Examination of amino acid hydrogen isotope measurements of scalp hair for region-of-origin studies.

RATIONALE: Hydrogen isotope (δ2 H) analysis of keratinaceous bulk tissues has been used in forensic science to reconstruct an individual's travel history or determine their region-of-origin. Here, we use a compound-specific approach to examine patterns of individual amino acid δ2 H values in relation to those of local tap water, bulk scalp hair tissues, and region-of-origin. METHODS: We measured δ2 H values of amino acids in anonymously collected scalp hair (n = 67) and tap water from 28 locations in the United States. Samples were hydrolyzed into their constituent amino acids, derivatized alongside in-house reference materials, and analyzed in triplicate using a GC-C-IRMS system. RESULTS: Non-essential amino acid (AANESS ) δ2 H values and their corresponding tap water samples varied systematically across continental regions. Hydrogen isotope values of alanine, glutamic acid, and glycine were significantly correlated with tap water and an estimated 42%-51% of the hydrogen atoms in these AANESS were derived from tap water. We used linear discriminate analysis (LDA) to explore regional patterns in scalp hair bulk tissue and amino acid δ2 H values. For the model that included AANESS data, 87% of the variance was explained by the first linear discriminant axis (LD1), and was driven by bulk hair tissue, alanine, and proline. This model had an overall 72% successful reclassification with samples from the south and northwest regions reclassifying correctly 92% and 78% of the time, respectively. For the model that included AAESS data, LD1 explained 81% of the variation and was driven bulk hair, threonine, valine, phenylalanine, and isoleucine. The overall reclassification rate for the model that included AAESS was 70%. CONCLUSIONS: Our findings suggest that δ2 H analyses of AANESS and AAESS could help improve geolocation models for human and wildlife forensics by simultaneously providing information about both dietary and tap water inputs of hydrogen to tissue synthesis.

Strontium isotope ratios (87 Sr/86 Sr) of human fingernail clippings reveal multiple location signals.

RATIONALE: Strontium isotope ratios (87 Sr/86 Sr) in human fingernail keratin tissues have been underexplored for region of origin and travel history reconstruction studies. Here we investigated 87 Sr/86 Sr ratios in fingernail keratin to establish baseline measurements in a resident group and to examine how 87 Sr/86 Sr ratios changed with relocation. METHODS: Fingernail clippings were collected from resident (n = 10) and non-resident/traveler groups (n = 4 and n = 4) that were part of a larger study in Salt Lake City (UT, USA) from 2015 to 2016. Strontium abundance and 87 Sr/86 Sr ratios were determined via multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS). 87 Sr/86 Sr and oxygen (δ18 O) isotope ratios from the traveler participants were compared to examine temporal patterns. RESULTS: Strontium abundance and 87 Sr/86 Sr ratios in fingernails from the resident group established a baseline against which we could evaluate potential differences in non-resident/traveler groups. Resident 87 Sr/86 Sr ratios remained constant over the study period and were consistent with previously measured tap waters for the area. 87 Sr/86 Sr ratio changes in non-resident/traveler groups were rapid and reflected the current location of the individual within 4-5 weeks of arrival. Lastly, δ18 O and 87 Sr/86 Sr ratios of the same fingernail clippings did not exhibit similar temporal patterns, since fingernail δ18 O ratios required more time to attain values characteristic of the new environment. CONCLUSIONS: Our findings suggest that strontium is incorporated into fingernail tissues differently from hair and this could be advantageous to forensic investigations. We found that 87 Sr/86 Sr and δ18 O ratios of the same fingernail clippings revealed two different time points reflecting an individual's residence over short- (4-5 weeks, 87 Sr/86 Sr ratios) and long-term (3-5 months, δ18 O values) time scales. It is likely that the 87 Sr/86 Sr ratios of fingernail clippings reflect exogenous signals that are incorporated through bathing waters and that these signals change rapidly with movement to a new location. Our results may aid future forensic studies in the determination of region of origin in unidentified remains.

Innate immune activation in neonatal tracheal aspirates suggests endotoxin-driven inflammation.

