Redirector of Vaccine-induced Effector Responses (RoVER) for specific killing of cellular targets.

BACKGROUND: In individuals with malignancy or HIV-1 infection, antigen-specific cytotoxic T lymphocytes (CTLs) often display an exhausted phenotype with impaired capacity to eliminate the disease. Existing cell-based immunotherapy strategies are often limited by the requirement for adoptive transfer of CTLs. We have developed an immunotherapy technology in which potent CTL responses are generated in vivo by vaccination and redirected to eliminate target cells using a bispecific Redirector of Vaccine-induced Effector Responses (RoVER). METHODS: Following Yellow fever (YF) 17D vaccination of 51 healthy volunteers (NCT04083430), single-epitope YF-specific CTL responses were quantified by tetramer staining and multi-parameter flow cytometry. RoVER-mediated redirection of YF-specific CTLs to kill antigen-expressing Raji-Env cells, autologous CD19+ B cells or CD4+ T cells infected in vitro with a full-length HIV-1-eGFP was assessed in cell killing assays. Moreover, secreted IFN-γ, granzyme B, and TNF-α were analyzed by mesoscale multiplex assays. FINDINGS: YF-17D vaccination induced strong epitope-specific CTL responses in the study participants. In cell killing assays, RoVER-mediated redirection of YF-specific CTLs to autologous CD19+ B cells or HIV-1-infected CD4+ cells resulted in 58% and 53% killing at effector to target ratio 1:1, respectively. INTERPRETATION: We have developed an immunotherapy technology in which epitope-specific CTLs induced by vaccination can be redirected to kill antigen-expressing target cells by RoVER linking. The RoVER technology is highly specific and can be adapted to recognize various cell surface antigens. Importantly, this technology obviates the need for adoptive transfer of CTLs. FUNDING: This work was funded by the Novo Nordisk Foundation (Hallas Møller NNF10OC0054577).

Myopathy as a cause of fatigue in long-term post-COVID-19 symptoms: Evidence of skeletal muscle histopathology.

BACKGROUND AND PURPOSE: Among post-COVID-19 symptoms, fatigue is reported as one of the most common, even after mild acute infection, and as the cause of fatigue, myopathy diagnosed by electromyography has been proposed in previous reports. This study aimed to explore the histopathological changes in patients with post-COVID-19 fatigue. METHODS: Sixteen patients (mean age = 46 years) with post-COVID-19 complaints of fatigue, myalgia, or weakness persisting for up to 14 months were included. In all patients, quantitative electromyography and muscle biopsies analyzed with light and electron microscopy were taken. RESULTS: Muscle weakness was present in 50% and myopathic electromyography in 75%, and in all patients there were histological changes. Muscle fiber atrophy was found in 38%, and 56% showed indications of fiber regeneration. Mitochondrial changes, comprising loss of cytochrome c oxidase activity, subsarcollemmal accumulation, and/or abnormal cristae, were present in 62%. Inflammation was found in 62%, seen as T lymphocytes and/or muscle fiber human leukocyte antigen ABC expression. In 75%, capillaries were affected, involving basal lamina and cells. In two patients, uncommon amounts of basal lamina were found, not only surrounding muscle fibers but also around nerves and capillaries. CONCLUSIONS: The wide variety of histological changes in this study suggests that skeletal muscles may be a major target of SARS-CoV-2, causing muscular post-COVID-19 symptoms. The mitochondrial changes, inflammation, and capillary injury in muscle biopsies can cause fatigue in part due to reduced energy supply. Because most patients had mild-moderate acute affection, the new variants that might cause less severe acute disease could still have the ability to cause long-term myopathy.

Helminth products modulate innate immune recognition of nucleic acids in systemic lupus erythematosus.

