Effective early antiretroviral therapy in perinatal-HIV infection reduces subsequent plasma inflammatory profile.

BACKGROUND: The long-term immunologic effects of antiretroviral therapy (ART) in children with perinatally-acquired HIV (PHIV) have not been fully elucidated. Here, we investigated how the timing of ART initiation affects the long-term immune profile of children living with PHIV by measuring immunomodulatory plasma cytokines, chemokines, and adenosine deaminases (ADAs). METHODS: 40 PHIV participants initiated ART during infancy. 39 participant samples were available; 30 initiated ART ≤6 months (early-ART treatment); 9 initiated ART >6 months and <2 years (late-ART treatment). We compared plasma cytokine and chemokine concentrations and ADA enzymatic activities between early-ART and late-ART treatment 12.5 years later and measured correlation with clinical covariates. RESULTS: Plasma concentrations of 10 cytokines and chemokines (IFNγ, IL-12p70, IL-13, IL-17A, IL-IRA, IL-5, IL-6, and IL-9 as well as CCL7, CXCL10), ADA1, and ADA total were significantly higher in late-ART compared to early-ART treatment. Furthermore, ADA1 was significantly positively correlated with IFNγ, IL-17A, and IL-12p70. Meanwhile, total ADA was positively correlated with IFNγ, IL-13, IL-17A, IL-1RA, IL-6, and IL-12p70 as well as CCL7. CONCLUSIONS: Elevation of several pro-inflammatory plasma analytes in late-ART despite 12.5 years of virologic suppression compared to early-ART treatment suggests that early treatment dampens the long-term plasma inflammatory profile in PHIV participants. IMPACT: This study examines differences in the plasma cytokine, chemokine, and ADA profiles 12.5 years after treatment between early (≤6months) and late (>6 months and <2 years) antiretroviral therapy (ART) treatment initiation in a cohort of European and UK study participants living with PHIV. Several cytokines and chemokines (e.g., IFNγ, IL-12p70, IL-6, and CXCL10) as well as ADA-1 are elevated in late-ART treatment in comparison to early-ART treatment. Our results suggest that effective ART treatment initiated within 6 months of life in PHIV participants dampens a long-term inflammatory plasma profile as compared to late-ART treatment.

Perinatally Human Immunodeficiency Virus-Infected Adolescents and Young Adults Demonstrate Distinct BNT162b2 Messenger RNA Coronavirus Disease 2019 Vaccine Immunogenicity.

BACKGROUND: Immunization of vulnerable populations with distinct immunity often results in suboptimal immunogenicity, durability, and efficacy. METHODS: Safety and immunogenicity profiles of BNT162b2 messenger RNA coronavirus disease 2019 (COVID-19) vaccine, among people living with human immunodeficiency virus (HIV), were evaluated in 28 perinatally HIV-infected patients under antiretroviral therapy (ART) and 65 healthy controls (HCs) with no previous history of COVID-19. Thus, we measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific humoral and CD4+ T cell responses. Samples were collected before vaccination (baseline, day [D] 0), at the second dose (D21), and at 4 weeks (D28) and 6 months (D180) after D0. Proteomic profiles at D0 and D28 were assessed with a multiplexed proximity extension assay (Olink) on plasma samples. RESULTS: All HIV-infected patients mounted similar anti-SARS-CoV-2 humoral responses to those of HCs, albeit with lower titers of anti-trimeric S at D28 (P = .01). Only peripheral blood mononuclear cells of HIV-infected patients demonstrated at D28 an impaired ability to expand their specific (CD40L+) CD4+ T-cell populations. Similar humoral titers were maintained between the 2 groups at 6-months follow-up. We additionally correlated baseline protein levels to either humoral or cellular responses, identifying clusters of molecules involved in immune response regulation with inverse profiles between the 2 study groups. CONCLUSIONS: Responses of ART-treated HIV-infected patients, compared to those of HCs, were characterized by distinct features especially within the proteomic compartment, supporting their eligibility to an additional dose, similarly to the HC schedule.

Innate Immune Responses and Gut Microbiomes Distinguish HIV-Exposed from HIV-Unexposed Children in a Population-Specific Manner.

