Cross-Platform Comparison of Highly Sensitive Immunoassays for Inflammatory Markers in a COVID-19 Cohort.

A variety of commercial platforms are available for the simultaneous detection of multiple cytokines and associated proteins, often employing Ab pairs to capture and detect target proteins. In this study, we comprehensively evaluated the performance of three distinct platforms: the fluorescent bead-based Luminex assay, the proximity extension-based Olink assay, and a novel proximity ligation assay platform known as Alamar NULISAseq. These assessments were conducted on human serum samples from the National Institutes of Health IMPACC study, with a focus on three essential performance metrics: detectability, correlation, and differential expression. Our results reveal several key findings. First, the Alamar platform demonstrated the highest overall detectability, followed by Olink and then Luminex. Second, the correlation of protein measurements between the Alamar and Olink platforms tended to be stronger than the correlation of either of these platforms with Luminex. Third, we observed that detectability differences across the platforms often translated to differences in differential expression findings, although high detectability did not guarantee the ability to identify meaningful biological differences. Our study provides valuable insights into the comparative performance of these assays, enhancing our understanding of their strengths and limitations when assessing complex biological samples, as exemplified by the sera from this COVID-19 cohort.

Cross-platform comparison of highly-sensitive immunoassays for inflammatory markers in a COVID-19 cohort 1.

A variety of commercial platforms are available for the simultaneous detection of multiple cytokines and associated proteins, often employing antibody pairs to capture and detect target proteins. In this study, we comprehensively evaluated the performance of three distinct platforms: the fluorescent bead-based Luminex assay, the proximity extension-based Olink assay, and a novel proximity ligation assay platform known as Alamar NULISAseq. These assessments were conducted on serum samples from the NIH IMPACC study, with a focus on three essential performance metrics: detectability, correlation, and differential expression. Our results reveal several key findings. Firstly, the Alamar platform demonstrated the highest overall detectability, followed by Olink and then Luminex. Secondly, the correlation of protein measurements between the Alamar and Olink platforms tended to be stronger than the correlation of either of these platforms with Luminex. Thirdly, we observed that detectability differences across the platforms often translated to differences in differential expression findings, although high detectability did not guarantee the ability to identify meaningful biological differences. Our study provides valuable insights into the comparative performance of these assays, enhancing our understanding of their strengths and limitations when assessing complex biological samples, as exemplified by the sera from this COVID-19 cohort.

Azithromycin promotes proliferation, and inhibits inflammation in nasal epithelial cells in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetic disorder associated with recurrent and chronic respiratory infections due to functional defects of motile cilia. In this study, we aimed to elucidate inflammatory and proliferative responses in PCD respiratory epithelium and evaluate the effect of Azithromycin (AZT) on these responses. Airway basal cells (BCs) were isolated from nasal samples of Wild-type (WT) epitope of healthy donors and PCD donors with bi-allelic mutations in DNAH5, DNAH11 and CCDC39. Cells were expanded in vitro and stimulated with either Lipopolysaccharide (LPS) or vehicle control. Post stimulation, cells were treated with either Azithromycin (AZT) or vehicle control. Cell proliferation was imaged in real-time. Separately, BCs from the same donors were expanded and grown at an air-liquid interface (ALI) to generate a multi-ciliated epithelium (MCE). Once fully mature, cells were stimulated with LPS, AZT, LPS + AZT or vehicle control. Inflammatory profiling was performed on collected media by cytokine Luminex assay. At baseline, there was a significantly higher mean production of pro-inflammatory cytokines by CCDC39 BCs and MCEs when compared to WT, DNAH11 and DNAH5 cells. AZT inhibited production of cytokines induced by LPS in PCD cells. Differences in cell proliferation were noted in PCD and this was also corrected with AZT treatment.

Acute Respiratory Illness Is Associated with Memory T Cell Differentiation and Other Immune Cell Changes in an Age-Associated Manner.