BACKGROUND: Tracheal aspirates (TAs) from critically ill neonates accumulate bacterial endotoxin and demonstrate mobilization of endotoxin-binding proteins, but the potential bioactivity of endotoxin in TAs is unknown. We characterized innate immune activation in TAs of mechanically ventilated neonates. METHODS: Innate immune activation in TAs of mechanically ventilated neonates was characterized using a targeted 84-gene quantitative real-time (qRT) PCR array. Protein expression of cytokines was confirmed by multiplex assay. Expression and localization of the endotoxin-inducible antimicrobial protein Calgranulin C (S100A12) was assessed by flow cytometry. Endotoxin levels were measured in TA supernatants using the Limulus amoebocyte lysate assay. RESULTS: Analyses by qRT-PCR demonstrated expression of pattern recognition receptors, Toll-like receptor-nuclear factor κB and inflammasome pathways, cytokines/chemokines and their receptors, and anti-infective proteins in TA cells. Endotoxin positivity increased with postnatal age. As compared with endotoxin-negative TAs, endotoxin-positive TAs demonstrated significantly greater tumor necrosis factor (TNF), interleukin (IL)-6, IL-10, and serpin peptidase inhibitor, clade E, member 1 (SERPINE1) mRNA, and IL-10, TNF, and IL-1ß protein. Expression of S100A12 protein was localized to TA neutrophils. CONCLUSION: Correlation of endotoxin with TA inflammatory responses suggests endotoxin bioactivity and the possibility that endotoxin antagonists could mitigate pulmonary inflammation and its sequelae in this vulnerable population.

Breath Stable Isotope Analysis Serves as a Non-invasive Analytical Tool to Demonstrate Dietary Changes in Adolescent Students Over Time.

Concern about adolescent diets, obesity, and the associated health risks have been growing in the United States. This inspired former First Lady Michelle Obama to spearhead the Healthy Hunger-Free Kids Act (HHFKA), which made changes to the national school lunch program by increasing servings of whole grains, fruits, and vegetables. Our study examined the variability of student carbohydrate sources throughout the day and before and after the implementation of HHFKA using a stable isotope dietary biomarker. This method uses carbon stable isotope values of exhaled CO2 breath (δ13Cbreath) and provides a quantitative, non-invasive measure. δ13Cbreath samples were collected throughout the day from students (n = 31) that attended a public high school in Salt Lake City, UT. δ13Cbreath measurements reflected the short-term carbohydrate inputs from the previous meal. Carbohydrate sources were not consistent throughout the day; most students had their lowest inputs of corn/sugar-based carbohydrates after lunch. We compared our results with an earlier study that had been conducted pre-HHFKA. After-lunch δ13Cbreath values decreased significantly between the two time points, suggesting an increase in whole grain, fruit, and vegetable carbohydrates in the lunch program. Our results demonstrated that δ13Cbreath measurements provide a valuable tool to examine carbohydrate sources in an individual's diet throughout the day. We believe that this tool could be beneficial to studies examining the relationship between sugar sweetened beverages, added sugars, and refined carbohydrates and health outcomes like diabetes and obesity in both adolescent and adult populations.

Traveling There and Back Again: A Fingernail's Tale.

This study focuses on the examination of stable isotope biomarkers in human fingernails of travelers who ventured away from Salt Lake City, UT and subsequently returned. Here, we describe three case studies examining: individual travelers (n = 4), paired travelers (n = 2), and a traveler (n = 1) who repeatedly left and returned. Our case studies examined the influences of dietary and drinking water inputs on fingernail stable isotope profiles for reconstructing travel histories. Stable isotope data were compared with theoretical and predictive models that laid the groundwork for anthropological and forensic geospatial reconstruction of travel histories. Drinking water inputs strongly influenced stable isotope profiles and the patterns observed were consistent with reported travel histories. Finally, observed fingernail stable isotope ratio data were consistent with modeled rates of stable isotope turnover and transition to expected values for the new environment. The results of our study further support the use of fingernail tissues for travel history reconstruction.

Resident and Nonresident Fingernail Isotopes Reveal Diet and Travel Patterns,.

Global travel has increased, and having a diagnostic tool to distinguish residents from visitors would be valuable. This study examined stable isotope biomarkers of fingernail tissues of resident (n = 26) and nonresident (n = 22) participants in Salt Lake City (SLC), UT, from 2015 to 2016. The purpose of this research was to determine whether fingernail isotopes could be used for reconstructing geolocation movements and to examine the convergence in nonresident fingernail isotopes to that of the resident signal following their arrival to SLC. Resident isotope values defined a baseline to make comparisons to. Initial nonresident hydrogen and oxygen isotope values were correlated with precipitation isotopes of their prior location. Fingernail isotope turnover rates were rapid and nonresident isotopes were indistinguishable from residents after ~71-90 days. The results of our study highlight the utility of stable isotope measurements of fingernail clippings to examine travel history reconstruction that could aid in identification of human remains.

