DNase analysed by a novel competitive assay in patients with complications after ChAdOx1 nCoV-19 vaccination and in normal unvaccinated blood donors.

Increased levels of neutrophil extracellular traps (NETs) have been detected in individuals with vaccine complications after the ChAdOx1 nCov vaccine with a correlation between the severity of vaccine side effects and the level of NETosis. DNases may disrupt NETs by degrading their content of DNA, and a balance has been reported between NETs and DNases. Because of this and since the inflammatory marker NETs may be used as a confirmatory test in diagnosing VITT, it is of interest to monitor levels of DNase in patients with increased NETs levels. The current novel rapid DNase ELISA was tested in blood samples of patients with known increased levels of NETs with or without VITT after ChAdOx1 nCoV-19 vaccination. DNase levels in VITT patients were significantly increased compared with normal unvaccinated blood donors and compared with patients with post-vaccination symptoms but not VITT. However, since EDTA was found to inhibit DNase, serum and not EDTA-plasma samples should be applied for DNase testing. The novel DNase assay may serve as a supplementary test to the NETs test when analysing samples from patients with suspected increased NETs levels.

Assessing IgE sensitization profiles to birch and timothy grass pollen allergens in birch pollen allergic blood donors using an oligoplex specific IgE assay.

IgE sensitization profiles to single birch allergens in birch-sensitized patients differ among European countries. The aim of this study was to determine the distribution of specific IgE antibodies to major and minor birch pollen allergens in a population of allergic Norwegian individuals by using a birch allergic blood donor population as a surrogate sample. Sixty blood donors were recruited and sampled based on birch allergy symptoms such as rhinitis, rhinoconjunctivitis and/or mild asthma in previous seasons. All sera were collected before start of the pollen season and tested using a line blot assay (Euroimmun AG, Lübeck, Germany) for IgE to birch and timothy pollen. Both extracts, single allergens, and cross-reacting carbohydrate determinants (CCD) were analysed. Only donors with specific IgE to birch and/or timothy grass were further evaluated. Specific IgE to birch pollen extract was found in 52 sera, and sensitization to timothy grass in 40 sera. Specific IgE to Bet v 1 was predominant in contrast to Bet v 4 which was absent. However, sensitization to the minor allergens Bet v 2 and 6 was always found together with high levels of IgE to Bet v 1. Subjects sensitized to the profilin Bet v 2 from birch were also sensitized to Phl p 12 from timothy grass. In conclusion, there was predominantly Bet v 1 sensitization in this cohort and low sensitization to minor allergens and cross-reactive allergens (Bet v 2, Bet v 4, Phl p 7 and Phl p 12).

COVID-19 patients treated with convalescent plasma. / Covid-19-pasienter behandlet med rekonvalesensplasma.

BACKGROUND: In Norway, treatment with COVID-19 convalescent plasma has been given through the NORPLASMA project. The treatment was initially offered to critically ill patients after an individual assessment, but from December 2020, the indication was limited to critically ill, immunocompromised patients. In this article we describe clinical characteristics, comorbidity and mortality in patients who received convalescent plasma in these two periods. MATERIAL AND METHOD: From 22 April 2020 to 30 March 2022, a total of 79 patients were included in the observational studies NORPLASMA MONITOR and the Norwegian SARS-CoV-2 study. The patients had received a total of 193 units of convalescent plasma at 15 Norwegian hospitals/nursing homes; 62 in South-Eastern Norway Regional Health Authority, 8 in Western Norway Regional Health Authority and 9 in Central Norway Regional Health Authority. Information on immune status, comorbidity and course of infection was retrieved from the patient records after informed written consent was obtained. RESULTS: Of 79 patients with a median age of 65 years (interquartile range 51-⁠73) who were treated with convalescent plasma, 31 (39 %) died during hospitalisation. A total of 59 patients were immunocompromised, and of these, 20 died in hospital compared to 11 of 20 who were assumed to be immunocompetent. Median number of comorbidities was 2 (interquartile range 1-4). The patients received a median of two plasma units (min.-max. 1-21). Two of the patients developed mild allergic skin reactions. INTERPRETATION: Convalescent plasma was well tolerated by patients with COVID-19. Immunocompromised patients may have benefitted from the treatment, with lower mortality than for those assumed to be immunocompetent.