In both high- and low-income countries, HIV-negative children born to HIV-positive mothers (HIV exposed, uninfected [HEU]) are more susceptible to severe infection than HIV-unexposed, uninfected (HUU) children, with altered innate immunity hypothesized to be a cause. Both the gut microbiome and systemic innate immunity differ across biogeographically distinct settings, and the two are known to influence each other. And although the gut microbiome is influenced by HIV infection and may contribute to altered immunity, the biogeography of immune-microbiome correlations among HEU children have not been investigated. To address this, we compared the innate response and the stool microbiome of 2-y-old HEU and HUU children from Belgium, Canada, and South Africa to test the hypothesis that region-specific immune alterations directly correlate to differences in their stool microbiomes. We did not detect a universal immune or microbiome signature underlying differences between HEU versus HUU that was applicable to all children. But as hypothesized, population-specific differences in stool microbiomes were readily detected and included reduced abundances of short-chain fatty acid-producing bacteria in Canadian HEU children. Furthermore, we did not identify innate immune-microbiome associations that distinguished HEU from HUU children in any population. These findings suggest that maternal HIV infection is independently associated with differences in both innate immunity and the stool microbiome in a biogeographical population-specific way.

Ontogeny of plasma cytokine and chemokine concentrations across the first week of human life.

INTRODUCTION/BACKGROUND & AIMS: Early life is marked by distinct and rapidly evolving immunity and increased susceptibility to infection. The vulnerability of the newborn reflects development of a complex immune system in the face of rapidly changing demands during the transition to extra-uterine life. Cytokines and chemokines contribute to this dynamic immune signaling network and can be altered by many factors, such as infection. Newborns undergo dynamic changes important to health and disease, yet there is limited information regarding human neonatal plasma cytokine and chemokine concentrations over the first week of life. The few available studies are limited by small sample size, cross-sectional study design, or focus on perturbed host states like severe infection or prematurity. To characterize immune ontogeny among healthy full-term newborns, we assessed plasma cytokine and chemokine concentrations across the first week of life in a robust longitudinal cohort of healthy, full-term African newborns. METHODS: We analyzed a subgroup of a cohort of healthy newborns at the Medical Research Council Unit in The Gambia (West Africa; N = 608). Peripheral blood plasma was collected from all study participants at birth (day of life (DOL) 0) and at one follow-up time point at DOL 1, 3, or 7. Plasma cytokine and chemokine concentrations were measured by bead-based cytokine multiplex assay. Unsupervised clustering was used to identify patterns in plasma cytokine and chemokine ontogeny during early life. RESULTS: We observed an increase across the first week of life in plasma Th1 cytokines such as IFNγ and CXCL10 and a decrease in Th2 and anti-inflammatory cytokines such as IL-6 and IL-10, and chemokines such as CXCL8. In contrast, other cytokines and chemokines (e.g. IL-4 and CCL5, respectively) remained unchanged during the first week of life. This robust ontogenetic pattern did not appear to be affected by gestational age or sex. CONCLUSIONS: Ontogeny is a strong driver of newborn plasma-based levels of cytokines and chemokines throughout the first week of life with a rising IFNγ axis suggesting post-natal upregulation of host defense pathways. Our study will prove useful to the design and interpretation of future studies aimed at understanding the neonatal immune system during health and disease.

Epigenetic basis for monocyte dysfunction in patients with severe alcoholic hepatitis.

BACKGROUND & AIMS: Severe forms of alcohol-related liver disease are associated with increased susceptibility to infections which are associated with poor prognosis. The cellular and molecular mechanisms responsible for this altered host defense are incompletely understood. METHODS: We performed whole blood phenotypic analysis and ex vivo stimulation with various pathogen-associated molecular patterns (PAMPs). We included 34 patients with alcohol-related cirrhosis (18 of whom had biopsy-proven severe alcoholic hepatitis [sAH]), 12 healthy controls and 11 patients with chronic alcohol consumption without significant liver disease. We also evaluated the transcriptomic (RNA-seq) and chromatin accessibility (ATAC-seq) profiles of CD14+ monocytes from a subset of patients. RESULTS: Circulating monocytes and conventional dendritic cells (DCs) from patients with sAH displayed complex alterations characterized by increased expression of both activating and inhibitory surface markers and an impaired pro-inflammatory response upon stimulation with PAMPs representative of gram-negative bacteria (lipopolysaccharide, Pam3CSK4) or fungal pathogens (Zymosan). Their decreased ability to produce more than 1 cytokine (polyfunctionality) upon PAMP stimulation correlated with the risk of developing infection at 28 days or mortality at 90 days. The presence of acute-on-chronic liver failure in patients with sAH did not significantly modify the immune profile of monocytes and DCs. Moreover, CD14+ monocytes of patients with sAH displayed altered transcriptional and epigenomic profiles characterized by downregulation of key innate immune and metabolic pathways and upregulation of important immunomodulatory factors. CONCLUSIONS: In patients with sAH, the altered transcriptional program and functional properties of monocytes that contribute to patients' susceptibility to infection have strong epigenetic determinants. LAY SUMMARY: Patients with severe alcoholic hepatitis are at increased risk of infections, which contribute to the poor prognosis associated with the disease. Herein, we show that epigenetic determinants underly the immune cell dysfunction and inappropriate responses to pathogens that are associated with severe alcoholic hepatitis.