Respiratory viruses such as influenza are encountered multiple times through infection and/or vaccination and thus have the potential to shape immune cell phenotypes over time. In particular, memory T cell compartments may be affected, as both CD4+ and CD8+ T cell responses likely contribute to viral control. In this study, we assessed immune phenotypes using cytometry by time of flight in the peripheral blood of 22 humans with acute respiratory illness and 22 age-matched noninfected controls. In younger infected individuals (1-19 y of age), we found decreased B and NK cell frequencies and a shift toward more effector-like CD4+ and CD8+ T cell phenotypes, compared with young healthy controls. Significant differences between noninfected and infected older individuals (30-74 y of age) were not seen. We also observed a decrease in naive CD4+ T cells and CD27+CD8+ T cells as well as an increase in effector memory CD8+ T cells and NKT cells in noninfected individuals with age. When cell frequencies were regressed against age for infected versus noninfected subjects, significant differences in trends with age were observed for multiple cell types. These included B cells and various subsets of CD4+ and CD8+ T cells. We conclude that acute respiratory illness drives T cell differentiation and decreases circulating B cell frequencies preferentially in young compared with older individuals.

Relationship of Heterologous Virus Responses and Outcomes in Hospitalized COVID-19 Patients.

The clinical trajectory of COVID-19 may be influenced by previous responses to heterologous viruses. We examined the relationship of Abs against different viruses to clinical trajectory groups from the National Institutes of Health IMPACC (Immunophenotyping Assessment in a COVID-19 Cohort) study of hospitalized COVID-19 patients. Whereas initial Ab titers to SARS-CoV-2 tended to be higher with increasing severity (excluding fatal disease), those to seasonal coronaviruses trended in the opposite direction. Initial Ab titers to influenza and parainfluenza viruses also tended to be lower with increasing severity. However, no significant relationship was observed for Abs to other viruses, including measles, CMV, EBV, and respiratory syncytial virus. We hypothesize that some individuals may produce lower or less durable Ab responses to respiratory viruses generally (reflected in lower baseline titers in our study), and that this may carry over into poorer outcomes for COVID-19 (despite high initial SARS-CoV-2 titers). We further looked at longitudinal changes in Ab responses to heterologous viruses, but found little change during the course of acute COVID-19 infection. We saw significant trends with age for Ab levels to many of these viruses, but no difference in longitudinal SARS-CoV-2 titers for those with high versus low seasonal coronavirus titers. We detected no difference in longitudinal SARS-CoV-2 titers for CMV seropositive versus seronegative patients, although there was an overrepresentation of CMV seropositives among the IMPACC cohort, compared with expected frequencies in the United States population. Our results both reinforce findings from other studies and suggest (to our knowledge) new relationships between the response to SARS-CoV-2 and Abs to heterologous viruses.

A taxonomy of multiple regression methods for immunologists.

Multiple regression is a powerful tool in the immunologist's toolbox. This paper defines multiple regression, discusses availability and accessibility, provides some additional helpful definitions, treats the topics of transformation and extreme value screening, and establishes the paper's scope and philosophy. Then eleven methods of multiple regression are detailed, giving strengths and limitations. Throughout an emphasis is placed on application to immunological assays. A flowchart to guide selection of multiple regression methods is provided.

Epigenetic and immunological indicators of IPEX disease in subjects with FOXP3 gene mutation.

BACKGROUND: Forkhead box protein 3 (FOXP3) is the master transcription factor in CD4+CD25hiCD127lo regulatory T (Treg) cells. Mutations in FOXP3 result in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome. Clinical presentation of IPEX syndrome is broader than initially described, challenging the understanding of the disease, its evolution, and treatment choice. OBJECTIVE: We sought to study the type and extent of immunologic abnormalities that remain ill-defined in IPEX, across genetic and clinical heterogeneity. METHODS: We performed Treg-cell-specific epigenetic quantification and immunologic characterization of severe "typical" (n = 6) and "atypical" or asymptomatic (n = 9) patients with IPEX. RESULTS: Increased number of cells with Treg-cell-Specific Demethylated Region demethylation in FOXP3 is a consistent feature in patients with IPEX, with (1) highest values in those with typical IPEX, (2) increased values in subjects with pathogenic FOXP3 but still no symptoms, and (3) gradual increase over the course of disease progression. Large-scale profiling using Luminex identified plasma inflammatory signature of macrophage activation and TH2 polarization, with cytokines previously not associated with IPEX pathology, including CCL22, CCL17, CCL15, and IL-13, and the inflammatory markers TNF-α, IL-1A, IL-8, sFasL, and CXCL9. Similarly, both Treg-cell and Teff compartments, studied by Mass Cytometry by Time-Of-Flight, were skewed toward the TH2 compartment, especially in typical IPEX. CONCLUSIONS: Elevated TSDR-demethylated cells, combined with elevation of plasmatic and cellular markers of a polarized type 2 inflammatory immune response, extends our understanding of IPEX diagnosis and heterogeneity.