Stable hydrogen and oxygen isotopes of tap water reveal structure of the San Francisco Bay Area's water system and adjustments during a major drought.

Water availability and sustainability in the Western United States is a major flashpoint among expanding communities, growing industries, and productive agricultural lands. This issue came to a head in 2015 in the State of California, when the State mandated a 25% reduction in urban water use following a multi-year drought that significantly depleted water resources. Water demands and challenges in supplying water are only expected to intensify as climate perturbations, such as the 2012-2015 California Drought, become more common. As a consequence, there is an increased need to understand linkages between urban centers, water transport and usage, and the impacts of climate change on water resources. To assess if stable hydrogen and oxygen isotope ratios could increase the understanding of these relationships within a megalopolis in the Western United States, we collected and analyzed 723 tap waters across the San Francisco Bay Area during seven collection campaigns spanning 21 months during 2013-2015. The San Francisco Bay Area was selected as it has well-characterized water management strategies and the 2012-2105 California Drought dramatically affected its water resources. Consistent with known water management strategies and previously collected isotope data, we found large spatiotemporal variations in the δ2H and δ18O values of tap waters within the Bay Area. This is indicative of complex water transport systems and varying municipality-scale management decisions. We observed δ2H and δ18O values of tap water consistent with waters originating from snowmelt from the Sierra Nevada Mountains, local precipitation, ground water, and partially evaporated reservoir sources. A cluster analysis of the isotope data collected in this study grouped waters from 43 static sampling sites that were associated with specific water utility providers within the San Francisco Bay Area and known management practices. Various management responses to the drought, such as source switching, bringing in new sources, and water conservation, were observed in the isotope data. Finally, we estimated evaporative loss from one utility's reservoir system during the 2015 water year using a modified Craig-Gordon model to estimate the consequences of the drought on this resource. We estimated that upwards of 6.6% of the water in this reservoir system was lost to evaporation.

TLR7/8 adjuvant overcomes newborn hyporesponsiveness to pneumococcal conjugate vaccine at birth.

Infection is the most common cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. However, newborns fail to respond optimally to most vaccines. Adjuvantation is a key approach to enhancing vaccine immunogenicity, but responses of human newborn leukocytes to most candidate adjuvants, including most TLR agonists, are functionally distinct. Herein, we demonstrate that 3M-052 is a locally acting lipidated imidazoquinoline TLR7/8 agonist adjuvant in mice, which, when properly formulated, can induce robust Th1 cytokine production by human newborn leukocytes in vitro, both alone and in synergy with the alum-adjuvanted pneumococcal conjugate vaccine 13 (PCV13). When admixed with PCV13 and administered i.m. on the first day of life to rhesus macaques, 3M-052 dramatically enhanced generation of Th1 CRM-197-specific neonatal CD4+ cells, activation of newborn and infant Streptococcus pneumoniae polysaccharide-specific (PnPS-specific) B cells as well as serotype-specific antibody titers, and opsonophagocytic killing. Remarkably, a single dose at birth of PCV13 plus 0.1 mg/kg 3M-052 induced PnPS-specific IgG responses that were approximately 10-100 times greater than a single birth dose of PCV13 alone, rapidly exceeding the serologic correlate of protection, as early as 28 days of life. This potent immunization strategy, potentially effective with one birth dose, could represent a new paradigm in early life vaccine development.

The effect of stable macromolecular complexes of ionic polyphosphazene on HIV Gag antigen and on activation of human dendritic cells and presentation to T-cells.

Neonates and infants are susceptible to infection due to distinct immune responses in early life. Therefore, development of vaccine formulation and delivery systems capable of activating human newborn leukocytes is of global health importance. Poly[di(carboxylatophenoxy)phosphazene] (PCPP) belongs to a family of ionic synthetic polyphosphazene polyelectrolyte compounds that can form non-covalent interactions with protein antigens and demonstrate adjuvant activity in animals and in human clinical trials. However, little is known about their ability to activate human immune cells. In this study, we characterized the effects of PCPP alone or in combination with a model antigen (recombinant HIV-Gag (Gag)), on the maturation, activation and antigen presentation by human adult and newborn dendritic cells (DCs) in vitro. PCPP treatment induced DC activation as assessed by upregulation of co-stimulatory molecules and cytokine production. Studies benchmarking PCPP to Alum, the most commonly used vaccine adjuvant, demonstrated that both triggered cell death and release of danger signals in adult and newborn DCs. When complexed with Gag antigen, PCPP maintained its immunostimulatory characteristics while permitting internalization and presentation of Gag by DCs to HIV-Gag-specific CD4(+) T cell clones. The PCPP vaccine formulation outlined here has intrinsic adjuvant activity, can facilitate effective delivery of antigen to DCs, and may be advantageous for induction of beneficial T cell-mediated immunity. Moreover, polyphosphazenes can further reduce cost of vaccine production and distribution through their dose-sparing and antigen-stabilizing properties, thus potentially eliminating the need for cold chain distribution.

