Whole blood cytokine release assays reveal disparity between capillary blood sampling methods.

INTRODUCTION: The use of whole blood in rapid cytokine release assays (CRAs) is becoming an established technique for screening immune responses following natural infection or vaccination, especially in the context of the SARS-CoV-2 pandemic. Establishing an accurate capillary blood sampling method to replace the need for venipuncture could make CRAs more accessible. In this study, capillary blood was collected via two different methods alongside traditional venipuncture to investigate whether the method of blood draw affects cytokine quantification when performing CRAs. METHODS: Adults previously vaccinated with SARS-CoV-2 vaccines donated three blood samples: one by venipuncture, one by finger prick, and one by a microneedle device. Whole blood was aliquoted and incubated overnight with SARS-CoV-2 peptides or left unstimulated. Cytokine release in plasma was measured by multiplex array. RESULTS: In unstimulated samples, little to no cytokines were detected in blood collected via venipuncture or by microneedle devices. Conversely, capillary blood collected by finger prick showed detectable levels of all cytokines analysed, with significantly inflated levels of TNFα, IL-10 (p < 0.0001), IL-2, GM-CSF, and IL-13 (p < 0.01), and 53% of these samples were also positive for IFN-γ. Following peptide stimulation, 25% of samples collected via finger prick showed dysregulated production of IFN-γ, TNFα, IL-2, and IL-10, with lower cytokine production than unstimulated controls. Contrastingly, this was seen in just 4% of venous blood samples and in none of the microneedle samples. CONCLUSIONS: Capillary blood draw via a microneedle device results in highly comparable immune responses to those seen via venipuncture at baseline and following peptide stimulation, suggesting this is a viable method for rapid whole blood CRAs. Conversely, differential cytokine production is observed following capillary blood draw via finger prick.

Differential cellular and humoral immune responses in immunocompromised individuals following multiple SARS-CoV-2 vaccinations.

Introduction: The heterogeneity of the immunocompromised population means some individuals may exhibit variable, weak or reduced vaccine-induced immune responses, leaving them poorly protected from COVID-19 disease despite receiving multiple SARS-CoV-2 vaccinations. There is conflicting data on the immunogenicity elicited by multiple vaccinations in immunocompromised groups. The aim of this study was to measure both humoral and cellular vaccine-induced immunity in several immunocompromised cohorts and to compare them to immunocompetent controls. Methods: Cytokine release in peptide-stimulated whole blood, and neutralising antibody and baseline SARS-CoV-2 spike-specific IgG levels in plasma were measured in rheumatology patients (n=29), renal transplant recipients (n=46), people living with HIV (PLWH) (n=27) and immunocompetent participants (n=64) post third or fourth vaccination from just one blood sample. Cytokines were measured by ELISA and multiplex array. Neutralising antibody levels in plasma were determined by a 50% neutralising antibody titre assay and SARS-CoV-2 spike specific IgG levels were quantified by ELISA. Results: In infection negative donors, IFN-γ, IL-2 and neutralising antibody levels were significantly reduced in rheumatology patients (p=0.0014, p=0.0415, p=0.0319, respectively) and renal transplant recipients (p<0.0001, p=0.0005, p<0.0001, respectively) compared to immunocompetent controls, with IgG antibody responses similarly affected. Conversely, cellular and humoral immune responses were not impaired in PLWH, or between individuals from all groups with previous SARS-CoV-2 infections. Discussion: These results suggest that specific subgroups within immunocompromised cohorts could benefit from distinct, personalised immunisation or treatment strategies. Identification of vaccine non-responders could be critical to protect those most at risk.

Longitudinal T Cell Responses against Ancestral, Delta, and Omicron SARS-CoV-2 Variants Determined by Rapid Cytokine Release Assay in Whole Blood.