Immunometabolic approaches to prevent, detect, and treat neonatal sepsis.

The first days of postnatal life are energetically demanding as metabolic functions change dramatically to accommodate drastic environmental and physiologic transitions after birth. It is increasingly appreciated that metabolic pathways are not only crucial for nutrition but also play important roles in regulating inflammation and the host response to infection. Neonatal susceptibility to infection is increased due to a functionally distinct immune response characterized by high reliance on innate immune mechanisms. Interactions between metabolism and the immune response are increasingly recognized, as changes in metabolic pathways drive innate immune cell function and activation and consequently host response to pathogens. Moreover, metabolites, such as acetyl-coenzyme A (acetyl-CoA) and succinate have immunoregulatory properties and serve as cofactors for enzymes involved in epigenetic reprogramming or "training" of innate immune cells after an initial infectious exposure. Highly sensitive metabolomic approaches allow us to define alterations in metabolic signatures as they change during ontogeny and as perturbed by immunization or infection, thereby linking metabolic pathways to immune cell effector functions. Characterizing the ontogeny of immunometabolism will offer new opportunities to prevent, diagnose, and treat neonatal sepsis.

Initiation of Antiretroviral Therapy Before Pregnancy Reduces the Risk of Infection-related Hospitalization in Human Immunodeficiency Virus-exposed Uninfected Infants Born in a High-income Country.

BACKGROUND: Epidemiological studies conducted in low- and high-income countries showed that infants exposed to maternal human immunodeficiency virus (HIV) have a high risk of severe infections. Immune alterations during fetal life have been proposed as a possible mechanism. METHODS: This prospective study assessed the relative risk of hospitalization for infection in HIV-exposed uninfected (HEU) infants as compared to HIV-unexposed (HU) infants born in a high-income country (HIC). Markers of monocyte activation and levels of pathogen-specific antibodies were measured at birth to identify correlates of infant susceptibility. RESULTS: There were 27 of 132 HEU infants and 14 of 123 HU infants hospitalized for infection during the first year of life (adjusted hazard ratio [aHR] 2.33, 95% confidence interval [CI] 1.10-4.97). Most of this increased risk was associated with the time of initiation of maternal antiretroviral therapy (ART). As compared to HU infants, the risk of hospitalization for infection of HEU infants was 4-fold higher when mothers initiated ART during pregnancy (aHR 3.84, 95% CI 1.69-8.71) and was not significantly increased when ART was initiated before pregnancy (aHR 1.42, 95% CI 0.58-3.48). The activation of newborn monocytes and the reduced transfer of maternal antibodies were most intense following ART initiation during pregnancy, and predicted the risk of infant hospitalization. CONCLUSIONS: These observations indicate that initiation of maternal ART before pregnancy reduces the susceptibility of HEU infants born in a HIC to severe infections, and that this effect could be related to the prevention of immune alterations during fetal life.

Absolute lymphocyte counts at end of induction correlate with distinct immune cell compartments in pediatric B cell precursor acute lymphoblastic leukemia.

Several retrospective studies in children with B cell precursor (BCP) acute lymphoblastic leukemia (ALL) provided clinical evidence that higher absolute lymphocyte counts (ALC) early into treatment significantly correlated with improved relapse-free and overall survival. It still remains unknown, however, whether the predictive role of higher ALCs reflects general bone marrow recovery or a more specific attribute of immune function. To investigate this question, we implemented a prospective observational cohort study in 20 children with BCP ALL on day 29 (D29) of induction chemotherapy and immunophenotyped their lymphoid (T, B and natural killer cells) and myeloid (neutrophils, monocytes, dendritic cells) compartments. In a first evaluation of a cohort treated with Children's Oncology Group-based induction chemotherapy, the immune cell compartments were differentially depleted at D29. Neither gender, risk status, minimal residual disease, nor bone marrow recovery markers correlated with D29 ALC. In contrast, both CD3+ T cell and dendritic cell compartments, which did not correlate with age, significantly correlated with D29 ALC (p < 0.0001). In addition, subset complexity of cellular immune compartments was preserved at D29. This study reveals that D29 ALC significantly correlates with distinct immune cell compartments but not with bone marrow recovery markers, suggesting that higher D29 ALCs may contribute to leukemia control by inducing specific host immune activity.