Birds with multiple homes. The annual cycle of the pallid swift (Apus pallidus brehmorum).

We tracked pallid swifts (Apus pallidus brehmorum) from a single breeding colony in Gibraltar over two years. Our results show movement of birds between specific regions within the non-breeding geographical area at specific times of the year. The tracking of a single individual showed remarkable fidelity to the areas visited between years. Furthermore, two pallid swifts tracked over the entire eight-month non-breeding period, while in Africa, gave no indication of coming to land, supporting previous findings of an airborne existence in swifts outside the breeding season. In addition, the crossing of the Sahara Desert to and from breeding grounds is remarkably fast, with one individual crossing it in just over a day. We discuss our findings in the context of bird migration evolutionary strategies.

Crag Martin neontology complements taphonomy at the Gorham's Cave Complex.

Species present in the fossil record may continue to exist at an archaeological site, allowing study that fine-tunes our picture of the ecological past. A large wintering population of Eurasian Crag Martins Ptyonoprogne rupestris (ECM) roosts at the 'Gorham's Cave Complex' UNESCO World Heritage site in Gibraltar, which is best known for its occupation by Neanderthals at times when ECMs were also present. Its complex geomorphology allows the study of use of different micro-sites (caves) within the roost. We used mark-recapture to test whether birds showed fidelity to micro-sites for roosting, and for differences in condition of birds across micro-sites. ECM showed very high fidelity towards micro-sites, within and between years, with > 90% chance of recapture at caves where they were first caught. Condition of birds differed between micro-sites, suggesting differences in roost quality between caves; birds were more likely to be recaptured at the micro-site where birds were in best condition, indicating higher survivorship. Our results demonstrate extremely fine-scale fidelity at the largest roosting site documented for ECM globally. Implications for conservation are discussed. The study provides current knowledge of a bird that has been using these caves since the Pleistocene and more generally on these caves as refuges.

Rigorous quantification of bacterial richness in ticks: A case study.

Kwan et al. (2017) published an informative study comparing results obtained by next-generation sequencing (NGS) of mean bacterial genera richness among different life stages, male and female adults, and rearing conditions (field vs. laboratory) for Ixodes pacificus. The current paper examines Kwan et al. (2017) as a case study to provide guidance on statistical design and analysis for estimation of richness, derived from next generation sequencing technology, of the bacterial microbiome in field-collected I. pacificus. Suggestions are provided to further strengthen quantification of microbiome richness in studies in ticks, with focus on sampling design. In-depth treatment is provided of the relative merits of estimating mean richness versus median richness. Research on microbiome diversity in ticks can be made quantitatively rigorous; although, more research on methods is needed.

Mass Cytometry Defines Virus-Specific CD4+ T Cells in Influenza Vaccination.

