Evaluating patient immunocompetence through antibody response to pneumococcal polysaccharide vaccine using a newly developed 23 serotype multiplexed assay.

Assessing T-cell independent antibody response to polysaccharide vaccines is crucial for diagnosing humoral immune deficiencies. However, immunocompetence criteria based on S. pneumoniae vaccination remain unclear. We evaluated IgG antibody vaccine response in healthy individuals to establish interpretive criteria. Pre- and 4-week post-vaccination sera were collected from 79 adults. Antibody concentrations to PNEUMOVAX 23 serotypes were measured using a multiplexed platform. Immunocompetence was determined by fold increase in post-vaccination response, percentage of serotypes achieving 4- or 2-fold antibody ratio, and post-vaccination concentration ≥ 1.3 µg/mL. Immunogenicity varied widely across the 23 serotypes (26.6% to 94.9% for ≥4-fold increase, 51.9% to 98.7% for ≥2-fold increase). Immunocompetence based on historic criteria of ≥4-fold increase in antibody ratio to ≥70% of serotypes was low (72.2%), but increased to 98.7% with criteria of at least a 2-fold increase and/or post-vaccination concentration ≥ 1.3 µg/mL. Current criteria for assessing immunocompetence may be overly stringent and require updating.

Reduced quantity and function of pneumococcal antibodies are associated with exacerbations of COPD in SPIROMICS.

While hypogammaglobulinemia is associated with COPD exacerbations, it is unknown whether frequent exacerbators have specific defects in antibody production/function. We hypothesized that reduced quantity/function of serum pneumococcal antibodies correlate with exacerbation risk in the SPIROMICS cohort. We measured total pneumococcal IgG in n = 764 previously vaccinated participants with COPD. In a propensity-matched subset of n = 200 with vaccination within five years (n = 50 without exacerbations in the previous year; n = 75 with one, n = 75 with ≥2), we measured pneumococcal IgG for 23 individual serotypes, and pneumococcal antibody function for 4 serotypes. Higher total pneumococcal IgG, serotype-specific IgG (17/23 serotypes), and antibody function (3/4 serotypes) were independently associated with fewer prior exacerbations. Higher pneumococcal IgG (5/23 serotypes) predicted lower exacerbation risk in the following year. Pneumococcal antibodies are inversely associated with exacerbations, supporting the presence of immune defects in frequent exacerbators. With further study, pneumococcal antibodies may be useful biomarkers for immune dysfunction in COPD.

Clinical significance of measuring serum cytokine levels as inflammatory biomarkers in adult and pediatric COVID-19 cases: A review.

Coronavirus disease 2019 (COVID-19) is a rapidly evolving infectious/inflammatory disorder which has turned into a global pandemic. With severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as its etiologic agent, severe COVID-19 cases usually develop uncontrolled inflammatory responses and cytokine storm-like syndromes. Measuring serum levels of pro-inflammatory cytokines (e.g., IL-6 and others) as inflammatory biomarkers may have several potential applications in the management of COVID-19, including risk assessment, monitoring of disease progression, determination of prognosis, selection of therapy and prediction of response to treatment.This is especially true for pediatric patients with COVID-19 associated Kawasaki-like disease and similar syndromes. In this report, we review the current knowledge of COVID-19 associated cytokines, their roles in host immune and inflammatory responses, the clinical significance and utility of cytokine immunoassays in adult and pediatric COVID-19 patients, as well as the challenges and pitfalls in implementation and interpretation of cytokine immunoassays. Given that cytokines are implicated in different immunological disorders and diseases, it is challenging to interpret the multiplex cytokine data for COVID-19 patients. Also, it should be taken into consideration that biological and technical variables may affect the commutability of cytokine immunoassays and enhance complexity of cytokine immunoassay interpretation. It is recommended that the same method, platform and laboratory should be used when monitoring differences in cytokine levels between groups of individuals or for the same individual over time. It may be important to correlate cytokine profiling data with the SARS-CoV-2 nucleic acid amplification testing and imaging observations to make an accurate interpretation of the inflammatory status and disease progression in COVID-19 patients.

