Accurate and reliable measurements of exposure to tobacco products are essential for identifying and confirming patterns of tobacco product use and for assessing their potential biological effects in both human populations and experimental systems. Due to the introduction of new tobacco-derived products and the development of novel ways to modify and use conventional tobacco products, precise and specific assessments of exposure to tobacco are now more important than ever. Biomarkers that were developed and validated to measure exposure to cigarettes are being evaluated to assess their use for measuring exposure to these new products. Here, we review current methods for measuring exposure to new and emerging tobacco products, such as electronic cigarettes, little cigars, water pipes, and cigarillos. Rigorously validated biomarkers specific to these new products have not yet been identified. Here, we discuss the strengths and limitations of current approaches, including whether they provide reliable exposure estimates for new and emerging products. We provide specific guidance for choosing practical and economical biomarkers for different study designs and experimental conditions. Our goal is to help both new and experienced investigators measure exposure to tobacco products accurately and avoid common experimental errors. With the identification of the capacity gaps in biomarker research on new and emerging tobacco products, we hope to provide researchers, policymakers, and funding agencies with a clear action plan for conducting and promoting research on the patterns of use and health effects of these products.

Rationale: Neutrophils are usually the first circulating leukocytes to respond during bacterial pneumonia. Their expression of oxidants, proteases, and other mediators present in granules is well documented, but their ability to produce mediators through transcription and translation after migration to an inflammatory site has been appreciated only more recently. Interferon (IFN)-g is a cytokine with many functions important in host defense and immunity. Objectives: To examine the expression and function of IFN-g in bacterial pneumonias. Methods: IFN-g mRNA and protein were measured in digests of mouse lungs with 24-hour bacterial pneumonia. Bacterial clearance was studied with IFN-g-deficient mice. Measurements and Main Results: Streptococcus pneumoniae and Staphylococcus aureus each induce expression of IFN-g mRNA and protein by neutrophils by 24 hours. Only neutrophils that have migrated into pneumonic tissue produce IFN-g. Deficiency of Hck/ Fgr/Lyn, Rac2, or gp91 phox prevents IFN-g production. IFN-g enhances bacterial clearance and is required for formation of neutrophil extracellular traps. In contrast, Pseudomonas aeruginosa and Escherichia coli induce production of IFN-g mRNA but not protein.During pneumonia induced by E. coli but not S. pneumoniae, neutrophils produce microRNAs that target the 39 untranslated region of the IFN-g gene. Conclusions: S. pneumoniae and S. aureus, but not P. aeruginosa and E. coli, induce emigrated neutrophils to produce IFN-g within 24 hours. Hck/Fgr/Lyn, Rac2, and NADPH oxidase are required for IFN-g production. IFN-g facilitates bacterial clearance at least in part through regulating formation of neutrophil extracellular traps. Differential expression by neutrophils of microRNAs that target the 39 untranslated region of the IFN-g gene may contribute to the pathogen-specific regulation of translation.

Bacterial pneumonia is a common public health problem associated with significant mortality, morbidity, and cost. Neutrophils are usually the earliest leukocytes to respond to bacteria in the lungs. Neutrophils rapidly sequester in the pulmonary microvasculature and migrate into the lung parenchyma and alveolar spaces, where they perform numerous effector functions for host defense. Previous studies showed that migrated neutrophils produce IFN-g early during pneumonia induced by Streptococcus pneumoniae and that early production of IFN-g regulates bacterial clearance. IFN-g production by neutrophils requires Rac2, Hck/Lyn/Fgr Src family tyrosine kinases, and NADPH oxidase. Our current studies examined the mechanisms that regulate IFN-g production by lung neutrophils during acute S. pneumoniae pneumonia in mice and its function. We demonstrate that IFN-g production by neutrophils is a tightly regulated process that does not require IL-12. The adaptor molecule MyD88 is critical for IFN-g production by neutrophils. The guanine nucleotide exchange factor CalDAG-GEFI modulates IFN-g production. The CD11/CD18 complex, CD44, Toll-like receptors 2 and 4, TRIF, and Nrf2 are not required for IFN-g production by neutrophils. The recently described neutrophil-dendritic cell hybrid cell, identified by its expression of Ly6G and CD11c, is present at low numbers in pneumonic lungs and is not a source of IFN-g. IFN-g produced by neutrophils early during acute S. pneumoniae pneumonia induces transcription of target genes in the lungs, which are critical for host defense. These studies underline the complexity of the neutrophil responses during pneumonia in the acute inflammatory response and in subsequent resolution or initiation of immune responses.

