Which factors are associated with COVID-19 infection incidence in care services for older people in Nordic countries? A cross-sectional survey.

AIMS: To investigate the differences between Sweden, Denmark, Finland and Norway regarding residential/home care units' and frontline managers' background factors, the resources allocated and measures taken during the initial phases of the COVID-19 pandemic, and whether and how these differences were associated with COVID-19 among older people in residential/home units. METHODS: Register- and survey-based data. Responses from managers in municipal and private residential/home units. Number of municipal COVID-19 cases from national registries. Multilevel logistic multivariate regression analysis with presence of COVID-19 among older people in residential/home units as the outcome variable. RESULTS: The proportions of residential/home units with client COVID-19 cases, mid-March-April 2020 were Denmark 22.7%, Finland 9.0%, Norway 9.7% and Sweden 38.8%, most cases found in clusters. The proportions were similar among employees. Client likelihood of having COVID-19 was six-fold higher if the employees had COVID-19. Mean client cases per residential/home unit were Denmark 0.78, Finland 0.46, Norway 0.22 and Sweden 1.23. For the same municipal infection incidence class, Sweden's mean client infection levels were three-fold those of other countries. The regression analysis variables country, municipal COVID-19 incidence proportion, and care type were associated with client cases at p ⩽ .001. Compared with Denmark, the odds ratios (ORs) for Sweden, Norway and Finland were 1.86, 0.41 and 0.35 respectively. The variable difficulties in preventive testing had an OR of 1.56, p ⩽ .05. CONCLUSIONS: Municipal COVID-19 incidence, employee cases, and the lack of testing resources somewhat explained the confirmed COVID-19 cases among older people in residential/home units. A two- to five-fold unexplained inter-country difference in ORs in the multivariate analyses was notable. The level of protection of vulnerable older clients in municipal and private residential/home units differed between the included countries.

Cytokine Autoantibodies Are Associated with Infection Risk and Self-Perceived Health: Results from the Danish Blood Donor Study.

The presence of naturally occurring cytokine-specific autoantibodies (c-aAb) in humans is well established, as well as associations to selected pathologies. However, the overall influence of c-aAb on immunocompetence remains largely unknown. In this paper, we performed a large-scale investigation of c-aAb association with infection risk. A cohort of healthy Danish blood donors was screened for c-aAb against IL-1α, IL-6, IL-10, IFNα, and GM-CSF using a Luminex-based multiplex assay, and results were linked to data from the Danish National Prescription Registry. The filing of an antimicrobial prescription following c-aAb measurement was used as a proxy for impaired immunocompetence. We found that c-aAb against pro-inflammatory cytokines IFNα and GM-CSF tended to associate with increased risk of prescription filings in women, whereas antibodies against anti-inflammatory IL-10 were associated with a lower predicted risk of antimicrobial prescriptions, as well as higher self-perceived health scores. We also observed an association of cumulative c-aAb presence with prescription risk. Our data show that cytokine autoantibodies in healthy individuals associate with various proxies for immunomodulation, with the exact association dependent on the pattern of pro- or anti-inflammatory cytokines targeted. This suggests that c-aAb may express cytokine-modulatory properties in healthy individuals and may be critical to further investigate as biomarkers of immunodeficiency.

IL-10-specific autoantibodies predict major adverse cardiovascular events in kidney transplanted patients - a retrospective cohort study.