Convalescent plasma from Norwegian blood donors to treat COVID-19. / Rekonvalesensplasma mot covid-19 fremstilt fra norske blodgivere.

BACKGROUND: At the start of the pandemic, the Norwegian Directorate of Health and Norwegian blood banks initiated the production of COVID-19 convalescent plasma within the framework of clinical studies. In this article we describe the blood donors who participated. MATERIAL AND METHOD: Blood donors who had recovered from COVID-19 were recruited to donate single donor plasma for the purpose of patient treatment. Data on the course of infection, leukocyte antibodies and antibody level against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) per plasma unit were registered after informed consent was obtained. We calculated a disease score defined as the total number of self-reported symptoms/findings and hospitalisation where relevant (score 0-⁠11). RESULTS: A total of 1644 plasma units were collected from 266 plasma donors at 12 blood banks. Median disease score was 5 (interquartile range 3-⁠6), and 15 donors had recovered from pneumonia and/or been hospitalised. A total of 599/1644 plasma units from 106/266 donors met our requirement for SARS-CoV-2 antibody content (> 60 % inhibition of virus binding to angiotensin-converting enzyme 2 (ACE2)) or positive virus neutralisation test. The antibody level in donors waned over time following infection, and showed no clear correlation with disease score. INTERPRETATION: The number of symptoms and findings in blood donors could not predict antibody response at individual level, and antibody testing was crucial for the production of effective convalescent plasma.

Rituximab-treated patients with lymphoma develop strong CD8 T-cell responses following COVID-19 vaccination.

B-cell depletion induced by anti-cluster of differentiation 20 (CD20) monoclonal antibody (mAb) therapy of patients with lymphoma is expected to impair humoral responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination, but effects on CD8 T-cell responses are unknown. Here, we investigated humoral and CD8 T-cell responses following two vaccinations in patients with lymphoma undergoing anti-CD20-mAb therapy as single agent or in combination with chemotherapy or other anti-neoplastic agents during the last 9 months prior to inclusion, and in healthy age-matched blood donors. Antibody measurements showed that seven of 110 patients had antibodies to the receptor-binding domain of the SARS-CoV-2 Spike protein 3-6 weeks after the second dose of vaccination. Peripheral blood CD8 T-cell responses against prevalent human leucocyte antigen (HLA) class I SARS-CoV-2 epitopes were determined by peptide-HLA multimer analysis. Strong CD8 T-cell responses were observed in samples from 20/29 patients (69%) and 12/16 (75%) controls, with similar median response magnitudes in the groups and some of the strongest responses observed in patients. We conclude that despite the absence of humoral immune responses in fully SARS-CoV-2-vaccinated, anti-CD20-treated patients with lymphoma, their CD8 T-cell responses reach similar frequencies and magnitudes as for controls. Patients with lymphoma on B-cell depleting therapies are thus likely to benefit from current coronavirus disease 2019 (COVID-19) vaccines, and development of vaccines aimed at eliciting T-cell responses to non-Spike epitopes might provide improved protection.

COVID-19 convalescent plasma: current status, lessons from the past and future perspectives.

When the COVID-19 pandemic hit, blood transfusion services worldwide started collection of convalescent plasma as early as possible, as exemplified by the response in Norway. There were challenges related to donor selection, donor safety, testing for relevant antibodies and indications for and dosing of the convalescent plasma. As more knowledge became available, the product quality was more standardised. Multiple case reports, observational studies and some randomized studies were published during the pandemic, as well as laboratory studies reporting different approaches to antibody testing. The results were conflicting and the importance of convalescent plasma was disputed. Even though there has been strong international collaboration with involvement of many key organisations, we may better prepare for the next pandemic. An even stronger, more formalised collaboration between these organisations could provide more clear evidence of the importance of convalescent plasma, based on the principles of passive immunisation.

Inflammatory markers calprotectin, NETs, syndecan-1 and neopterin in COVID-19 convalescent blood donors.