AIM: Current treatment of Systemic Lupus Erythematosus (SLE) is suboptimal and causes broad immunosuppression. Therapeutic use of helminths or helminth products has been suggested for autoimmune diseases such as SLE. In the present study, we evaluated possible immunomodulating effects of adult body fluid (ABF) from Ascaris suum on peripheral blood mononuclear cells (PBMCs) from SLE patients in an ex vivo setup. METHODS: PBMCs from SLE patients and healthy controls (HC) were isolated and stimulated ex vivo with ABF and Toll-like receptor agonists or activators of the stimulator of interferon genes (STING) or mitochondrial antiviral signaling protein (MAVS) pathways. After 24 h of incubation, the cytokine profile was analyzed using ELISA and Meso Scale Discovery techniques. RESULTS: ABF suppressed production of IL-6, TNF-α, CXCL10, and IL-10 by PBMCs from SLE patients and HCs following stimulation with specific agonists. ABF also reduced IFN-у production by stimulated PBMCs from HCs. CONCLUSIONS: Our data show that ABF has an immunomodulatory effect on the production of key cytokines in the pathogenesis of SLE. These results suggest that ABF or ABF components hold potential as a novel treatment option for SLE.

Diagnostic performance, user acceptability, and safety of unsupervised SARS-CoV-2 rapid antigen-detecting tests performed at home.

BACKGROUND: One strategy for reducing spread of COVID-19 is to contain the infection with broad screening, isolating infected individuals, and tracing contacts. This strategy requires widely available, reliable SARS-CoV-2 testing. To increase testing, rapid antigen detection tests (RADTs) were developed for self-sampling, self-testing, and self-interpretation. This study examined diagnostic performance, user acceptability, and safety of nasal self-RADTs compared with polymerase chain reaction (PCR) testing. METHODS: Self-RADT kits were distributed at a public COVID-19 test center in Aarhus, Denmark or delivered to participants. Participants reported test results and test preferences. During enrollment, participants reported occurrence and duration of symptoms consistent with COVID-19. Sensitivity and specificity of self-RADT, relative to oropharyngeal PCR testing, were calculated. RESULTS: Among 827 participants, 102 showed positive PCR test results. Sensitivities of the self-RADTs were 65.7% (95% confidence interval [CI]: 49.2-79.2; DNA Diagnostic) and 62.1% (95% CI: 50.1-72.9; Hangzhou), and specificities were 100% (95% CI: 99.0-100; DNA Diagnostic) and 100% (95% CI: 98.9-100; Hangzhou). The sensitivities of both self-RADTs appeared higher in symptomatic participants than in asymptomatic participants. Two of every 3 participants preferred self-RADT over PCR test. CONCLUSION: Self-performed RADTs were reliable, user-acceptable, and safe among laypeople as a supplement to professionally collected oropharyngeal PCR testing.

Efficacy of the TMPRSS2 inhibitor camostat mesilate in patients hospitalized with Covid-19-a double-blind randomized controlled trial.

BACKGROUND: The trans-membrane protease serine 2 (TMPRSS2) is essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry and infection. Efficacy and safety of TMPRSS2 inhibitors in patients with coronavirus disease 2019 (Covid-19) have not been evaluated in randomized trials. METHODS: We conducted an investigator-initiated, double-blind, randomized, placebo-controlled multicenter trial in patients hospitalized with confirmed SARS-CoV-2 infection from April 4, to December 31, 2020. Within 48 h of admission, participants were randomly assigned in a 2:1 ratio to receive the TMPRSS2 inhibitor camostat mesilate 200 mg three times daily for 5 days or placebo. The primary outcome was time to discharge or clinical improvement measured as ≥2 points improvement on a 7-point ordinal scale. Other outcomes included 30-day mortality, safety and change in oropharyngeal viral load. FINDINGS: 137 patients were assigned to receive camostat mesilate and 68 to placebo. Median time to clinical improvement was 5 days (interquartile range [IQR], 3 to 7) in the camostat group and 5 days (IQR, 2 to 10) in the placebo group (P = 0·31). The hazard ratio for 30-day mortality in the camostat compared with the placebo group was 0·82 (95% confidence interval [CI], 0·24 to 2·79; P = 0·75). The frequency of adverse events was similar in the two groups. Median change in viral load from baseline to day 5 in the camostat group was -0·22 log10 copies/mL (p <0·05) and -0·82 log10 in the placebo group (P <0·05). INTERPRETATION: Under this protocol, camostat mesilate treatment was not associated with increased adverse events during hospitalization for Covid-19 and did not affect time to clinical improvement, progression to ICU admission or mortality. ClinicalTrials.gov Identifier: NCT04321096. EudraCT Number: 2020-001200-42.