The antiviral response to influenza virus is complex and multifaceted, involving many immune cell subsets. There is an urgent need to understand the role of CD4+ T cells, which orchestrate an effective antiviral response, to improve vaccine design strategies. In this study, we analyzed PBMCs from human participants immunized with influenza vaccine, using high-dimensional single-cell proteomic immune profiling by mass cytometry. Data were analyzed using a novel clustering algorithm, denoised ragged pruning, to define possible influenza virus-specific clusters of CD4+ T cells. Denoised ragged pruning identified six clusters of cells. Among these, one cluster (Cluster 3) was found to increase in abundance following stimulation with influenza virus peptide ex vivo. A separate cluster (Cluster 4) was found to expand in abundance between days 0 and 7 postvaccination, indicating that it is vaccine responsive. We examined the expression profiles of all six clusters to characterize their lineage, functionality, and possible role in the response to influenza vaccine. Clusters 3 and 4 consisted of effector memory cells, with high CD154 expression. Cluster 3 expressed cytokines like IL-2, IFN-γ, and TNF-α, whereas Cluster 4 expressed IL-17. Interestingly, some participants had low abundance of Clusters 3 and 4, whereas others had higher abundance of one of these clusters compared with the other. Taken together, we present an approach for identifying novel influenza virus-reactive CD4+ T cell subsets, a method that could help advance understanding of the immune response to influenza, predict responsiveness to vaccines, and aid in better vaccine design.

Generalized mathematical model for immunoassay interference.

As part of a landmark review of the antigen excess, Jacobs et al. (Autoimmunity Reviews 2015;14:160-167) contrast how immunoassay "interference" by non-target biomolecules can cause spurious readings for clinical diagnostic tests. The purpose of the present Letter is to complement and analytically extend this description by Jacobs et al. by briefly presenting a generalized mathematical model of immunoassay interference that is derived from a simple mechanistic system of differential equations. Derivation includes expressions for the location of the peak concentration of bound reporter as well as that maximum concentration of target biomolecule at which immunoassay interference drives concentration of bound reporter to zero. This parsimonious, mechanistic, and generalized model may prove foundational to analytic study of immunoassay interference. More comprehensive models for specific classes of immunoassays have and can be developed.

Penalized Supervised Star Plots: Example Application in Influenza-Specific CD4+ T Cells.

An immune cell's phenotype expresses through its high-dimensional marker signature. Cluster analyses of data from high-throughput mass and flow cytometry marker panels permit discovery of previously undescribed immune cell phenotypes. Impactful reporting of new phenotypes demands low-dimensional visualization tools that preserve with integrity phenotypes' original high-dimensional structure. For this purpose, we introduce penalized supervised star plots. As designed and as we demonstrate, penalized supervised star plots are two-dimensional projections that tend to preserve separation of clusters as well as information on the relative contributions of various markers in differentiating phenotypes. The new method is robust to markers that do not differentiate phenotypes at all, as shown in a challenge data set. Results include comparison with other popular procedures. Penalized supervised star plots incorporate cross-validation to permit portability of estimated optimal projections to new samples. Supervised star plots are further illustrated with a featured influenza-specific T cell data set as well as a peripheral blood mononuclear cell phenotyping data set.

Diminished B-Cell Response After Repeat Influenza Vaccination.

Annual vaccination with influenza vaccines is recommended for protection against influenza in the United States. Past clinical studies and meta-analysis, however, have reported conflicting results on the benefits of annual vaccination. B-cell responses elicited following repeat influenza vaccinations over multiple seasons have not been examined in detail. We analyzed the B-cell and antibody (Ab) responses in volunteers vaccinated yearly, from 2010 or 2011 through 2014, with seasonal trivalent inactivated influenza vaccines. Statistical analyses were designed to help correct for possible bias due to reduced sample size in the later years of the study. We show that, after the second annual vaccination, the frequency of vaccine-specific plasmablasts and the binding reactivity of plasmablast-derived polyclonal Abs are reduced and do not increase in subsequent years. Similar trends are observed with the serum hemagglutination inhibition Ab response after each annual vaccination, as well as the binding reactivity of plasmablast-derived polyclonal Abs to the hemagglutinin of influenza A virus vaccine components, even with changes in the seasonal vaccine components during the study. Our findings indicate a diminished B-cell response to annual vaccination with seasonal trivalent influenza vaccine. These results emphasize the need for developing improved strategies to enhance the immunogenicity and efficacy of annual influenza vaccination.

Differences in multiple immune parameters between Indian and U.S. infants.