End-stage renal failure is associated with persistent systemic inflammation. The aim of this study was to investigate if systemic inflammation at the time of kidney transplantation is linked to poor graft survival, major adverse cardiovascular events (MACE), and increased mortality, and if these processes are modulated by naturally occurring cytokine-specific autoantibodies (c-aAbs), which have been shown to regulate cytokine activity in vitro. Serum levels of cytokines, high-sensitivity C-reactive protein (hsCRP) and c-aAbs specific for interleukin (IL)-1α, tumor necrosis factor (TNF)-α, IL-6, and IL-10 were measured at the time of transplantation in a retrospective cohort study of 619 kidney transplanted patients with a median follow-up of 4.9 years (range 1.2-8.2 years). Systemic inflammation was associated with all-cause mortality in simple and multiple Cox regression analyses. IL-10-specific c-aAbs were associated with MACE after transplantation, suggesting that IL-10 may be a protective factor. Similarly, patients with a history of MACE before transplantation had lower levels of TNF-α-specific c-aAbs, hence we hypothesized that TNF may be a risk factor of MACE. These findings support that pro-inflammatory activity before transplantation is a pathological driver of MACE and all-cause mortality after transplantation. This information adds to pretransplantation risk estimation in renal transplant candidates.

Identified metabolic signature for assessing red blood cell unit quality is associated with endothelial damage markers and clinical outcomes.

BACKGROUND: There has been interest in determining whether older red blood cell (RBC) units have negative clinical effects. Numerous observational studies have shown that older RBC units are an independent factor for patient mortality. However, recently published randomized clinical trials have shown no difference of clinical outcome for patients receiving old or fresh RBCs. An overlooked but essential issue in assessing RBC unit quality and ultimately designing the necessary clinical trials is a metric for what constitutes an old or fresh RBC unit. STUDY DESIGN AND METHODS: Twenty RBC units were profiled using quantitative metabolomics over 42 days of storage in SAGM with 3- to 4-day time intervals. Metabolic pathway usage during storage was assessed using systems biology methods. The detected time intervals of the metabolic states were compared to clinical outcomes. RESULTS: Using multivariate statistics, we identified a nonlinear decay process exhibiting three distinct metabolic states (Days 0-10, 10-17, and 17-42). Hematologic variables traditionally measured in the transfusion setting (e.g., pH, hemolysis, RBC indices) did not distinguish these three states. Systemic changes in pathway usage occurred between the three states, with key pathways changing in both magnitude and direction. Finally, an association was found between the time periods of the metabolic states with the clinical outcomes of more than 280,000 patients in the country of Denmark transfused over the past 15 years and endothelial damage markers in healthy volunteers undergoing autologous transfusions. CONCLUSION: The state of RBC metabolism may be a better indicator of cellular quality than traditional hematologic variables.

Enzyme-Activatable Cell-Penetrating Peptides through a Minimal Side Chain Modification.

Activatable cell-penetrating peptides are of great interest in drug delivery because of their enhanced selectivity which can be controlled by the external stimuli that trigger their activation. The use of a specific enzymatic reaction to trigger uptake of an inert peptide offers a relevant targeting strategy because the activation process takes place in a short time and only in areas where the specific cell surface enzyme is present. To this aim, the lysine side chain of Tat peptides was modified with an enzyme-cleavable domain of minimal size. This yielded blocked Tat-peptides which were inactive but that could be activated by coincubation with the selected enzymes.

Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism.

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies.

Evaluation of the quality of blood components obtained after automated separation of whole blood by a new multiunit processor.

BACKGROUND: The Reveos system (Terumo BCT) is a fully automated device able to process four whole blood (WB) units simultaneously into a plasma unit, a red blood cell (RBC) unit, and an interim platelet (PLT) unit (IPU). Multiple IPUs can be pooled to form a transfusable PLT product. The aim of our study was to evaluate the quality of components made with the Reveos system from either fresh (2-8 hr) or overnight-held WB. STUDY DESIGN AND METHODS: A prototype of the Reveos system was used to process WB. RBCs were resuspended in SAGM, leukoreduced, and assayed for in vitro quality variables during a 42-day storage period at 2 to 6 °C. Twenty-four-hour in vivo recovery was determined on Day 42. Plasma was assayed for cellular contamination and activation variables. IPUs were pooled with SSP+ additive solution for in vitro quality assessments during a 7-day storage period at room temperature. RESULTS: Reveos-produced RBCs and plasma units met the predefined requirements. RBC recovery was superior to control units. On Day 42, hemolysis was below 0.8% and in vivo recovery was above 75% for all RBCs. Cellular contamination was lower for Reveos-produced plasma. PLT yield was higher with overnight-stored WB. PLT quality was well maintained during storage with no significant differences between the two groups. CONCLUSION: Blood components prepared with the Reveos from fresh or overnight-held WB meet quality criteria without any relevant difference between the two groups. The Reveos system has the potential to increase efficacy and standardization of blood component preparation.