Single-cell analysis of innate cytokine responses to pattern recognition receptor stimulation in children across four continents.

Innate immunity instructs adaptive immunity, and suppression of innate immunity is associated with an increased risk for infection. We showed previously that whole-blood cellular components from a cohort of South African children secreted significantly lower levels of most cytokines following stimulation of pattern recognition receptors compared with whole blood from cohorts of Ecuadorian, Belgian, or Canadian children. To begin dissecting the responsible molecular mechanisms, we set out to identify the relevant cellular source of these differences. Across the four cohorts represented in our study, we identified significant variation in the cellular composition of whole blood; however, a significant reduction in the intracellular cytokine production on the single-cell level was only detected in South African children's monocytes, conventional dendritic cells, and plasmacytoid dendritic cells. We also uncovered a marked reduction in polyfunctionality for each of these cellular compartments in South African children compared with children from the other continents. Together, our data identify differences in cell composition, as well as profoundly lower functional responses of innate cells, in our cohort of South African children. A possible link between altered innate immunity and increased risk for infection or lower response to vaccines in South African infants needs to be explored.

Pattern recognition receptor-mediated cytokine response in infants across 4 continents.

BACKGROUND: Susceptibility to infection as well as response to vaccination varies among populations. To date, the underlying mechanisms responsible for these clinical observations have not been fully delineated. Because innate immunity instructs adaptive immunity, we hypothesized that differences between populations in innate immune responses may represent a mechanistic link to variation in susceptibility to infection or response to vaccination. OBJECTIVE: Determine whether differences in innate immune responses exist among infants from different continents of the world. METHODS: We determined the innate cytokine response following pattern recognition receptor (PRR) stimulation of whole blood from 2-year-old infants across 4 continents (Africa, North America, South America, and Europe). RESULTS: We found that despite the many possible genetic and environmental exposure differences in infants across 4 continents, innate cytokine responses were similar for infants from North America, South America, and Europe. However, cells from South African infants secreted significantly lower levels of cytokines than did cells from infants from the 3 other sites, and did so following stimulation of extracellular and endosomal but not cytosolic PRRs. CONCLUSIONS: Substantial differences in innate cytokine responses to PRR stimulation exist among different populations of infants that could not have been predicted. Delineating the underlying mechanism(s) for these differences will not only aid in improving vaccine-mediated protection but possibly also provide clues for the susceptibility to infection in different regions of the world.

Longitudinal plasma proteomic analysis of 1117 hospitalized patients with COVID-19 identifies features associated with severity and outcomes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by highly heterogeneous manifestations ranging from asymptomatic cases to death for still incompletely understood reasons. As part of the IMmunoPhenotyping Assessment in a COVID-19 Cohort study, we mapped the plasma proteomes of 1117 hospitalized patients with COVID-19 from 15 hospitals across the United States. Up to six samples were collected within ~28 days of hospitalization resulting in one of the largest COVID-19 plasma proteomics cohorts with 2934 samples. Using perchloric acid to deplete the most abundant plasma proteins allowed for detecting 2910 proteins. Our findings show that increased levels of neutrophil extracellular trap and heart damage markers are associated with fatal outcomes. Our analysis also identified prognostic biomarkers for worsening severity and death. Our comprehensive longitudinal plasma proteomics study, involving 1117 participants and 2934 samples, allowed for testing the generalizability of the findings of many previous COVID-19 plasma proteomics studies using much smaller cohorts.

Integrated longitudinal multiomics study identifies immune programs associated with acute COVID-19 severity and mortality.