T cell immunity to natural SARS-CoV-2 infection may be more robust and longer lived than Ab responses. Accurate assessment of T cell responses is critical for understanding the magnitude and longevity of immunity across patient cohorts, and against emerging variants. By establishing a simple, accurate, and rapid whole blood test, natural and vaccine-induced SARS-CoV-2 immunity was determined. Cytokine release in whole blood stimulated with peptides specific for SARS-CoV-2 was measured in donors with previous PCR-confirmed infection, suspected infection, or with no exposure history (n = 128), as well as in donors before and after vaccination (n = 32). Longitudinal assessment of T cell responses following initial vaccination and booster vaccination was also conducted (n = 50 and n = 62, respectively). Cytokines were measured by ELISA and multiplex array. IL-2 and IFN-γ were highly elevated in PCR-confirmed donors compared with history-negative controls, with median levels ∼33-fold and ∼48-fold higher, respectively. Receiver operating curves showed IL-2 as the superior biomarker (area under the curve = 0.9950). Following vaccination, all donors demonstrated a positive IL-2 response. Median IL-2 levels increased ∼32-fold from prevaccination to postvaccination in uninfected individuals. Longitudinal assessment revealed that T cell responses were stable up to 6 mo postvaccination. No significant differences in cytokine production were observed between stimulations with Wuhan, Delta, or Omicron peptides. This rapid, whole blood-based test can be used to make comparable longitudinal assessments of vaccine-induced T cell immunity across multiple cohorts and against variants of concern, thus aiding decisions on public health policies.

Simultaneous quantification of specific food allergen proteins using a fluorescent multiplex array.

The aim was to develop a fluorescent multiplex array for simultaneously measuring regulated food allergens using specific allergen protein molecules from peanut, tree nut, cow's milk, egg, soy, fish, shellfish, sesame, mustard and celery. Microspheres coupled to specific monoclonal antibodies were used for allergen detection, with purified allergens as reference standards.Standard curves for 17 allergens covered a 5-log dynamic range. Intra- and inter-assay acceptance criteria were within 70-130% recovery and a CV of ≤15%. Food reference materials contained high levels of their respective major allergens (2000-175,000 µg/g), Similar high allergen levels were found in 10 selected foods analysed using a 9-plex array. Egg, milk, peanut, hazelnut and walnut allergens were detectable in chocolate bars with incurred allergens at 3, 10, 30, and 100 ppm. The multiplex array is an efficient tool for measuring specific food allergens, with applications for risk assessment and standardization of therapeutic products for food allergy.

Bos d 11 in baked milk poses a risk for adverse reactions in milk-allergic patients.

BACKGROUND: Patients are commonly challenged with foods containing baked milk, for example muffins, yet little is known about the specific allergen content of muffins used in milk challenges or of the effect that baking has on allergenicity. OBJECTIVE: Our objective was to compare the levels of major milk allergens in uncooked and baked muffins using monoclonal immunoassays and IgE antibody binding before and after baking. METHODS: Uncooked and baked muffins were prepared using recipes from Mount Sinai and Imperial College. Allergen levels were compared by ELISA for Bos d 5 (ß-lactoglobulin) and Bos d 11 (ß-casein). IgE reactivity was assessed using sera from milk-sensitized donors in direct binding and inhibition ELISA. RESULTS: Bos d 5 was reduced from 680 µg/g in uncooked muffin mix to 0.17 µg/g in baked muffins, representing a >99% decrease after baking. Conversely, Bos d 11 levels in baked muffin remained high and only decreased by 30% from a mean of 4249 µg/g in uncooked muffin mix to 2961 µg/g when baked (~181 mg Bos d 11 per muffin). Baked muffins retained ~70% of the IgE binding to uncooked muffin mix. Baked muffin extract inhibited IgE binding to uncooked muffin mix by up to 80%, demonstrating retention of in vitro IgE reactivity. CONCLUSIONS AND CLINICAL RELEVANCE: High levels of Bos d 11 in baked muffins pose a risk for adverse reactions, especially in patients who have high anti-casein IgE antibodies.

Binding of complement regulatory proteins to group A Streptococcus.