Persisting inflammation has been discovered in lungs and other parenchymatous organs of some COVID-19 convalescents. Calprotectin, neutrophil extracellular traps (NETs), syndecan-1 and neopterin are general key inflammatory markers, and systemically enhanced levels of them may remain after the COVID-19 infection. These inflammatory markers were therefore measured in serum samples of 129 COVID-19 convalescent and 27 healthy blood donors or employees at Oslo Blood bank, Norway. Also antibodies against SARS-CoV-2 nucleocapsid antigen were measured, and timing of sampling and severity of infection noted. Whereas neopterin and NETs values remained low and those for syndecan-1 were not raised to statistically significant level, concentrations for calprotectin, as measured by a novel mixed monoclonal assay, were significantly increased in the convalescents. Antibodies against SARS-CoV-2 nucleocapsid antigen were elevated, but did not correlate with levels of inflammatory markers. Difference between the groups in only one biomarker makes evaluation of ongoing or residual inflammation in the convalescents difficult. If there is a low-grade inflammation, it would in that case involve neutrophils.

NETs analysed by novel calprotectin-based assays in blood donors and patients with multiple myeloma or rheumatoid arthritis: A pilot study.

Two novel enzyme-linked immunosorbent assays (ELISAs), designed to detect complexes containing DNA, leucocyte calprotectin and S100A12 proteins, were generated for improved specificity and rapid measurement of neutrophil extracellular traps (NETs). The assays were applied on plasma and serum samples from blood donors for establishment of reference values, and from patients with multiple myeloma (MM) or rheumatoid arthritis (RA) in order to examine putatively increased values in the two different inflammatory conditions. Although NETs were hardly detectable in healthy individuals, NET levels were as expected highly and statistically significantly increased in RA patients. The detection of statistically significantly increased NET levels in MM is a novel finding.

IgE-sensitization to food and inhalant allergens in IBD patients compared with normal blood donors at Oslo University Hospital, Norway.

Objectives: Crohn's disease (CD) and ulcerative colitis (UC) have been regarded as autoimmune Th-1/Th-17- and Th-2-associated conditions, respectively. The aim of the study was to examine possible differences in allergen sensitization between these diseases and relative to normal blood donors (BD). Materials and methods: Plasma from 29 UC and 37 CD patients with moderate disease activity and 100 healthy age- and gender-matched BD, were analyzed for specific IgE to 22 food- and 28 inhalation allergens using EUROLINE atopy screen. Results: There was significantly higher proportion of allergen sensitized patients in UC compared to BD. Corresponding mean percentages for UC, CD and BD were 8.5, 8.9 (p = .2) and 5.9 (p = .04). There was no intergroup difference in sensitization to food allergens. Most prominent result was the double level of sensitization to inhalants in CD (15%) compared to BD (8%) (p = .03). Overall highest levels of sensitization to inhalants were for grass pollens. Interestingly, the number of allergens (n = 50) the subjects were sensitized to, was significantly lower among UC (n = 20; 40%) (p = .0005) than CD (n = 31; 62%) and BD (n = 38; 76%). Conclusions: The percentage of individuals sensitized to inhalants in CD and to inhalants and foods in UC, were higher than corresponding results in BD. However, whereas allergen positive reactions in CD were comparable to those in BD, they were reduced in UC because of the few UC reactions to food allergens. This contrasts previous data and the study also points to sensitization to inhalants as a potential factor in the complex pathogenesis of IBD.

The effect of donation frequency on donor health in blood donors donating plasma by plasmapheresis: study protocol for a randomized controlled trial.

BACKGROUND: The demand for plasma products is growing, necessitating an increase in plasma collection by plasmapheresis. While the 20th edition of the European Guidelines permits plasma donors in Europe to donate with 96-h donation intervals, the potential short- and long-term consequences of high-frequency plasma donations on donor health remain unknown. This study aims to measure the effect of plasma donation frequency on plasma protein composition, including total serum protein (TSP) and immunoglobulin G (IgG), in Norwegian male blood donors. METHODS: This randomized controlled trial (RCT) included 120 male blood donors who were randomized into two intervention groups and one control group: high-frequency plasma donors (HFPDs) who donated 650 mL of plasma 3 times every 2 weeks, whereas regular-frequency plasma donors (RFPDs) who donated 650 mL of plasma 1 time every 2 weeks. The control group consisted of whole blood donors. The primary outcomes are the concentrations of TSP and IgG. DISCUSSION: The findings from this study may have implications for recommendations related to donor health and plasma donation frequencies and may contribute to supporting the strategic independence of plasma products in Norway and Europe without compromising donor health. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05179200 . Registered December 20th, 2021.