BACKGROUNDPatients hospitalized for COVID-19 exhibit diverse clinical outcomes, with outcomes for some individuals diverging over time even though their initial disease severity appears similar to that of other patients. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity.METHODSWe performed deep immunophenotyping and conducted longitudinal multiomics modeling, integrating 10 assays for 1,152 Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study participants and identifying several immune cascades that were significant drivers of differential clinical outcomes.RESULTSIncreasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, formation of neutrophil extracellular traps, and T cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma Igs and B cells and dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to failure of viral clearance in patients with fatal illness.CONCLUSIONOur longitudinal multiomics profiling study revealed temporal coordination across diverse omics that potentially explain the disease progression, providing insights that can inform the targeted development of therapies for patients hospitalized with COVID-19, especially those who are critically ill.TRIAL REGISTRATIONClinicalTrials.gov NCT04378777.FUNDINGNIH (5R01AI135803-03, 5U19AI118608-04, 5U19AI128910-04, 4U19AI090023-11, 4U19AI118610-06, R01AI145835-01A1S1, 5U19AI062629-17, 5U19AI057229-17, 5U19AI125357-05, 5U19AI128913-03, 3U19AI077439-13, 5U54AI142766-03, 5R01AI104870-07, 3U19AI089992-09, 3U19AI128913-03, and 5T32DA018926-18); NIAID, NIH (3U19AI1289130, U19AI128913-04S1, and R01AI122220); and National Science Foundation (DMS2310836).

A simple, time- and cost-effective, high-throughput depletion strategy for deep plasma proteomics.

We introduce a cost-effective, robust high-throughput-compatible plasma depletion method enabling in-depth profiling of plasma that detects >1300 proteins per run with a throughput of 60 samples per day. The method has been fully validated by processing >3000 samples with no apparent batch effect at a cost for the depletion step of ~$2.5 per sample.

Early cellular and molecular signatures correlate with severity of West Nile virus infection.

Infection with West Nile virus (WNV) drives a wide range of responses, from asymptomatic to flu-like symptoms/fever or severe cases of encephalitis and death. To identify cellular and molecular signatures distinguishing WNV severity, we employed systems profiling of peripheral blood from asymptomatic and severely ill individuals infected with WNV. We interrogated immune responses longitudinally from acute infection through convalescence employing single-cell protein and transcriptional profiling complemented with matched serum proteomics and metabolomics as well as multi-omics analysis. At the acute time point, we detected both elevation of pro-inflammatory markers in innate immune cell types and reduction of regulatory T cell activity in participants with severe infection, whereas asymptomatic donors had higher expression of genes associated with anti-inflammatory CD16+ monocytes. Therefore, we demonstrated the potential of systems immunology using multiple cell-type and cell-state-specific analyses to identify correlates of infection severity and host cellular activity contributing to an effective anti-viral response.

Integrated longitudinal multi-omics study identifies immune programs associated with COVID-19 severity and mortality in 1152 hospitalized participants.

Hospitalized COVID-19 patients exhibit diverse clinical outcomes, with some individuals diverging over time even though their initial disease severity appears similar. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity. In this study, we carried out deep immunophenotyping and conducted longitudinal multi-omics modeling integrating ten distinct assays on a total of 1,152 IMPACC participants and identified several immune cascades that were significant drivers of differential clinical outcomes. Increasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, NETosis, and T-cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma immunoglobulins and B cells, as well as dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to the failure of viral clearance in patients with fatal illness. Our longitudinal multi-omics profiling study revealed novel temporal coordination across diverse omics that potentially explain disease progression, providing insights that inform the targeted development of therapies for hospitalized COVID-19 patients, especially those critically ill.

Plasma Proteomic Analysis Distinguishes Severity Outcomes of Human Ebola Virus Disease.