Identification of single nucleotide polymorphisms in hematopoietic cell transplant patients affecting early recognition of, and response to, endotoxin.

Hematopoietic cell transplant (HCT) is a life-saving therapy for many malignant and non-malignant bone marrow diseases. Associated morbidities are often due to transplant-related toxicities and infections, exacerbated by regimen-induced immune suppression and systemic incursion of bacterial products. Patients undergoing myeloablative conditioning for HCT become endotoxemic and display blood/plasma changes consistent with lipopolysaccharide (LPS)-induced systemic innate immune activation. Herein, we addressed whether patients scheduled for HCT display differences in recognition/response to LPS ex vivo traceable to specific single nucleotide polymorphisms (SNPs). Two SNPs of LPS binding protein (LBP) were associated with changes in plasma LBP levels, with one LBP SNP also associating with differences in efficiency of extraction and transfer of endotoxin to myeloid differentiation factor-2 (MD-2), a step needed for activation of TLR4. None of the examined SNPs of CD14, bactericidal/permeability-increasing protein (BPI), TLR4 or MD-2 were associated with corresponding protein plasma levels or endotoxin delivery to MD-2, but CD14 and BPI SNPs significantly associated with differences in LPS-induced TNF-α release ex vivo and infection frequency, respectively. These findings suggest that specific LBP, CD14 and BPI SNPs might be contributory assessments in studies where clinical outcome may be affected by host response to endotoxin and bacterial infection.

A neonatal model of intravenous Staphylococcus epidermidis infection in mice <24 h old enables characterization of early innate immune responses.

Staphylococcus epidermidis (SE) causes late onset sepsis and significant morbidity in catheterized preterm newborns. Animal models of SE infection are useful in characterizing disease mechanisms and are an important approach to developing improved diagnostics and therapeutics. Current murine models of neonatal bacterial infection employ intraperitoneal or subcutaneous routes at several days of age, and may, therefore, not accurately reflect distinct features of innate immune responses to bacteremia. In this study we developed, validated, and characterized a murine model of intravenous (IV) infection in neonatal mice <24 hours (h) old to describe the early innate immune response to SE. C57BL/6 mice <24 h old were injected IV with 10(6), 10(7), 10(8) colony-forming units (CFU) of SE 1457, a clinical isolate from a central catheter infection. A prospective injection scoring system was developed and validated, with only high quality injections analyzed. Newborn mice were euthanized between 2 and 48 h post-injection and spleen, liver, and blood collected to assess bacterial viability, gene expression, and cytokine production. High quality IV injections demonstrated inoculum-dependent infection of spleen, liver and blood. Within 2 h of injection, SE induced selective transcription of TLR2 and MyD88 in the liver, and increased systemic production of plasma IL-6 and TNF-α. Despite clearance of bacteremia and solid organ infection within 48 h, inoculum-dependent impairment in weight gain was noted. We conclude that a model of IV SE infection in neonatal mice <24 h old is feasible, demonstrating inoculum-dependent infection of solid organs and a pattern of bacteremia, rapid and selective innate immune activation, and impairment of weight gain typical of infected human neonates. This novel model can now be used to characterize immune ontogeny, evaluate infection biomarkers, and assess preventative and therapeutic modalities.

17(R)-Resolvin D1 differentially regulates TLR4-mediated responses of primary human macrophages to purified LPS and live E. coli.