Vitamin D sufficiency associates with an increase in anti-inflammatory cytokines after intense exercise in humans.

The purpose of this study was to identify the influence of vitamin D status (insufficient vs. sufficient) on circulating cytokines and skeletal muscle strength after muscular injury. To induce muscular injury, one randomly selected leg (SSC) performed exercise consisting of repetitive eccentric-concentric contractions. The other leg served as the control. An averaged serum 25(OH)D concentration from two blood samples collected before exercise and on separate occasions was used to establish vitamin D insufficiency (<30ng/mL, n=6) and sufficiency (>30ng/mL, n=7) in young, adult males. Serum cytokine concentrations, single-leg peak isometric force, and single-leg peak power output were measured before and during the days following the exercise protocol. The serum IL-10 and IL-13 responses to muscular injury were significantly (both p<0.05) increased in the vitamin D sufficient group. The immediate and persistent (days) peak isometric force (p<0.05) and peak power output (p<0.05) deficits in the SSC leg after the exercise protocol were not ameliorated with vitamin D sufficiency. We conclude that vitamin D sufficiency increases the anti-inflammatory cytokine response to muscular injury.

Severely depressed interleukin-17 production by human neonatal mononuclear cells.

BACKGROUND: The role of T-helper 17 cells (Th17) in neonatal host defense remains to be fully elucidated. Interleukin (IL)-17 plays an important role in the immune response to bacterial and fungal pathogens by promoting inflammation. METHODS: We examined neonatal production of IL-17 in mixed mononuclear cells (MMCs) isolated from umbilical cord blood for comparison with adult peripheral blood mononuclear cell controls. RESULTS: IL-17 production was profoundly diminished in MMCs isolated from cord blood when compared with MMCs from adult blood. This was associated with a marked reduction in the population of CCR6+ IL-17(+) T-cells in the neonatal cord blood. We also found diminished intracellular formation of IL-17, and diminished IL-17 responses to both group B streptococci (GBS) and Escherichia coli. Neonatal mononuclear cells were found to adequately phosphorylate signal transducer and activator of transcription 3, pY705, and pS727. We and others have reported markedly reduced interferon-γ production by neonate mononuclear cells exposed to GBS. Here, we correct that profound abnormality with added IL-17. CONCLUSION: Our results suggest that profound deficiency of IL-17 production associated with a marked decrease in Th17 cells likely contributes significantly to the increased susceptibility of human neonates to invasive bacterial and fungal infections.

Specimen type validation and establishment of normal cytokine reference intervals in cerebrospinal fluid.

Cerebrospinal fluid (CSF) is a critical body fluid to examine in attempts to discover potential biomarkers for neuroinflammatory and other disorders of the central nervous system (CNS). Serum and/or plasma cytokine levels have been associated with a variety of inflammatory conditions, and some have been shown to be actionable therapeutic targets. Less is known, however, about cytokine levels in CSF. Serum and plasma cytokine testing is widely available in clinical and research laboratories, but cytokine testing in CSF is extremely limited and if performed, accompanied by a disclaimer that it is an unvalidated specimen type. In this study, we validate CSF as a suitable specimen type and determine normal reference intervals for multiple cytokines as well as a soluble cytokine receptor. CSF was validated as a specimen type for testing using a laboratory developed multiplexed cytokine assay previously validated to measure 13 cytokines/markers in serum and plasma. Performance parameters including specimen dilution, specimen interference, linearity and precision were examined. Reference intervals were established using 197 normal and control CSF specimens by non-parametric quantile-based methods. CSF cytokine analysis demonstrated within and between run precision of <10% and < 20% CV, respectively and linearity of ±15% for all analytes throughout the analytical measurement range of the assay. Reference intervals for the 13 cytokines/markers were established from 197 normal and control CSF specimens (78 Male; mean 44.8 y ± 21.7 SD, 119 Female; mean 42.8 y ± 20.3 SD). Cytokine concentrations in CSF from normal donors and controls were less than the lower limit of quantitation of our assay for 6 of the 13 measured cytokines/markers. The chemokine IL8 demonstrated the highest concentration of all analytes measured. CSF demonstrated acceptable performance as a specimen type in our multiplexed cytokine assay. By validating CSF as a specimen type and establishing normal reference intervals for cytokine concentrations in CSF, their potential as biomarkers for infectious, autoimmune and other inflammatory CNS disorders can be more appropriately investigated.