Quantitative live-cell imaging of lipidated peptide transport through an epithelial cell layer.

Oral drug delivery increases patient compliance and is thus the preferred administration route for most drugs. However, for biologics the intestinal barrier greatly limits the absorption and reduces their bioavailability. One strategy employed to improve on this is chemical modification of the biologic through the addition of lipid side chains. While it has been established that lipidation of peptides can increase transport, a mechanistic understanding of this effect remains largely unexplored. To pursue this mechanistic understanding, end-point detection of biopharmaceuticals transported through a monolayer of fully polarized epithelial cells is typically used. However, these methods are time-consuming and tedious. Furthermore, most established methods cannot be combined easily with high-resolution live-cell fluorescence imaging that could provide a mechanistic insight into cellular uptake and transport. Here we address this challenge by developing an axial PSF deconvolution scheme to quantify the transport of peptides through a monolayer of Caco-2 cells using single-cell analysis with live-cell confocal fluorescence microscopy. We then measure the known cross-barrier transport of several compounds in our model and compare the results with results obtained in an established microfluidic model finding similar transport phenotypes. This verifies that already after two days the Caco-2 cells in our model form a tight monolayer and constitute a functional barrier model. We then apply this assay to investigate the effects of side chain lipidation of the model peptide drug salmon calcitonin (sCT) modified with 4­carbon and 8­carbon-long fatty acid chains. Furthermore, we compare that with experiments performed at lower temperature and using inhibitors for some endocytotic pathways to pinpoint how lipidation length modifies the main avenues for the transport. We thus show that increasing the length of the lipid chain increases the transport of the drug significantly but also makes endocytosis the primary transport mechanism in a short-term cell culture model.

Applying flow cytometry to identify the modes of action of membrane-active peptides in a label-free and high-throughput fashion.

Membrane-active peptides (MAPs) have several potential therapeutic uses, including as antimicrobial drugs. Many traditional methods used to evaluate the membrane interactions of MAPs have limited applicability. Low-throughput methods, such as microscopy, provide detailed information but often rely on fluorophore-labeled MAPs, and high-throughput assays, such as the calcein release assay, cannot assess the mechanism behind the disruption of vesicular-based lipid membranes. Here we present a flow cytometric assay that provides detailed information about the peptide-lipid membrane interactions on single artificial lipid vesicles while being high-throughput (1000-2000 vesicles/s) and based on label-free MAPs. We synthesized and investigated six MAPs with different modes of action to evaluate the versatility of the assay. The assay is based on the flow cytometric readouts from artificial lipid vesicles, including the fluorescence from membrane-anchored and core-encapsulated fluorophores, and the vesicle concentration. From these parameters, we were able to distinguish between MAPs that induce vesicle solubilization, permeation (pores/membrane distortion), and aggregation or fusion. Our flow cytometry findings have been verified by traditional methods, including the calcein release assay, dynamic light scattering, and fluorescence microscopy on giant unilamellar vesicles. We envision that the presented flow cytometric assay can be used for various types of peptide-lipid membrane studies, e.g. to identify new antibiotics. Moreover, the assay can easily be expanded to derive additional valuable information.

Quantifying the transport of biologics across intestinal barrier models in real-time by fluorescent imaging.