Elevated NET, Calprotectin, and Neopterin Levels Discriminate between Disease Activity in COVID-19, as Evidenced by Need for Hospitalization among Patients in Northern Italy.

Coronavirus disease 2019 (COVID-19) displays clinical heterogeneity, but little information is available for patients with mild or very early disease. We aimed to characterize biomarkers that are useful for discriminating the hospitalization risk in a COVID-19 cohort from Northern Italy during the first pandemic wave. We enrolled and followed for four weeks 76 symptomatic SARS-CoV-2 positive patients and age/sex-matched healthy controls. Patients with mild disease were discharged (n.42), and the remaining patients were hospitalized (n.34). Blood was collected before any anti-inflammatory/immunosuppressive therapy and assessed for soluble C5b-9/C5a, H3-neutrophil extracellular traps (NETs), calprotectin, and DNase plasma levels via ELISA and a panel of proinflammatory cytokines via ELLA. Calprotectin and NET levels discriminate between hospitalized and non-hospitalized patients, while DNase negatively correlates with NET levels; there are positive correlations between calprotectin and both NET and neopterin levels. Neopterin levels increase in patients at the beginning of the disease and do so more in hospitalized than non-hospitalized patients. C5a and sC5b-9, and other acute phase proteins, correlate with neopterin, calprotectin, and DNase. Both NET and neopterin levels negatively correlate with platelet count. We show that calprotectin, NETs, and neopterin are important proinflammatory parameters potentially useful for discriminating between COVID-19 patients at risk of hospitalization.

Prevalent and immunodominant CD8 T cell epitopes are conserved in SARS-CoV-2 variants.

The emergence of SARS-CoV-2 variants of concern (VOC) is driven by mutations that mediate escape from neutralizing antibodies. There is also evidence that mutations can cause loss of T cell epitopes. However, studies on viral escape from T cell immunity have been hampered by uncertain estimates of epitope prevalence. Here, we map and quantify CD8 T cell responses to SARS-CoV-2-specific minimal epitopes in blood drawn from April to June 2020 from 83 COVID-19 convalescents. Among 37 HLA ligands eluted from five prevalent alleles and an additional 86 predicted binders, we identify 29 epitopes with an immunoprevalence ranging from 3% to 100% among individuals expressing the relevant HLA allele. Mutations in VOC are reported in 10.3% of the epitopes, while 20.6% of the non-immunogenic peptides are mutated in VOC. The nine most prevalent epitopes are conserved in VOC. Thus, comprehensive mapping of epitope prevalence does not provide evidence that mutations in VOC are driven by escape of T cell immunity.

Elevated NETs and Calprotectin Levels after ChAdOx1 nCoV-19 Vaccination Correlate with the Severity of Side Effects.

ChAdOx1 nCoV-19 vaccination has been associated with the rare side effect; vaccine-induced immune thrombotic thrombocytopenia (VITT). The mechanism of thrombosis in VITT is associated with high levels of neutrophil extracellular traps (NETs). The present study examines whether key markers for NETosis, such as H3-NETs and calprotectin, as well as syndecan-1 for endotheliopathy, can be used as prognostic factors to predict the severity of complications associated with ChAdOx1 vaccination. Five patients with VITT, 10 with prolonged symptoms and cutaneous hemorrhages but without VITT, and 15 with only brief and mild symptoms after the vaccination were examined. Levels of H3-NETs and calprotectin in the vaccinated individuals were markedly increased in VITT patients compared to vaccinees with milder vaccination-associated symptoms, and a strong correlation (r ≥ 0.745, p < 0.001) was found with severity of vaccination side effects. Syndecan-1 levels were also positively correlated (r = 0.590, p < 0.001) in vaccinees to side effects after ChAdOx1 nCoV-19 vaccination. We hypothesize that the inflammatory markers NETs and calprotectin may be used as confirmatory tests in diagnosing VITT.

Antitumor, Anti-Inflammatory and Antiallergic Effects of Agaricus blazei Mushroom Extract and the Related Medicinal Basidiomycetes Mushrooms, Hericium erinaceus and Grifola frondosa: A Review of Preclinical and Clinical Studies.