Circulating pro-inflammatory cytokines are elevated and peak power output correlates with 25-hydroxyvitamin D in vitamin D insufficient adults.

The purpose of this study was to identify circulating cytokines, skeletal muscle strength, and peak power output in young adults with contrasting serum 25-hydroxyvitamin D (25(OH)D) concentrations. Serum 25(OH)D, inflammatory cytokines, muscle strength, and peak power output were, therefore, measured in young adults (25-42 years). Data were collected during the winter to avoid the seasonal influence on serum 25(OH)D. After serum 25(OH)D concentration measurements, subjects were separated into one of two groups: (1) vitamin D insufficient [serum 25(OH)D ≤32 ng/mL, n = 14], or (2) vitamin D sufficient [serum 25(OH)D >32 ng/mL, n = 14]. Following group allocation, serum 25(OH)D concentrations were significantly (p < 0.05) lower and pro-inflammatory cytokines [interleukin (IL)-2, IL-1ß, tumor necrosis factor-α, and interferon-γ] were significantly (all p < 0.05) greater in vitamin D insufficient adults. An anti-inflammatory cytokine (i.e., IL-10; p > 0.05), peak isometric forces (p > 0.05), and peak power outputs (p > 0.05) were not significantly different between vitamin D groups. However, peak power outputs correlated with serum 25(OH)D concentrations in vitamin D insufficient (r = 0.55, p < 0.05) but not in vitamin D sufficient adults (r = -0.27, p = 0.36). Based on these data, we conclude that vitamin D insufficiency, in part, could result in pro-inflammatory stress without altering muscular strength or function in young adults. Future research investigating the causality of the correlation between low-serum 25(OH)D and peak power output in young adults is required.

Circulating interferon-γ correlates with 1,25(OH)D and the 1,25(OH)D-to-25(OH)D ratio.

The mechanism responsible for the decrease in vitamin D status (i.e., plasma or serum 25-hydroxyvitamin D (25(OH)D) concentration) during inflammatory stress is unknown in humans. Interferon (IFN)-γ is an inflammatory cytokine that regulates vitamin D metabolism in isolated immune cells, but data suggesting this regulation exists in vivo is lacking. The purpose of this study, therefore, was to associate circulating IFN-γ perturbations with 25(OH)D and 1,25-dihydroxyvitamin D (1,25(OH)D) alterations during inflammatory stress in young adults recovering from anterior cruciate ligament (ACL) reconstruction. Plasma 25(OH)D, 1,25(OH)D and IFN-γ concentrations were measured in fasting blood draw samples obtained from twelve-male patients pre-surgery and 90-m, 3-d and 7-d post-surgery. 25(OH)D decreased significantly (p<0.05) after surgery, and strikingly, tended to inversely correlate (r=-0.32, p=0.058) with IFN-γ changes from pre- to post- (i.e., 90-m, 3-d, and 7-d) surgery. Additionally, 1,25(OH)D (r=0.37, p<0.05) and the 1,25(OH)D-to-25(OH)D ratio (r=0.52, p<0.05) changes from pre- to post- (i.e., 90-m, 3-d, and 7-d) surgery correlated with those of IFN-γ. These are the first reported in vivo findings suggesting that the 25(OH)D decrease and conversion to 1,25(OH)D increase with increasing IFN-γ in the circulation. We conclude that IFN-γ contributes to the decrease in vitamin D and the conversion of vitamin D to its active hormonal form in the circulation during inflammatory insult in humans.

Plasma cytokine levels reveal deficiencies in IL-8 and gamma interferon in Long-COVID.