Ebola virus (EBV) disease (EVD) is a highly virulent systemic disease characterized by an aggressive systemic inflammatory response and impaired vascular and coagulation systems, often leading to uncontrolled hemorrhaging and death. In this study, the proteomes of 38 sequential plasma samples from 12 confirmed EVD patients were analyzed. Of these 12 cases, 9 patients received treatment with interferon beta 1a (IFN-ß-1a), 8 survived EVD, and 4 died; 2 of these 4 fatalities had received IFN-ß-1a. Our analytical strategy combined three platforms targeting different plasma subproteomes: a liquid chromatography-mass spectrometry (LC-MS)-based analysis of the classical plasma proteome, a protocol that combines the depletion of abundant plasma proteins and LC-MS to detect less abundant plasma proteins, and an antibody-based cytokine/chemokine multiplex assay. These complementary platforms provided comprehensive data on 1,000 host and viral proteins. Examination of the early plasma proteomes revealed protein signatures that differentiated between fatalities and survivors. Moreover, IFN-ß-1a treatment was associated with a distinct protein signature. Next, we examined those proteins whose abundances reflected viral load measurements and the disease course: resolution or progression. Our data identified a prognostic 4-protein biomarker panel (histone H1-5, moesin, kininogen 1, and ribosomal protein L35 [RPL35]) that predicted EVD outcomes more accurately than the onset viral load. IMPORTANCE As evidenced by the 2013-2016 outbreak in West Africa, Ebola virus (EBV) disease (EVD) poses a major global health threat. In this study, we characterized the plasma proteomes of 12 individuals infected with EBV, using two different LC-MS-based proteomics platforms and an antibody-based multiplexed cytokine/chemokine assay. Clear differences were observed in the host proteome between individuals who survived and those who died, at both early and late stages of the disease. From our analysis, we derived a 4-protein prognostic biomarker panel that may help direct care. Given the ease of implementation, a panel of these 4 proteins or subsets thereof has the potential to be widely applied in an emergency setting in resource-limited regions.

Assessing the safety of bioactive ingredients in infant formula that affect the immune system: recommendations from an expert panel.

Bioactive ingredients for infant formula have been sought to reduce disparities in health outcomes between breastfed and formula-fed infants. Traditional food safety methodologies have limited ability to assess some bioactive ingredients. It is difficult to assess the effects of nutrition on the infant immune system because of coincident developmental adaptations to birth, establishment of the microbiome and introduction to solid foods, and perinatal environmental factors. An expert panel was convened to review information on immune system development published since the 2004 Institute of Medicine report on evaluating the safety of new infant formula ingredients and to recommend measurements that demonstrate the safety of bioactive ingredients intended for that use. Panel members participated in a 2-d virtual symposium in November 2020 and in follow-up discussions throughout early 2021. Key topics included identification of immune system endpoints from nutritional intervention studies, effects of human milk feeding and human milk substances on infant health outcomes, ontologic development of the infant immune system, and microbial influences on tolerance. The panel explored how "nonnormal" conditions such as preterm birth, allergy, and genetic disorders could help define developmental immune markers for healthy term infants. With consideration of breastfed infants as a reference, ensuring proper control groups, and attention to numerous potential confounders, the panel recommended a set of standard clinical endpoints including growth, response to vaccination, infection and other adverse effects related to inflammation, and allergy and atopic diseases. It compiled a set of candidate markers to characterize stereotypical patterns of immune system development during infancy, but absence of reference ranges, variability in methods and populations, and unreliability of individual markers to predict disease prevented the panel from including many markers as safety endpoints. The panel's findings and recommendations are applicable for industry, regulatory, and academic settings, and will inform safety assessments for immunomodulatory ingredients in foods besides infant formula.

Urine Proteomics for Noninvasive Monitoring of Biomarkers in Bronchopulmonary Dysplasia.

INTRODUCTION: Current techniques to diagnose and/or monitor critically ill neonates with bronchopulmonary dysplasia (BPD) require invasive sampling of body fluids, which is suboptimal in these frail neonates. We tested our hypothesis that it is feasible to use noninvasively collected urine samples for proteomics from extremely low gestational age newborns (ELGANs) at risk for BPD to confirm previously identified proteins and biomarkers associated with BPD. METHODS: We developed a robust high-throughput urine proteomics methodology that requires only 50 µL of urine. We utilized the methodology with a proof-of-concept study validating proteins previously identified in invasively collected sample types such as blood and/or tracheal aspirates on urine collected within 72 h of birth from ELGANs (gestational age [26 ± 1.2] weeks) who were admitted to a single Neonatal Intensive Care Unit (NICU), half of whom eventually developed BPD (n = 21), while the other half served as controls (n = 21). RESULTS: Our high-throughput urine proteomics approach clearly identified several BPD-associated changes in the urine proteome recapitulating expected blood proteome changes, and several urinary proteins predicted BPD risk. Interestingly, 16 of the identified urinary proteins are known targets of drugs approved by the Food and Drug Administration. CONCLUSION: In addition to validating numerous proteins, previously found in invasively collected blood, tracheal aspirate, and bronchoalveolar lavage, that have been implicated in BPD pathophysiology, urine proteomics also suggested novel potential therapeutic targets. Ease of access to urine could allow for sequential proteomic evaluations for longitudinal monitoring of disease progression and impact of therapeutic intervention in future studies.