Unsuccessful clinical translation of orally delivered biological drugs remains a challenge in pharmaceutical development and has been linked to insufficient mechanistic understanding of intestinal drug transport. Live cell imaging could provide such mechanistic insights by directly tracking drug transport across intestinal barriers at subcellular resolution, however traditional intestinal in vitro models are not compatible with the necessary live cell imaging modalities. Here, we employed a novel microfluidic platform to develop an in vitro intestinal epithelial barrier compatible with advanced widefield- and confocal microscopy. We established a quantitative, multiplexed and high-temporal resolution imaging assay for investigating the cellular uptake and cross-barrier transport of biologics while simultaneously monitoring barrier integrity. As a proof-of-principle, we use the generic model to monitor the transport of co-administrated cell penetrating peptide (TAT) and insulin. We show that while TAT displayed a concentration dependent difference in its transport mechanism and efficiency, insulin displayed cellular internalization, but was restricted from transport across the barrier. This illustrates how such a sophisticated imaging based barrier model can facilitate mechanistic studies of drug transport across intestinal barriers and aid in vivo and clinical translation in drug development.

A modular and noncovalent transduction system for leucine-zipper-tagged proteins.

Modes of transport: A leucine-zipper-tagged GFP was transported into cells by "zipping" it (red) to it's complementary leucine zipper (blue) functionalized with a cell-penetrating peptide (CPP). This transport system has an inherent modularity as the CPP is "clicked" to the leucine zipper, and then noncovalently bound to the protein, thus making it system particularly useful for targeting studies.

Bacterial genotoxins induce T cell senescence.

Several types of pathogenic bacteria produce genotoxins that induce DNA damage in host cells. Accumulating evidence suggests that a central function of these genotoxins is to dysregulate the host's immune response, but the underlying mechanisms remain unclear. To address this issue, we investigated the effects of the most widely expressed bacterial genotoxin, the cytolethal distending toxin (CDT), on T cells-the key mediators of adaptive immunity. We show that CDT induces premature senescence in activated CD4 T cells in vitro and provide evidence suggesting that infection with genotoxin-producing bacteria promotes T cell senescence in vivo. Moreover, we demonstrate that genotoxin-induced senescent CD4 T cells assume a senescence-associated secretory phenotype (SASP) which, at least partly, is orchestrated by the ATM-p38 signaling axis. These findings provide insight into the immunomodulatory properties of bacterial genotoxins and uncover a putative link between bacterial infections and T cell senescence.

Interleukin-6 signaling requires only few IL-6 molecules: Relation to physiological concentrations of extracellular IL-6.

INTRODUCTION: The aim of this study was to give quantitative insight into the number of cytokine molecules needed to activate a target cell and relate this to the physiological consequences of the amounts of cytokines typically detectable in humans. As a model system blood interleukin-6 (IL-6) was chosen since this cytokine is one of the most studied and clinically monitored cytokines, and because of the tools for the present investigations such as fully bioactive iodinated recombinant IL-6, cellular cytokine binding assays, and bioassays have been thoroughly validated. METHODS: The key intermediates of the basic equilibrium principles that govern cytokine binding and exchange were deduced and applied on concrete estimations of cellular and extracellular IL-6 binding in the bloodstream based on experimental binding data and data from the literature. In parallel, in vitro cellular IL-6 binding data was substantiated by paired measurements of IL-6 bioactivity on IL-6 sensitive B9 hybridoma cells. RESULTS: Blood leucocytes and B9 cells expressed 50 to 300, 10 to 20 picomolar affinity, IL-6 binding sites per cell and at physiological concentrations of IL-6 less than 10 IL-6 molecules seemed to be bound to blood cells. Nonetheless, binding off as few as four IL-6 molecules per cell seemed to result in statistically significant bioactivity, whereas binding of 16 IL-6 molecules triggered extensive cellular responses. CONCLUSION: Together, the estimations and the measurements support the notion that target cells with more than 100 bioactive cytokine receptors per cell, such as T cells and hepatocytes, are likely to be under steady and substantial cytokine-induced endocrine activation.

