] and beta [B.1.351]), and anti-S1-IgG avidity. T-cell reactivity was measured by IFN-γ release assay.

We determined the effect of inhaled corticosteroid, budesonide, on the release of the anti-inflammatory cytokine, interleukin-10 (IL-10), and of pro-inflammatory cytokines, macrophage inflammatory protein-1alpha (MIP-1alpha), interferon-gamma (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF), from blood monocytes and alveolar macrophages of mild asthmatic subjects in a double-blind, cross-over, placebo-controlled study. Budesonide reduced bronchial hyperresponsiveness and improved baseline FEV1. Alveolar macrophages were obtained by bronchoalveolar lavage performed at the end of each treatment phase. IL-10 from blood monocytes was not altered, but both IL-10 mRNA and protein expression from alveolar macrophages stimulated by lipopolysaccharide and IL-1beta were increased after corticosteroid therapy. By contrast, alveolar macrophages released significantly less MIP-1alpha, IFN-gamma, and GM-CSF after steroid treatment. In comparison to alveolar macrophages from normal nonasthmatic volunteers, those from asthmatic patients released more MIP-1alpha, IFN-gamma, and GM-CSF but lower amounts of IL-10 particularly at baseline and after IL-1beta stimulation. The ability of steroids to inhibit pro-inflammatory cytokines but to enhance the anti-inflammatory cytokine such as IL-10 may contribute to their beneficial actions in asthma. Asthma is characterized by alveolar macrophages exhibiting both an enhanced capacity to release pro-inflammatory cytokines and a reduced capacity to produce IL-10.

1 The cyclic AMP phosphodiesterases (PDE) expressed by CD4+ and CD8+ T-lymphocytes purified from the peripheral blood of normal adult subjects were identified and characterized, and their role in modulating proliferation and the biosynthesis of interleukin (IL)-2 and interferon (IFN)-y evaluated. 2 In lysates prepared from both subsets, SK&F 95654 (PDE3 inhibitor) and rolipram (PDE4 inhibitor) suppressed cyclic AMP hydrolysis indicating the presence of PDE3 and PDE4 isoenzymes in these cells. Differential centrifugation and subsequent inhibitor and kinetic studies revealed that the particulate fraction contained, predominantly, a PDE3 isoenzyme. In contrast, the soluble fraction contained a PDE4 (-65% of total activity) and, in addition, a novel enzyme that had the kinetic characteristics of the recently identified PDE7. 3 Reverse transcription-polymerase chain reaction (RT-PCR) studies with primer pairs designed to recognise unique sequences in the human PDE4 and PDE7 genes amplified cDNA fragments that corresponded to the predicted sizes of HSPDE4A, HSPDE4B, HSPDE54D and HSPDE7. No message was detected for HSPDE4C after 35 cycles of amplification. 4 Functionally, rolipram inhibited phytohaemagglutinin-(PHA) and anti-CD3-induced proliferation of CD4+ and CD8+ T-lymphocytes, and the elaboration of IL-2, which was associated with a three to four fold increase in cyclic AMP mass. In all experiments, however, rolipram was approximately 60 fold more potent at suppressing IL-2 synthesis than at inhibiting mitogenesis. In contrast, SK&F 95654 failed to suppress proliferation and cytokine generation, and did not elevate the cyclic AMP content in T-cells. Although inactive alone, SK&F 95654 potentiated the ability of rolipram to suppress PHA-and anti-CD3-induced T-cell proliferation, and PHA-induced IL-2 release. 5 When a combination of phorbol myristate acetate (PMA) and ionomycin were used as a co-mitogen, rolipram did not affect proliferation but, paradoxically, suppressed IL-2 release indicating that cyclic AMP can inhibit mitogenesis by acting at, or proximal to, the level of inositol phospholipid hydrolysis. 6 Collectively, these data suggest that PDE3 and PDE4 isoenzymes regulate the cyclic AMP content, IL-2 biosynthesis and proliferation in human CD4+ and CD8+ T-lymphocytes. However, the ability of rolipram to suppress markedly mitogen-induced IL-2 generation without affecting T-cell proliferation suggests that growth and division of T-lymphocytes may be governed by mediators in addition to IL-2. Finally, T-cells have the potential to express PDE7, although elucidating the functional role of this enzyme must await the development of selective inhibitors. Keywords: CD4+ T-lymphocytes; CD8+ T-lymphocytes; phosphodiesterase inhibitors; proliferation; interleukin-2 generation; phosphodiesterase 3; phosphodiesterase 4; phosphodiesterase 7 IntroductionAlthough glucocorticosteroids are considered the most effective agents currently available for the treatment of asthmatic inflammation, they are not without adverse effe...

