Excitatory cholinergic responses in mouse primary bronchial smooth muscle require both Ca2+ entry via l-type Ca2+ channels and store operated Ca2+ entry via Orai channels.

Malfunctions in airway smooth muscle Ca2+-signalling leads to airway hyperresponsiveness in asthma and chronic obstructive pulmonary disease. Ca2+-release from intracellular stores is important in mediating agonist-induced contractions, but the role of influx via l-type Ca2+ channels is controversial. We re-examined roles of the sarcoplasmic reticulum Ca2+ store, refilling of this store via store-operated Ca2+ entry (SOCE) and l-type Ca2+ channel pathways on carbachol (CCh, 0.1-10 µM)-induced contractions of mouse bronchial rings and intracellular Ca2+ signals of mouse bronchial myocytes. In tension experiments, the ryanodine receptor (RyR) blocker dantrolene (100 µM) reduced CCh-responses at all concentrations, with greater effects on sustained rather than initial components of contraction. 2-Aminoethoxydiphenyl borate (2-APB, 100 µM), in the presence of dantrolene, abolished CCh-responses, suggesting the sarcoplasmic reticulum Ca2+ store is essential for contraction. The SOCE blocker GSK-7975A (10 µM) reduced CCh-contractions, with greater effects at higher (e.g. 3 and 10 µM) CCh concentrations. Nifedipine (1 µM), abolished remaining contractions in GSK-7975A (10 µM). A similar pattern was observed on intracellular Ca2+-responses to 0.3 µM CCh, where GSK-7975A (10 µM) substantially reduced Ca2+ transients induced by CCh, and nifedipine (1 µM) abolished remaining responses. When nifedipine (1 µM) was applied alone it had less effect, reducing tension responses at all CCh concentrations by 25% - 50%, with greater effects at lower (e.g. 0.1 and 0.3 µM) CCh concentrations. When nifedipine (1 µM) was examined on the intracellular Ca2+-response to 0.3 µM CCh, it only modestly reduced Ca2+ signals, while GSK-7975A (10 µM) abolished remaining responses. In conclusion, Ca2+-influx from both SOCE and l-type Ca2+ channels contribute to excitatory cholinergic responses in mouse bronchi. The contribution of l-type Ca2+ channels was especially pronounced at lower doses of CCh, or when SOCE was blocked. This suggests l-type Ca2+ channels might be a potential target for bronchoconstriction under certain circumstances.

Examining Relationships Between Groundwater Nitrate Concentrations in Drinking Water and Landscape Characteristics to Understand Health Risks.

Nitrate ingested from drinking water has been linked to adverse health outcomes (e.g., cancer, birth defects) at levels as low as ∼2 mg/L NO3-N, far below the regulatory limits of 10 mg/L. In many areas, groundwater is a common drinking water source and may contain elevated nitrate, but limited data on the patterns and concentrations are available. Using an extensive regulatory data set of over 100,000 nitrate drinking water well samples, we developed new maps of groundwater nitrate concentrations from 76,724 wells in Michigan's Lower Peninsula, USA for the 2006-2015 period. Kriging, a geostatistical method, was used to interpolate concentrations and quantify probability of exceeding relevant thresholds (>0.4 [common detection limit], >2 mg/L NO3-N). We summarized this probability in small watersheds (∼80 km2) to identify correlated variables using the machine learning method classification and regression trees (CARTs). We found 79% of wells had concentrations below the detection limit in this analysis (<0.4 mg/L NO3-N). In the shallow aquifer (focus of study), 13% of wells exceeded 2 mg/L NO3-N and 2% exceeded the EPA maximum contaminant level of 10 mg/L. CART explained 40%-45% of variation in each model and identified three categories of critical correlated variables: source (high agricultural nitrogen inputs), vulnerable soil conditions (low soil organic carbon and high hydraulic conductivity), and transport mechanisms (high aquifer recharge). These findings add to the body of literature seeking to identify communities at risk of elevated nitrate and study associated adverse health outcomes.