Vaccination with IL-6 analogues induces autoantibodies to IL-6 and influences experimentally induced inflammation.

IL-6 is involved in inflammation and a therapeutic target. 0.1% of Danish blood donors have nanomolar plasma concentrations of polyclonal, picomolar affinity and in vitro as well as in vivo neutralizing IgG autoantibodies to IL-6 (aAb-IL-6). Such donors are assumed to be severely IL-6 deficient; yet they appear healthy and do not exhibit overt clinical or laboratory abnormalities. We induced comparable levels of aAb-IL-6 in different mouse strains by vaccination with immunogenic IL-6 analogues. We observed that the induced aAb-IL-6 protected against collagen-induced arthritis and experimental allergic encephalitis. Furthermore, aAb-IL-6 carrying mice displayed increased plasma TNFalpha concentrations upon challenge with LPS. Taken together, induction of IL-6 autoantibodies was possible in different mouse strains. The autoantibodies influenced experimental inflammation. This immunotherapeutic principle might be a viable alternative in immune competent humans suffering from disorders driven by IL-6.

Results of Automated Retinal Image Analysis for Detection of Diabetic Retinopathy from the Nakuru Study, Kenya.

OBJECTIVE: Digital retinal imaging is an established method of screening for diabetic retinopathy (DR). It has been established that currently about 1% of the world's blind or visually impaired is due to DR. However, the increasing prevalence of diabetes mellitus and DR is creating an increased workload on those with expertise in grading retinal images. Safe and reliable automated analysis of retinal images may support screening services worldwide. This study aimed to compare the Iowa Detection Program (IDP) ability to detect diabetic eye diseases (DED) to human grading carried out at Moorfields Reading Centre on the population of Nakuru Study from Kenya. PARTICIPANTS: Retinal images were taken from participants of the Nakuru Eye Disease Study in Kenya in 2007/08 (n = 4,381 participants [NW6 Topcon Digital Retinal Camera]). METHODS: First, human grading was performed for the presence or absence of DR, and for those with DR this was sub-divided in to referable or non-referable DR. The automated IDP software was deployed to identify those with DR and also to categorize the severity of DR. MAIN OUTCOME MEASURES: The primary outcomes were sensitivity, specificity, and positive and negative predictive value of IDP versus the human grader as reference standard. RESULTS: Altogether 3,460 participants were included. 113 had DED, giving a prevalence of 3.3% (95% CI, 2.7-3.9%). Sensitivity of the IDP to detect DED as by the human grading was 91.0% (95% CI, 88.0-93.4%). The IDP ability to detect DED gave an AUC of 0.878 (95% CI 0.850-0.905). It showed a negative predictive value of 98%. The IDP missed no vision threatening retinopathy in any patients and none of the false negative cases met criteria for treatment. CONCLUSIONS: In this epidemiological sample, the IDP's grading was comparable to that of human graders'. It therefore might be feasible to consider inclusion into usual epidemiological grading.

Hyper-inflammation and skin destruction mediated by rosiglitazone activation of macrophages in IL-6 deficiency.

Injury initiates recruitment of macrophages to support tissue repair; however, excessive macrophage activity may exacerbate tissue damage causing further destruction and subsequent delay in wound repair. Here we show that the peroxisome proliferation-activated receptor-γ agonist, rosiglitazone (Rosi), a medication recently reintroduced as a drug to treat diabetes and with known anti-inflammatory properties, paradoxically generates pro-inflammatory macrophages. This is observed in both IL-6-deficient mice and control wild-type mice experimentally induced to produce high titers of auto-antibodies against IL-6, mimicking IL-6 deficiency in human diseases. IL-6 deficiency when combined with Rosi-mediated upregulation of suppressor of cytokine signaling 3 leads to an altered ratio of nuclear signal transducer and activator of transcription 3/NF-κB that allows hyper-induction of inducible nitric oxide synthase (iNOS). Macrophages activated in this manner cause de novo tissue destruction, recapitulating human chronic wounds, and can be reversed in vivo by recombinant IL-6, blocking macrophage infiltration, or neutralizing iNOS. This study provides insight into an unanticipated paradoxical role of Rosi in mediating hyper-inflammatory macrophage activation significant for diseases associated with IL-6 deficiency.