Detection and clearance of bacterial infection require balanced effector and resolution signals to avoid chronic inflammation. Detection of GNB LPS by TLR4 on m induces inflammatory responses, contributing to chronic inflammation and tissue injury. LXs and Rvs are endogenous lipid mediators that enhance resolution of inflammation, and their actions on primary human m responses toward GNB are largely uncharacterized. Here, we report that LXA(4), LXB(4), and RvD1, tested at 0.1-1 µM, inhibited LPS-induced TNF production from primary human m, with ATL and 17(R)-RvD1, demonstrating potent inhibition at 0.1 µM. In addition, 17(R)-RvD1 inhibited LPS-induced primary human m production of IL-7, IL-12p70, GM-CSF, IL-8, CCL2, and MIP-1α without reducing that of IL-6 or IL-10. Remarkably, when stimulated with live Escherichia coli, m treated with 17(R)-RvD1 demonstrated increased TNF production and enhanced internalization and killing of the bacteria. 17(R)-RvD1-enhanced TNF, internalization, and killing were not evident for an lpxM mutant of E. coli expressing hypoacylated LPS with reduced inflammatory activity. Furthermore, 17(R)-RvD1-enhanced, E. coli-induced TNF production was evident in WT but not TLR4-deficient murine m. Thus, Rvs differentially modulate primary human m responses to E. coli in an LPS- and TLR4-dependent manner, such that this Rv could promote resolution of GNB/LPS-driven inflammation by reducing m proinflammatory responses to isolated LPS and increasing m responses important for clearance of infection.

Age-dependent maturation of Toll-like receptor-mediated cytokine responses in Gambian infants.

The global burden of neonatal and infant mortality due to infection is staggering, particularly in resource-poor settings. Early childhood vaccination is one of the major interventions that can reduce this burden, but there are specific limitations to inducing effective immunity in early life, including impaired neonatal leukocyte production of Th1-polarizing cytokines to many stimuli. Characterizing the ontogeny of Toll-like receptor (TLR)-mediated innate immune responses in infants may shed light on susceptibility to infection in this vulnerable age group, and provide insights into TLR agonists as candidate adjuvants for improved neonatal vaccines. As little is known about the leukocyte responses of infants in resource-poor settings, we characterized production of Th1-, Th2-, and anti-inflammatory-cytokines in response to agonists of TLRs 1-9 in whole blood from 120 Gambian infants ranging from newborns (cord blood) to 12 months of age. Most of the TLR agonists induced TNFα, IL-1ß, IL-6, and IL-10 in cord blood. The greatest TNFα responses were observed for TLR4, -5, and -8 agonists, the highest being the thiazoloquinoline CLO75 (TLR7/8) that also uniquely induced cord blood IFNγ production. For most agonists, TLR-mediated TNFα and IFNγ responses increased from birth to 1 month of age. TLR8 agonists also induced the greatest production of the Th1-polarizing cytokines TNFα and IFNγ throughout the first year of life, although the relative responses to the single TLR8 agonist and the combined TLR7/8 agonist changed with age. In contrast, IL-1ß, IL-6, and IL-10 responses to most agonists were robust at birth and remained stable through 12 months of age. These observations provide fresh insights into the ontogeny of innate immunity in African children, and may inform development of age-specific adjuvanted vaccine formulations important for global health.

Bactericidal/permeability-increasing protein (rBPI21) and fluoroquinolone mitigate radiation-induced bone marrow aplasia and death.

Identification of safe, effective treatments to mitigate toxicity after extensive radiation exposure has proven challenging. Only a limited number of candidate approaches have emerged, and the U.S. Food and Drug Administration has yet to approve any agent for a mass-casualty radiation disaster. Because patients undergoing hematopoietic stem cell transplantation undergo radiation treatment that produces toxicities similar to radiation-disaster exposure, we studied patients early after such treatment to identify new approaches to this problem. Patients rapidly developed endotoxemia and reduced plasma bactericidal/permeability-increasing protein (BPI), a potent endotoxin-neutralizing protein, in association with neutropenia. We hypothesized that a treatment supplying similar endotoxin-neutralizing activity might replace the BPI deficit and mitigate radiation toxicity and tested this idea in mice. A single 7-Gy radiation dose, which killed 95% of the mice by 30 days, was followed 24 hours later by twice-daily, subcutaneous injections of the recombinant BPI fragment rBPI21 or vehicle alone for 14 or 30 days, with or without an oral fluoroquinolone antibiotic with broad-spectrum antibacterial activity, including that against endotoxin-bearing Gram-negative bacteria. Compared to either fluoroquinolone alone or vehicle plus fluoroquinolone, the combined rBPI21 plus fluoroquinolone treatment improved survival, accelerated hematopoietic recovery, and promoted expansion of stem and progenitor cells. The observed efficacy of rBPI21 plus fluoroquinolone initiated 24 hours after lethal irradiation, combined with their established favorable bioactivity and safety profiles in critically ill humans, suggests the potential clinical use of this radiation mitigation strategy and supports its further evaluation.