Up to half of individuals who contract SARS-CoV-2 develop symptoms of long-COVID approximately three months after initial infection. These symptoms are highly variable, and the mechanisms inducing them are yet to be understood. We compared plasma cytokine levels from individuals with long-COVID to healthy individuals and found that those with long-COVID had 100% reductions in circulating levels of interferon gamma (IFNγ) and interleukin-8 (IL-8). Additionally, we found significant reductions in levels of IL-6, IL-2, IL-17, IL-13, and IL-4 in individuals with long-COVID. We propose immune exhaustion as the driver of long-COVID, with the complete absence of IFNγ and IL-8 preventing the lungs and other organs from healing after acute infection, and reducing the ability to fight off subsequent infections, both contributing to the myriad of symptoms suffered by those with long-COVID.

Cytokine Deficiencies in Patients with Long-COVID.

Up to half of individuals who contract SARS-CoV-2 develop symptoms of long-COVID approximately three months after initial infection. These symptoms are highly variable, and the mechanisms inducing them are yet to be understood. We compared plasma cytokine levels from individuals with long-COVID to healthy individuals and found that those with long-COVID had 100% reductions in circulating levels of Interferon Gamma (IFNγ) and Interleukin-8 (IL-8). Additionally, we found significant reductions in levels of IL-6, IL-2, IL-17, IL-13, and IL-4 in individuals with long-COVID. We propose immune exhaustion as the driver of long-COVID, with the complete absence of IFNγ and IL-8preventing the lungs and other organs from healing after acute infection, and reducing the ability to fight off subsequent infections, both contributing to the myriad of symptoms suffered by those with long-COVID.

Comprehensive analysis of antibody responses to streptococcal and tissue antigens in patients with acute rheumatic fever.

Acute rheumatic fever (ARF) is an autoimmune disease occurring in individuals following untreated group A streptococcal infection believed to be triggered by antibodies to bacterial components that cross-react with human tissues. We developed a multiplexed immunoassay for the simultaneous quantitation of antibodies to nine streptococcal-related antigens including streptolysin O (SLO), DNase B, collagen I and IV, fibronectin, myosin, group A carbohydrate, M6 protein and streptococcal C5a peptidase. Utilizing this method, we examined serum from 49 ARF, 58 pharyngitis patients and age- and sex-matched controls in samples collected at initial disease onset, and at 4 weeks, 6 months and 1 year after diagnosis. Antibody responses were significantly higher for SLO, DNase B, M6 protein, group A carbohydrate and the cross-reactive antigens collagen I and myosin in ARF compared with pharyngitis patients (P

Binding of anti-HLA class I antibody to endothelial cells produce an inflammatory cytokine secretory pattern.

Current methods are inadequate for the diagnosis of early chronic allograft rejection. The goal of this study was to determine whether ligation of anti-HLA antibodies to endothelial cells is associated with a distinctive cytokine secretory pattern. Human iliac artery endothelial cells (HIAEC) cultured in vitro were incubated with w6/32, an anti-HLA class I mAb. Culture supernatants collected daily for up to 4 days were tested for secretion of 13 cytokines using a multiplexed fluorescent microsphere immunoassay. Culture of HIAEC with medium containing mAb w6/32 supported the growth of HIAEC during the 4-day study period. Levels of the pro-inflammatory cytokines IL-1beta, IL-6, IL-8, and TNF-alpha became significantly increased in supernatants of HIAEC incubated with the mAb w6/32. We conclude that ligation of anti-HLA class I antibodies to HLA class I antigens in endothelial cells initiates an acute inflammatory process and detecting an inflammatory cytokine secretory pattern might be useful to diagnose sub-clinical chronic allograft rejection.

Laboratory Evaluation of Antiphospholipid Syndrome.