Since the 1980s, medicinal effects have been documented in scientific studies with the related Basidiomycota mushrooms Agaricus blazei Murill (AbM), Hericium erinaceus (HE) and Grifola frondosa (GF) from Brazilian and Eastern traditional medicine. Special focus has been on their antitumor effects, but the mushrooms' anti-inflammatory and antiallergic properties have also been investigated. The antitumor mechanisms were either direct tumor attack, e.g., apoptosis and metastatic suppression, or indirect defense, e.g., inhibited tumor neovascularization and T helper cell (Th) 1 immune response. The anti-inflammatory mechanisms were a reduction in proinflammatory cytokines, oxidative stress and changed gut microbiota, and the antiallergic mechanism was amelioration of a skewed Th1/Th2 balance. Since a predominant Th2 milieu is also found in cancer, which quite often is caused by a local chronic inflammation, the three conditions-tumor, inflammation and allergy-seem to be linked. Further mechanisms for HE were increased nerve and beneficial gut microbiota growth, and oxidative stress regulation. The medicinal mushrooms AbM, HE and GF appear to be safe, and can, in fact, increase longevity in animal models, possibly due to reduced tumorigenesis and oxidation. This article reviews preclinical and clinical findings with these mushrooms and the mechanisms behind them.

Agaricus blazei-Based Mushroom Extract Supplementation to Birch Allergic Blood Donors: A Randomized Clinical Trial.

Since Agaricus blazei Murill (AbM) extract reduced specific IgE and ameliorated a skewed Th1/Th2 balance in a mouse allergy model, it was tested in blood donors with self-reported, IgE-positive, birch pollen allergy and/or asthma. Sixty recruited donors were randomized in a placebo-controlled, double-blinded study with pre-seasonal, 7-week, oral supplementation with the AbM-based extract AndosanTM. Before and after the pollen season, questionnaires were answered for allergic rhino-conjunctivitis, asthma, and medication; serum IgE was measured, and Bet v 1-induced basophil activation was determined by CD63 expression. The reported general allergy and asthma symptoms and medication were significantly reduced in the AbM compared to the placebo group during pollen season. During the season, there was significant reduction in specific IgE anti-Bet v 1 and anti-t3 (birch pollen extract) levels in the AbM compared with the placebo group. While the maximal allergen concentrations needed for eliciting basophil activation before the season, changed significantly in the placebo group to lower concentrations (i.e., enhanced sensitization) after the season, these concentrations remained similar in the AndosanTM AbM extract group. Hence, the prophylactic effect of oral supplementation before the season with the AbM-based AndosanTM extract on aeroallergen-induced allergy was associated with reduced specific IgE levels during the season and basophils becoming less sensitive to allergen activation.

Osteopenia, decreased bone formation and impaired osteoblast development in Sox4 heterozygous mice.

The transcription factor Sox4 is vital for fetal development, as Sox4(-/-) homozygotes die in utero. Sox4 mRNA is expressed in the early embryonic growth plate and is regulated by parathyroid hormone, but its function in bone modeling/remodeling is unknown. We report that Sox4(+/-) mice exhibit significantly lower bone mass (by dual-energy X-ray absorptiometry) from an early age, and fail to obtain the peak bone mass of wild-type (WT) animals. Microcomputed tomography (muCT), histomorphometry and biomechanical testing of Sox4(+/-) bones show reduced trabecular and cortical thickness, growth plate width, ultimate force and stiffness compared with WT. Bone formation rate (BFR) in 3-month-old Sox4(+/-) mice is 64% lower than in WT. Primary calvarial osteoblasts from Sox4(+/-) mice demonstrate markedly inhibited proliferation, differentiation and mineralization. In these cultures, osterix (Osx) and osteocalcin (OCN) mRNA expression was reduced, whereas Runx2 mRNA was unaffected. No functional defects were found in osteoclasts. Silencing of Sox4 by siRNA in WT osteoblasts replicated the defects observed in Sox4(+/-) cells. We demonstrate inhibited formation and altered microarchitecture of bone in Sox4(+/-) mice versus WT, without apparent defects in bone resorption. Our results implicate the transcription factor Sox4 in regulation of bone formation, by acting upstream of Osx and independent of Runx2.