A STING antagonist modulating the interaction with STIM1 blocks ER-to-Golgi trafficking and inhibits lupus pathology.

BACKGROUND: Nucleic acids are potent stimulators of type I interferon (IFN-I) and antiviral defense, but may also promote pathological inflammation. A range of diseases are characterized by elevated IFN-I, including systemic lupus erythematosus (lupus). The DNA-activated cGAS-STING pathway is a major IFN-I-inducing pathway, and activation of signaling is dependent on trafficking of STING from the ER to the Golgi. METHODS: Here we used cell culture systems, a mouse lupus model, and material from lupus patients, to explore the mode of action of a STING antagonistic peptide, and its ability to modulate disease processes. FINDINGS: We report that the peptide ISD017 selectively inhibits all known down-stream activities of STING, including IFN-I, inflammatory cytokines, autophagy, and apoptosis. ISD017 blocks the essential trafficking of STING from the ER to Golgi through a mechanism dependent on the STING ER retention factor STIM1. Importantly, ISD017 blocks STING activity in vivo and ameliorates disease development in a mouse model for lupus. Finally, ISD017 treatment blocks pathological cytokine responses in cells from lupus patients with elevated IFN-I levels. INTERPRETATION: These data hold promise for beneficial use of STING-targeting therapy in lupus. FUNDING: The Novo Nordisk Foundation, The European Research Council, The Lundbeck Foundation, European Union under the Horizon 2020 Research, Deutsche Forschungsgemeinschaft, Chulalongkorn University.

[Inattentional blindness in the diagnostic process].

When engaged in a demanding task of visual perception we tend to become blind to stimuli, which are not part of our primary task - a bias known as inattentional blindness. This article studies inattentional blindness in radiologists' interpretation of scans. Furthermore, it is discussed, how attention - being a limited resource - is distributed in the diagnostic process. Does focus on one diagnosis make us blind to findings supporting a differential diagnosis? Does focus on typing patient data into electronic health records draw attention away from the general clinical view of the patient?

Risk and prognosis of bacteremia and fungemia among first-time kidney transplant recipients: a population-based cohort study.

BACKGROUND: Bacterial infections are common complications in kidney transplant recipients (KTRs). Little is known about incidence rates of bacteremia and fungemia (BAF) in KTRs. METHODS: In this population-based cohort study, we used medical and administrative registries to identify episodes of BAF among KTRs in the Central Denmark and North Denmark Regions during 1995-2010. KTRs were followed from the date of their first transplantation to the earliest of BAF, graft loss, death, emigration or 31 December 2010. We calculated incidence rates of first BAF episode overall and stratified by time from transplantation. Potential risk factors were assessed using Cox regression analysis. The Kaplan-Meier analysis was used to estimate 30- and 90-day mortality. RESULTS: Among 612 KTRs, we identified 138 first episodes of bacteremia during 2397 person-years of follow-up (PYFU). The overall incidence rate (IR) was 5.8 BAF episodes per 100 PYFU (95% confidence interval [CI]: 4.9-6.8). The incidence rate declined from 84.0 per 100 PYFU (95% CI: 61.6-114.5) during post-transplant day 0-30 to 2.3 per 100 PYFU (95% CI: 1.7-3.0) from post-transplant day 365 and onwards. Hospital-onset BAF comprised 39% of the episodes of BAF. The most frequently isolated microorganisms were Escherichia coli and Klebsiella species causing 49 (35.5%) and 29 (21.0%) episodes of BAF, respectively. The 30-day mortality was 2.1% (95% CI: 0.7-6.6). CONCLUSIONS: While the risk of BAF in KTRs was high, thirty-day mortality was low. After the first post-transplant year, the IR of bacteremia was substantially lower than in the immediate post-transplant period.

Real-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves.