OBJECTIVES: Anti-ß2 glycoprotein I domain I (anti-domain I) and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies are present in patients with antiphospholipid syndrome (APS); however, their use in evaluation remains unclear. METHODS: Diagnostic attributes of lupus anticoagulant (LAC), anti-domain I IgG, anti-cardiolipin, anti-ß2 glycoprotein I (anti-ß2GPI), and aPS/PT IgG and IgM antibodies were assessed in 216 patients evaluated for APS. RESULTS: LAC had the best odds ratio (OR, 14.2) while that for anti-domain 1 IgG was comparable to anti-ß2GPI IgG (OR, 8.3 vs 9.4) but higher than all others. Significant correlations were observed for thrombosis (P = .03) and pregnancy-related morbidity (P = .001) with anti-domain IgG and for any thrombosis with aPS/PT IgG (P = .006). Use of noncriteria antiphospholipid with or without criteria markers did not significantly increase the probability to diagnose APS. CONCLUSIONS: Noncriteria tests can contribute to diagnosis and stratification of APS but do not improve diagnostic yield. Optimal strategies for implementation require prospective investigation.

Evaluation of a multiplex fluorescent microsphere immunoassay for the determination of epstein-barr virus serologic status.

Epstein-Barr virus (EBV), a human herpesvirus, affects up to 95% of adults. Diagnosis of acute EBV infection can be challenging and often relies on the serologic antibody pattern to 3 distinct antigens, most often determined by indirect fluorescent antibody (IFA), enzyme-linked immunosorbent assays (ELISAs), and, more recently, multiplex assays. We compared a multiplex assay for the simultaneous detection of antibodies to viral capsid (VCA), nuclear (EBNA), and early (EA) EBV antigens with ELISAs using IFA for discrepancy resolution. Concordance of the multiplex assay was good for all 4 antigens: VCA IgM, 86.6% vs ELISA and 92.9% vs IFA; VCA IgG, 92.8% vs ELISA and 98.0% vs IFA; EBNA IgG, 90.3% vs ELISA and 98.1% vs IFA; and EA IgG, 83.8% vs ELISA and 92.8% vs IFA. After IFA resolution, correlation between the multiplex assay and ELISA for serologic disease stage, based on the antibody profile of all 4 analytes, was 90%. The multiplex assay showed good correlation with an established ELISA and even better correlation with the "gold standard" IFA. Advantages of the multiplex assay over traditional methods include multiple results per assay, inclusion of internal controls for each assay, and well-to-well monitoring of assay drift.

Assessment of diagnostic methods for the detection of anticardiolipin and anti-ßeta2 glycoprotein I antibodies in patients under routine evaluation for antiphospholipid syndrome.

BACKGROUND: We assessed the performance characteristics and correlations of the traditional enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (CIA) for detecting IgG and IgM antibodies to cardiolipin (aCL) and beta2 glycoprotein (anti-ß2GPI) antibodies in patients under routine evaluation for APS. METHODS: Patients (n = 216) referred to ARUP Laboratories for lupus anticoagulant (LAC) and/or aCL or anti-ß2GPI IgG/IgM antibodies evaluation were assessed by ELISA and CIA methods. Diagnostic accuracies, correlations between methods and specific clinical manifestations in APS were investigated. RESULTS: The areas under the curve (%) for APS using LAC with CIA (74, 95% CI: 65-82) or ELISA (70, 95% CI: 61-79) aPLs were comparable. The overall agreements and linear regression correlations between methods for aPL antibody of the same specificity were variable: aCL IgG 87.3%; R2 = 0.7491, aCL IgM 71.6%; R2 = 0.2656, anti-ß2GPI IgG 77.2%; R2 = 0.7688 and anti-ß2GPI IgM 81.7%; R2 = 0.3305. CONCLUSIONS: With inclusion of LAC, the ELISA and CIA show comparable performance for the diagnosis of APS. However, correlations of APS-specific manifestations were dependent on method of detecting the aPL antibodies suggesting platforms may not be used interchangeable.

Comparison of three multiplex immunoassays for detection of antibodies to extractable nuclear antibodies using clinically defined sera.