To compare immune phenotypes across two geographic and ethnic communities, we examined umbilical cord blood by flow cytometry and Luminex in parallel cohorts of 53 newborns from New Delhi, India, and 46 newborns from Stanford, California. We found that frequencies of a B cell subset suggested to be B-1-like, and serum IgM concentration were both significantly higher in the Stanford cohort, independent of differences in maternal age. While serum IgA levels were also significantly higher in the Stanford cohort, IgG1, IgG2, and IgG4 were significantly higher in the New Delhi samples. We found that neutrophils, plasmacytoid dendritic cells, CD8+ T cells, and total T cells were higher in the U.S. cohort, while dendritic cells, patrolling monocytes (CD14dimCD16+), natural killer cells, CD4+ T cells, and naïve B cells were higher in the India cohort. Within the India cohort, we also identified cell types whose frequency was positively or negatively predictive of occurrence of infection(s) in the first six months of life. Monocytes, total T cells, and memory CD4+ T cells were most prominent in having an inverse relationship with infection. We suggest that these data provide impetus for follow-up studies linking phenotypic differences to environmental versus genetic factors, and to infection outcomes.

Deception in clinical trials and its impact on recruitment and adherence of study participants.

Deceptive practices by participants in clinical research are prevalent. It has been shown that as high as 75% of participants withheld information to avoid exclusion from studies. Self-reported adherence has been found to be largely inaccurate. Overcoming deception is a critical issue, since the safety of study participants, the integrity of research data and research resources are at risk. In this review article, we examine deception from the perspective of investigators conducting clinical trials; we describe the types (concealment, fabrication, drug holidays and collusion), prevalence, risks, and predictors of deception, and propose an approach to reduce the impact of deception, especially on adherence, in clinical trials.

Genetic Characterization of Toxoplasma gondii DNA Samples Isolated From Humans Living in North America: An Unexpected High Prevalence of Atypical Genotypes.

Background: Whereas in Europe most of Toxoplasma gondii genotypes belong to the type II lineage, in Latin America, type II is rare and atypical strains predominate. In North America, data on T. gondii genotypes in humans are scarce. Methods: In this study, T. gondii DNA samples from 67 patients with diagnosed toxoplasmosis in the United States were available for genotyping. Discriminant analysis of principal components was used to infer each atypical genotype to a geographic area where patients were probably infected. Associations between genotype, disease severity, immune status, and geographic region were also estimated. Results: Of 67 DNA samples, 41 were successfully genotyped: 18 (43.9%) and 5 (12.2%) were characterized as types II and III, respectively. The remaining 18 genotypes (43.9%) were atypical and were assigned to a geographic area. Ten genotypes originated from Latin America, 7 from North America, and 1 from Asia (China). In North America, unlike in Europe, T. gondii atypical genotypes are common in humans and, unlike in Latin America, type II strains are still present with significant frequency. Conclusions: Clinicians should be aware that atypical genotypes are common in North America and have been associated with severe ocular and systemic disease and unusual presentations of toxoplasmosis in immunocompetent patients.

Linear-rank testing of a non-binary, responder-analysis, efficacy score to evaluate pharmacotherapies for substance use disorders.

The design of pharmacological trials for management of substance use disorders is shifting toward outcomes of successful individual-level behavior (abstinence or no heavy use). While binary success/failure analyses are common, McCann and Li (CNS Neurosci Ther 2012; 18: 414-418) introduced "number of beyond-threshold weeks of success" (NOBWOS) scores to avoid dichotomized outcomes. NOBWOS scoring employs an efficacy "hurdle" with values reflecting duration of success. Here, we evaluate NOBWOS scores rigorously. Formal analysis of mathematical structure of NOBWOS scores is followed by simulation studies spanning diverse conditions to assess operating characteristics of five linear-rank tests on NOBWOS scores. Simulations include assessment of Fisher's exact test applied to hurdle component. On average, statistical power was approximately equal for five linear-rank tests. Under none of conditions examined did Fisher's exact test exhibit greater statistical power than any of the linear-rank tests. These linear-rank tests provide good Type I and Type II error control for comparing distributions of NOBWOS scores between groups (e.g. active vs. placebo). All methods were applied to re-analyses of data from four clinical trials of differing lengths and substances of abuse. These linear-rank tests agreed across all trials in rejecting (or not) their null (equality of distributions) at ≤ 0.05.