Abstract-Endothelial hyperpermeability induced by inflammatory mediators is a hallmark of sepsis and adult respiratory distress syndrome. Increased levels of the regulatory peptide adrenomedullin (ADM) have been found in patients with systemic inflammatory response. We analyzed the effect of ADM on the permeability of cultured human umbilical vein endothelial cell (HUVEC) and porcine pulmonary artery endothelial cell monolayers. ADM dose-dependently reduced endothelial hyperpermeability induced by hydrogen peroxide (H 2 O 2 ), thrombin, and Escherichia coli hemolysin. Moreover, ADM pretreatment blocked H 2 O 2 -related edema formation in isolated perfused rabbit lungs and increased cAMP levels in lung perfusate. ADM bound specifically to HUVECs and porcine pulmonary artery endothelial cells and increased cellular cAMP levels. Simultaneous inhibition of cAMP-degrading phosphodiesterase isoenzymes 3 and 4 potentiated ADM-dependent cAMP accumulation and synergistically enhanced ADM-dependent reduction of thrombininduced hyperpermeability. However, ADM showed no effect on endothelial cGMP content, basal intracellular Ca 2ϩ levels, or the H 2 O 2 -stimulated, thrombin-stimulated, or Escherichia coli hemolysin-stimulated Ca 2ϩ increase. ADM diminished thrombin-and H 2 O 2 -related myosin light chain phosphorylation as well as stimulus-dependent stress fiber formation and gap formation in HUVECs, suggesting that ADM may stabilize the barrier function by cAMP-dependent relaxation of the microfilament system. These findings identify a new function of ADM and point to ADM as a potential interventional agent for the reduction of vascular leakage in sepsis and adult respiratory distress syndrome. (Circ Res. 2002;91:618-625.)Key Words: adrenomedullin Ⅲ cultured endothelial cells Ⅲ endothelial permeability Ⅲ endothelial barrier dysfunction T he incidence of sepsis and ensuing multiple organ failure has increased over the past two decades and has caused multiple deaths in intensive care units. The development of adult respiratory distress syndrome (ARDS) characterized by noncardiogenic pulmonary edema contributes substantially to a fatal outcome. 1 Increased microvascular permeability is a hallmark of an inflammatory reaction, including ARDSrelated pulmonary edema formation. Circumstantial evidence has suggested that endothelial hyperpermeability is related to alterations of the cellular cytoskeleton. 2 Endothelial cells have been shown to contain an elaborate microfilament system allowing active actin-and myosin-based cell contraction. Activation of cell contraction and disturbance of junctional organization subsequently result in the induction of interendothelial gaps followed by enhanced paracellular endothelial permeability. [2][3][4][5][6] Major initiators of this process are polymorphonuclear leukocyte-derived oxygen metabolites, 7-9 pore-forming bacterial exotoxins, 9,10 and endogenous proinflammatory mediators, such as thrombin. 5,9,11 Exposure of endothelial cells to hydrogen peroxide (H 2 O 2 ) results in the activat...

The two cyclooxygenase (COX) isoforms convert arachidonic acid to precursor prostaglandins (PGs). Upregulation of COX-2 is responsible for increased PG production in inflammation and is antagonized by corticosteriods such as dexamethasone. In human pulmonary A549 cells, interleukin-1␤ (IL-1␤) increases prostaglandin E 2 (PGE 2 ) synthesis via dexamethasone-sensitive induction of COX-2. Nuclear run-off assays showed that COX-2 transcription rate was repressed 25-40% by dexamethasone, while PGE 2 release, COX activity, and COX-2 protein were totally repressed. At the mRNA level, complete repression of COX-2 was only observed at later (6 h) time points. Preinduced COX-2 mRNA was also potently repressed by dexamethasone, yet suppression of transcription by actinomycin D showed little effect. This dexamethasone-dependent repression involved a reduced COX-2 mRNA half-life, was blocked by actinomycin D or cycloheximide, and was antagonized by the steroid antagonist RU38486. Repression of IL-1␤-induced PGE 2 release, COX activity, and COX-2 protein by actinomycin D was only effective within the first hour following IL-1␤ treatment, while dexamethasone was effective when added up to 10 h later, suggesting a functional role for post-transcriptional mechanisms of repression. Following dexamethasone treatment, shortening of the average length of COX-2 mRNA poly(A) tails was observed. Finally, ligation of the COX-2 3-UTR to a heterologous reporter failed to confer dexamethasone sensitivity. In conclusion, these data indicate a major role for post-transcriptional mechanisms in the dexamethasone-dependent repression of COX-2 that require de novo glucocorticoid receptor-dependent transcription and translation. This mechanism involves shortening of the COX-2 poly(A) tail and requires determinants other than just the 3-UTR for specificity.