BACKGROUND: Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we compared RIMS-based drqPCR with classical quantifications based on external standard curves and the "comparative Ct method". Specifically, we used a raw real-time PCR dataset as the basis for more than two-and-a-half million simulated quantifications with various user-defined conditions. Compared with classical approaches, we found that RIMS-based drqPCR provided superior precision and comparable accuracy. CONCLUSIONS/SIGNIFICANCE: The obviation of referencing to control samples is attractive whenever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, copy number variations etc. Also, lab-to-lab comparability can be greatly simplified.

[Toll-like receptors, a new way of treatment?]. / Toll-like receptorer, nye behandlingsstrategier?

Toll-like receptors (TLRs) comprise a group of recently discovered receptors which are essential molecular structures in the activation of immunity. The discovery of TLRs has provided a substantial increase in the knowledge of immunologic aspects of disease pathology and is presently giving rise to new treatment strategies. This review summarizes the current knowledge on TLRs functioning in infections, their possible roles in inflammatory bowl disease and the pivotal role for TLRs in endotoxic shock, an area which is currently subject to development of a new farmakon.

Chlamydia pneumoniae and cardiovascular disease.

Chlamydia pneumoniae has been detected in atherosclerotic plaques, while seropositivity to this organism confers a slightly increased risk of coronary events. However, no aetiological link has been established; a major difficulty when investigating this link is the lack of a gold standard for diagnosing chronic vessel infection. The outcomes of case-control studies and prospective trials of macrolides in treatment and prevention of cardiovascular disease have been ambiguous but suggest a short-term preventive effect. Whether this is due to the antimicrobial or anti-inflammatory activity of the macrolides is unknown. Larger and longer prospective trials currently under way may provide better insight into the association of C. pneumoniae with cardiovascular disease. At present, there is no justification for treating cardiovascular disease with antibiotics.

[Reduction of the expansion rate of small abdominal aortic aneurysms with roxithromycin. Results from a randomized controlled trial]. / Haemning af små abdominale aortaaneurismers ekspansion med roxithromycin. Resultater fra et randomiseret klinisk kontrolleret forsøg.

INTRODUCTION: Macrolide treatment has been reported to reduce the risk of recurrent ischaemic heart disease. The influence of a macrolide on the expansion rate of small abdominal aortic aneurysms (AAA) is unknown at present. The aim of this study was to investigate the effect of roxithromycin on the expansion rate of small AAA. MATERIALS AND METHODS: A total of 92 patients with a small AAA were recruited from two populations. One population consisted of 6.339 men aged 65-73 years who were offered participation in a mass screening programme for AAA at the local hospital. From this population 66 subjects were recruited. The remaining 26 were recruited from among 49 subjects diagnosed at interval screening for an initial aortic diameter between 25 mm and 29 mm. The patients were randomized to receive either oral roxithromycin 300 mg once daily for 28 days or matching placebo, and followed for a mean of 1.5 years. RESULTS: During the first year the mean annual expansion rate of AAA was reduced by 44% in the macrolid group (1.56 mm/year) compared to 2.80 mm/year after placebo (p = 0.02). During the second year the difference was only 5%. Multiple linear regression analysis showed that roxithromycin treatment and initial AAA size were significantly related to AAA expansion when adjusted for smoking, diastolic blood pressure, and IgA level > or = 20. The logistic regression analysis confirmed a significant difference in expansion rates above 2 mm annually between the intervention and placebo groups, OR = 0.09 (95% CI: 0.01-0.83). DISCUSSION: In comparison to placebo, roxithromycin 300 mg daily for four weeks reduced the expansion rate of AAA.

[Atherosclerosis and Chlamydia pneumoniae]. / Aterosklerose og Chlamydia pneumoniae.

Chlamydia pneumoniae could be associated with the risk of developing atherosclerosis and an increased risk of thromboembolic complications. However, the evidence of an association seems to be declining and there is no evidence of causality. The effect of antibiotic treatment in cardiovascular disease has been explored in epidemiologic studies and in randomised controlled trials. Data suggest a protective but short-lasting effect of macrolide antibiotics on cardiovascular disease. The effect could be the result of anti-bacterial as well as anti-inflammatory properties. Ongoing larger and longer lasting treatment trials could provide better measures of the effects of antibiotic treatment, although they will not clarify the role of C. pneumoniae. Currently, there is no indication for treating cardiovascular disease with antibiotics.