We set out to determine the agreement of three multiplex immunoassays for the detection of autoantibodies involved in connective tissue disease using clinically defined sera. Usefulness of the immunoassays will be defined by correlation to disease state. Using the immunoassays from Inova Diagnostics, Biomedical Diagnostics (BMD), and AtheNA, reactivity, to Smith (Sm), ribonucleic protein (RNP), SSA (Ro), SSB (La), Scl-70, and dsDNA or chromatin, we tested 273 clinically defined sera consisting of 57 systemic lupus erythrematosus (SLE) sera, 69 rheumatoid arthritis (RA) sera, 47 sera defined as various other connective tissue diseases, and 100 normal donor sera. Samples were also tested for anti-nuclear antibody (ANA) oN HEp-2 cells by IFA for analysis of discrepant results. Inova, BMD, and AtheNA assays demonstrated 57%, 89%, and 80% concordance, respectively, in the 47 connective tissue disease sera. The BMD assay was the most sensitive in detecting Scl-70. The immunoassays did not correlate well in the 57 SLE-defined sera; however, each had a variety of antibodies positive for each serum. The AtheNA assay demonstrated the highest degree of nonspecificity. Inova, BMD, AtheNA, and the Inova ANA HEp-2 IFA demonstrated 97%, 98%, 97%, and 99% specificity, respectively, using normal sera. Thus, all three assays showed a 97% or better negative predictive value. Positive correlation varied from 83% to 98%. Antibodies in SLE sera did not compare well among the three immunoassays. Significant variation in specificity and sensitivity due to individual characteristics of each assay was demonstrated, with the BMD assay showing the highest correlation with clinical diagnosis.

Development of a multiplexed fluorescent immunoassay for the quantitation of antibody responses to group A streptococci.

The host immunologic response to group A streptococcal infections gives rise to numerous antibodies directed against cellular and extracellular bacterial antigens. For determining individual immune status, or studying the pathogenesis of group A streptococcal associated diseases, such as acute rheumatic fever (ARF), an assay capable of determining antibodies responses to multiple antigens would be of great advantage. We have developed a microsphere based, multiplexed immunoassay for the simultaneous quantitation of antibodies to nine different extracellular, ARF related tissue and group A streptococci specific antigens using only 5 microl of sample. Through the selection of microspheres and serum diluent, non-specific antibody binding was reduced by 17%. Different formulations of the coupling buffer were found to greatly influence the efficiency of coupling antigens to the carboxylated microspheres. Monoclonal antibodies against the different antigens demonstrated assay specificity as well as sensitivities of less than 1 ng/ml of antibody. This multiplexed assay should be a powerful research and clinical tool in determining antibody responses to group A streptococcal infections and in potentially determining the role of a variety of cross-reactive antigens in rheumatic fever and rheumatic heart disease.

Performance characteristics of six IMMULITE 2000 TORCH assays.

TORCH is an acronym for Toxoplasma gondii (Toxo), other microorganisms (eg, syphilis), rubella virus (RV), cytomegalovirus (CMV), and herpes simplex virus (HSV) that are associated with congenital abnormalities from maternal infection. We evaluated linearity, imprecision, and comparison with commercially available methods of the IMMULITE 2000 (Diagnostic Products, Los Angeles, CA) Toxo IgG, Toxo IgM, RV IgG, RV IgM, CMV IgG, and HSV IgG assays. Linearity and imprecision results were acceptable. The IMMULITE 2000 assays show good concordance with other commercially available methods except for Toxo IgM and RV IgM. Toxo IgM showed better concordance with a consensus of 3 of 4 (Access, Beckman Coulter, Fullerton, CA; IMMULITE 2000; Platelia, Bio-Rad Laboratories Diagnostics Group, Redmond, WA; and Vidas, bioMerieux, Hazelwood, MO) assays than with Access alone. The RV IgM assay showed better concordance with the Zeus method than with the Diamedix method (Diamedix, Miami, FL). The IMMULITE 2000 TORCH assays studied show acceptable performance and are suitable for routine clinical use. Some commercial assays for Toxo IgM and RV IgM show rather poor concordance.