Thrombelastography and rotational thromboelastometry early amplitudes in 182 trauma patients with clinical suspicion of severe injury.

BACKGROUND: Viscoelastic hemostatic assays may provide means for earlier detection of trauma-induced coagulopathy (TIC). METHODS: This is a prospective observational study of 182 trauma patients admitted to a Level 1 trauma center. Clinical data, thrombelastography (TEG), and rotational thromboelastometry (ROTEM) parameters were recorded upon arrival. Citrated kaolin (CK), rapid TEG (rTEG), and functional fibrinogen curves were extracted, and early amplitudes A5 and A10 were registered. Patients were stratified according to international normalized ratio of 1.2 or less and international normalized ratio greater than 1.2 (TIC patients) as well as transfusion needs (no red blood cells [RBCs], 1-9 RBCs, and ≥10 RBC in 6 hours). Correlations were analyzed by Spearman's correlation. RESULTS: TIC patients had lower amplitudes than non-TIC patients in ROTEM/TEG as follows: EXTEM, INTEM, and FIBTEM: A5, A10, and maximum clot firmness (MCF); rTEG: A10; CK: maximum amplitude (MA); and functional fibrinogen: A5, A10, and MA (p < 0.05). Furthermore, A5 and A10 had a strong correlation with MA/MCF (ρ > 0.7 and p < 0.01). The A10 amplitudes were significantly lower in patients transfused with 10 or more units of RBC compared with nontransfused patients (p < 0.02). Fibrinogen concentration and platelet count had moderate correlation with A10 compared with A5 and MA/MCF (0.3 < ρ < 0.7 and p < 0.01). Time (median [interquartile range], in minutes) to obtain a reading was faster for A10 than MA/MCF (p < 0.001) (CK, 16 [15-17] vs. 27 [25-30]; rTEG, 11 [11-11] vs. 18 [17-20]; EXTEM, 11 [11-11] vs. 29 [26-31]; and INTEM 13[12-13] vs. 25 [22-29]). CONCLUSION: Early amplitudes were lower in TIC patients, had significant correlations with MA/MCF, and differentiated between nontransfused and patients receiving one to nine RBC units or 10 or more RBC units within 6 hours. A10's superior correlation with platelet count and fibrinogen concentration suggests that A10 reflects a more dynamic part of the hemostatic process rather than MA/MCF. Early amplitudes may translate into earlier goal-directed transfusion therapy and may allow refinement of existing transfusion algorithms. LEVEL OF EVIDENCE: Prognostic and diagnostic study, level III.

Constrained and UV-activatable cell-penetrating peptides for intracellular delivery of liposomes.

Herein we report on the development of a novel method of constraining a cell-penetrating peptide, which can be used to trigger transport of liposomes into cells upon in this case radiation with UV-light. A cell-penetrating peptide, which was modified on both termini with an alkyl chain, was anchored to the liposomal surface in a constrained and deactivated form. Since one of the two alkyl chains was connected to the peptide via a UV-cleavable linker, disconnection of this alkyl chain upon irradiation led to the exposure of the cell-penetrating peptide, and mediated the transport of the entire liposome particle into cells.

Simple and efficient solid-phase preparation of azido-peptides.

A shelf-stable and easily prepared diazotransfer reagent, imidazole-1-sulfonyl azide hydrochloride, was used to transform the N-terminus of a model peptide on solid phase into an azide moiety. It is demonstrated that this conversion was accomplished within 30 min with high efficiency under aqueous conditions on a NovaPEG resin or in DMF on polystyrene beads.