Background: Reliable point-of-care (POC) diagnostics not requiring laboratory infrastructure could be a game changer in the COVID-19 pandemic, particularly in the Global South. We assessed performance, limit of detection (LOD) and ease-of-use of three antigen-detecting, rapid POC diagnostics (Ag-RDT) for SARS-CoV-2. Methods: This prospective, multi-centre diagnostic accuracy study, recruited participants suspected to have SARS-CoV2 in Germany and UK. Paired nasopharyngeal swabs (NP) or NP and/or oropharyngeal swabs (OP) were collected from participants (one for clinical real-time reverse transcription polymerase chain reaction (RT-PCR) and one for Ag-RDT testing). Performance of each of three Ag-RDTs was compared to RT-PCR overall, and according to predefined subcategories e.g. cycle threshold (CT)-value, days from symptom onset, etc. In addition, limited verification of analytical limit-of-detection (LOD) was determined. To understand the usability of each Ag-RDT a System Usability Scale (SUS) questionnaire and ease-of-use assessment were performed. Results: Between April 17th and August 25th, 2020, 2417 participants were enrolled, with 70 (3.0%) testing positive by RT-PCR. The best-performing test (SD Biosensor, Inc. STANDARD Q) was 76.6% [95% Confidence Interval (CI) 62.8-86.4] sensitive and 99.3% [CI 98.6-99.6] specific. A sub-analysis showed all samples with RT-PCR CT-values <25 were detectable by STANDARD Q. The test was considered easy-to-use (SUS 86/100) and suitable for POC. Bioeasy and Coris showed specificity of 93.1% [CI 91.0%-94.8%] and 95.8% [CI 93.4%-97.4%], respectively, not meeting the predefined target of ≥98%. Conclusion: There is large variability in performance of Ag-RDT tests with one test showing promise. Given the usability at POC, these tests are likely to have impact despite imperfect sensitivity; however further research and modelling are needed.

The pleiotropic cytokine tumor necrosis factor-␣ (TNF-␣) and thrombin lead to increased endothelial permeability in sepsis. Numerous studies demonstrated the significance of intracellular cyclic nucleotides for the maintenance of endothelial barrier function. Actions of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are terminated by distinct cyclic nucleotide phosphodiesterases (PDEs). We hypothesized that TNF-␣ could regulate PDE activity in endothelial cells, thereby impairing endothelial barrier function. In cultured human umbilical vein endothelial cells (HUVECs), we found a dramatic increase of PDE2 activity following TNF-␣ stimulation, while PDE3 and PDE4 activities remained unchanged. Significant PDE activities other than PDE2, PDE3, and PDE4 were not detected. TNF-␣ increased PDE2 expression in a p38 mitogen-activated protein kinase (MAPK)-dependent manner. Endothelial barrier function was investigated in HUVECs and in isolated mice lungs. Selective PDE2 up-regulation sensitized HUVECs toward the permeability-increasing agent thrombin. In isolated mice lungs, we demonstrated that PDE2 inhibition was effective in preventing thrombin-induced lung edema, as shown with a reduction in both lung wet-to-dry ratio and albumin flux from the vascular to bronchoalveolar compartment. Our findings suggest that TNF-␣-mediated upregulation of PDE2 may destabilize endothelial barrier function in sepsis. Inhibition of PDE2 is therefore of potential therapeutic interest in sepsis and acute respiratory distress syndrome (ARDS). IntroductionSepsis-the systemic inflammatory response to infection-is the most common cause of death among patients in noncoronary intensive care units. 1 Laboratory markers of inflammation include high circulating levels of tumor necrosis factor ␣ (TNF-␣) and other cytokines, as well as activation of the coagulation cascade. 1 Endothelial hyperpermeability is a hallmark of sepsis. TNF-␣ increases endothelial cell permeability 2 with subsequent vascular leakage contributing to severe organ dysfunction such as adult respiratory distress syndrome (ARDS). 3 Half of the sepsis patients develop disseminated intravascular coagulation 2,4 due to a shift toward a procoagulant state with excessive thrombin and fibrin generation. 1 On a cellular level, thrombin changes the shape of endothelial cells and increases endothelial permeability, 5 thereby impairing endothelial barrier function synergistically with TNF-␣. 1,6 Thrombin, the main effector protease of the coagulation cascade, activates endothelial cells directly via protease-activated receptor 1 (PAR1) and PAR4, 7 thereby inducing hyperpermeability and promoting adhesion of platelets and leukocytes, as well as secretion of plateletactivating factor (PAF) and inflammatory cytokines. 8 After binding to one of the various receptors, TNF-␣ mediates activation of diverse signaling pathways, such as the p38 mitogenactivated protein kinase (p38 MAPK), JUN N-terminal kinase (JNK), and nuclear factor kappa B (NF-B). TNF recepto...

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