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Nrf2 regulates the transcriptional response to oxidative stress. These studies tested the role of Nrf2 during S. pneumoniae pneumonia and identified Nrf2-dependent genes and pathways in lung tissue and in recruited neutrophils. Nrf2 null and WT mice were studied at 6 and 24 h following instillation of S. pneumoniae or PBS. At 6 h, fewer neutrophils were recruited and the number of bacterial remaining in the lungs tended to be less (p=0.06) in the Nrf2 null compared to WT mice. In uninfected lungs, 53 genes were already differentially expressed in Nrf2 null compared to WT mouse lungs and gene sets involved in phagocytosis, Fc receptor function, complement and immunoglobulin regulation are enhanced in PBS-treated Nrf2 null gene profiles compared to those of WT mice. These results suggest that initial host defense is enhanced in Nrf2 null mice, resulting in less recruitment of neutrophils. At 24 h, neutrophil recruitment was greater. The percentages of early apoptotic and late apoptotic/necrotic neutrophils were similar. At increasing inoculum numbers, mortality strikingly increased from 15% to 31% and 100% in Nrf2 null mice, whereas all WT mice survived, and Nrf2 null mice had a defect in clearance, particularly at the intermediate dose. The mortality was due to enhanced lung injury and greater systemic response. Gene profiling identified differentially regulated genes and pathways in neutrophils and lung tissue, including those involved in redox stress response, metabolism, inflammation, immunoregulatory pathways and tissue repair, providing insight into the mechanisms for the greater tissue damage and increased neutrophil accumulation.

Microsatellite markers derived from simple sequence repeats have been useful in studying a number of human pathogens, including the human malaria parasite Plasmodium falciparum. Genetic markers for P. vivax would likewise help elucidate the genetics and population characteristics of this other important human malaria parasite. We have identified a locus in a P. vivax telomeric clone that contains simple sequence repeats. Primers were designed to amplify this region using a two-step semi-nested polymerase chain reaction protocol. The primers did not amplify template obtained from non-infected individuals, nor DNA from primates infected with the other human malaria parasites (P. ovale, P. malariae, or P. falciparum). The marker was polymorphic in P. vivax-infected field isolates obtained from Papua New Guinea, Indonesia and Guyana. This microsatellite marker may be useful in genetic and epidemiologic studies of P. vivax malaria.

The complex role of neutrophils in modulating the inflammatory response is increasingly appreciated. Our studies profiled the expression of mRNAs and microRNAs (miRs) in lung neutrophils in mice during S. pneumoniae pneumonia and performed in depth in silico analyses. Lung neutrophils were isolated 24 hours after intratracheal instillation of PBS or S. pneumoniae, and differentially expressed (DE) mRNAs and miRs were identified. Lung neutrophils from mice with S. pneumoniae pneumonia contained 4127 DE mRNAs, 36% of which were upregulated at least 2-fold. During pneumonia, lung neutrophils increase expression of pattern recognition receptors, receptors for inflammatory mediators, transcription factors including NF-κB and AP-1, Nrf2 targets, cytokines, chemokines and other inflammatory mediators. Interestingly, neutrophils responded to Type I interferons, whereas they both produced and responded to Type II interferon. Expression of regulators of the inflammatory and immune response was verified at the mRNA and protein level. Of approximately 1100 miRs queried, 31 increased and 67 decreased more than 2-fold in neutrophils from S. pneumoniae pneumonia. Network analyses of potential DE miR-target DE mRNA interactions revealed candidate key regulatory miRs. Thus, S. pneumoniae modulates mRNA and miR expression by lung neutrophils, increasing their ability to respond and facilitating host defense.

Neutrophil numbers must be tightly controlled to maintain host protection and prevent neutrophil-mediated tissue injury. CD18 deficiency leads to neutrophilia and myeloid hyperplasia in the bone marrow (BM). These studies examined the role of CD18 in regulating neutrophil production and determined whether the defects in neutrophil production that are observed in CD18 deficiency persist in the presence of wild type (WT) leukocytes that confer host protection. Neutrophil production was evaluated in CD18−/− mice and lethally irradiated WT mice reconstituted with mixtures of CD18−/− and WT stem cells. Neutrophil kinetic studies suggest that CD18 may facilitate the release of the most mature neutrophils into the circulation. The proportion of CD18−/− neutrophils in chimeric animals was greater than the proportion of CD18−/− donor cells used to reconstitute the mice, and the percentage of CD18−/− leukocytes that were neutrophils was greater than for WT leukocytes, indicating that CD18 may regulate the lineage distribution of hematopoietic cells in the blood and BM. The proportion of annexin V-positive Gr-1+ cells in both the BM of chimeric animals and in vitro cultures of WT and CD18−/− hematopoietic stem cells was lower in CD18−/− than in WT cells, suggesting that CD18 modulates apoptosis. These data suggest that CD18 directly regulates neutrophil production, in part by limiting the survival of neutrophils and their precursors. Thus, the granulocytosis observed in CD18−/− mice and CD18 deficient patients is due both to defects in host defense and to BM-intrinsic roles of CD18 in regulating neutrophil production.