Streptococcus pyogenes or Group A Streptococcus (GAS) is the etiologic agent of important human infections such as acute pharyngitis, impetigo, rheumatic fever and the streptococcal toxic shock syndrome. Binding of the complement regulatory proteins factor H, factor H-like protein 1 (FHL-1), C4b-binding protein (C4BP), or CD46 is a crucial step in the pathogenesis of these infections. M protein is the GAS protein that generally mediates these interactions. However, a detailed analysis of the reports that have investigated the binding of complement regulatory components to GAS indicates that this microorganism has evolved alternative mechanisms for the recruitment of complement regulatory proteins to the bacterial surface. This article summarizes these data to provide a starting point for future research aimed at the characterization of additional mechanisms developed by GAS to evade the immune system.

Evaluation of the ability of erythromycin-resistant and -susceptible pharyngeal group A Streptococcus isolates from Spain to enter and persist in human keratinocytes.

The macrolide-resistance rate among group A Streptococcus (GAS) isolates has increased in some European countries. To investigate the reasons for this increase, the ability of 60 erythromycin-resistant and 61 erythromycin-susceptible, genetically unrelated, pharyngeal GAS isolates from Spain to enter and persist within human keratinocytes was evaluated. It was observed that erythromycin resistance was associated with the presence of prtF1, a gene related to invasiveness, whereas no association was observed with the ability to enter human keratinocytes. However, the ability to enter human keratinocytes was not associated with the presence of prtF1 or with the emm type, suggesting that interaction with keratinocytes depends on several characteristics of the isolate. Almost all strains (95.9 %) were capable of persisting within human keratinocytes. However, most of them (91.7 %) exhibited a decline in viability over time. Interestingly, the ability to persist within keratinocytes protected from the action of the beta-lactams was higher among erythromycin-resistant isolates and correlated with their ability to avoid the induction of cellular lysis. These observations suggest that if the carrier state results from intracellular GAS survival, the association between erythromycin resistance and intracellular persistence may represent a serious problem for the eradication of these isolates.

Detection of QTL affecting fatty acid composition in the pig.

We present a QTL genome scan for fatty acid composition in pigs. An F2 cross between Iberian x Landrace pigs and a regression approach fitting the carcass weight as a covariate for QTL identification was used. Chromosomes (Chrs) 4, 6, 8, 10, and 12 showed highly significant effects. The Chr 4 QTL influenced the linoleic content and both the fatty acid double-bond index and peroxidability index. In Chr 6 we found significant associations with the double-bond index and the unsaturated index of fatty acids. Chr 8 showed clear effects on the percentages of palmitic and palmitoleic fatty acids as well as the average chain length of fatty acids. In Chr 10 we detected a significant QTL for the percentage of myristic fatty acid, with an F value that was slightly above the genomewide threshold. The percentage of linolenic fatty acid was affected by a region on Chr 12. A nearly significant QTL for the content of gadoleic fatty acid was also detected in Chr 12. We also analyzed the genomic QTL distribution by a regression model that fits the backfat thickness as a covariate. Some of the QTL that were detected in our analysis could not be detected when the data were corrected by backfat thickness. This work shows how critical the selection of a covariate can be in the interpretation of results. This is the first report of a genome scan detection of QTL directly affecting fatty acid composition in pigs.

Exploring alternative models for sex-linked quantitative trait loci in outbred populations: application to an iberian x landrace pig intercross.

We present a very flexible method that allows us to analyze X-linked quantitative trait loci (QTL) in crosses between outbred lines. The dosage compensation phenomenon is modeled explicitly in an identity-by-descent approach. A variety of models can be fitted, ranging from considering alternative fixed alleles within the founder breeds to a model where the only genetic variation is within breeds, as well as mixed models. Different genetic variances within each founder breed can be estimated. We illustrate the method with data from an F(2) cross between Iberian x Landrace pigs for intramuscular fat content and meat color component a*. The Iberian allele exhibited a strong overdominant effect for intramuscular fat in females. There was also limited evidence of one or more regions affecting color component a*. The analysis suggested that the QTL alleles were fixed in the Iberian founders, whereas there was some evidence of segregation in Landrace for the QTL affecting a* color component.