Inflammatory and immunological biomarkers are not related to survival in adults with Cystic Fibrosis.

BACKGROUND: Chronic Pseudomonas aeruginosa pulmonary infection is associated with a decline in lung function and reduced survival in people with Cystic Fibrosis (CF). Damaging inflammatory and immunological mediators released in the lungs can be used as markers of chronic infection, inflammation and lung tissue damage. METHODS: Clinical samples were collected from CF patients and healthy controls. Serum IgG and IgA anti-Pseudomonas antibodies, sputum IL-8 and TNFα, plasma IL-6 and urine TNFr1 were measured by ELISA. Sputum neutrophil elastase (NE), cathepsin S and cathepsin B were measured by spectrophotometric and fluorogenic assays. The relationship between IgG and IgA, inflammatory mediators and long-term survival was determined. RESULTS: IgG and IL-6 positively correlated with mortality. However, multivariate analysis demonstrated that after adjusting for FEV(1), IgG was not independently related to mortality. A relationship was observed between IgG and IL-6, TNFα, TNFr1 and between IgA and IL8, cathepsin S and cathepsin B. CONCLUSIONS: These data indicate that biomarkers of inflammation are not independent predictors of survival in people with CF.

Medicinal chemistry of the epigenetic diet and caloric restriction.

The pronounced effects of the epigenetic diet (ED) and caloric restriction (CR) have on epigenetic gene regulation have been documented in many pre-clinical and clinical studies. Understanding epigenetics is of high importance because of the concept that external factors such as nutrition and diet may possess the ability to alter gene expression without modifying the DNA sequence. The ED introduces bioactive medicinal chemistry compounds such as sulforaphane (SFN), curcumin (CCM), epigallocatechin gallate (EGCG) and resveratrol (RSV) that are thought to aid in extending the human lifespan. CR, although similar to ED in the target of longevity, mildly reduces the total daily calorie intake while concurrently providing all beneficial nutrients. Both CR and ED may act as epigenetic modifiers to slow the aging process through histone modification, DNA methylation, and by modulating microRNA expression. CR and ED have been proposed as two important mechanisms that modulate and potentially slow the progression of age-related diseases such as cardiovascular disease (CVD), cancer, obesity, Alzheimer's and osteoporosis to name a few. While many investigators have examined CR and ED as separate entities, this review will primarily focus on both as they relate to age-related diseases, their epigenetic effects and their medicinal chemistry.

Chymotrypsin-like serine proteinases are involved in the maintenance of cell viability.

An increasing number of studies have implicated serine proteinases in the development of apoptosis. In this study, we assessed the ability of a set of highly specific irreversible inhibitors (activity probes), incorporating an α-amino alkane diphenyl phosphonate moiety, to modulate cell death. In an initial assessment of the cellular toxicity of these activity probes, we discovered that one example, N-α-tetramethylrhodamine phenylalanine diphenylphosphonate {TMR-Phe(P)(OPh)(2)} caused a concentration-dependent decrease in the viability of HeLa and U251 mg cells. This reduced cell viability was associated with a time-dependent increase in caspase-3 activity, PARP cleavage and phosphatidylserine translocation, establishing apoptosis as the mechanism of cell death. SDS-PAGE analysis of cell lysates prepared from the HeLa cells treated with TMR-Phe(P)(OPh)(2), revealed the presence of a fluorescent band of molecular weight 58 kDa. Given that we have previously reported on the use of this type of activity probe to reveal active proteolytic species, we believe that we have identified a chymotrypsin-like serine proteinase activity integral to the maintenance of cell viability.

Association of airway cathepsin B and S with inflammation in cystic fibrosis.

SUMMARY: Irreversible tissue damage within the cystic fibrosis (CF) lung is mediated by proteolytic enzymes during an inflammatory response. Serine proteinases, in particular neutrophil elastase (NE), have been implicated however, members of the cysteine proteinase family may also be involved. The aim of this study was to determine cathepsin B and S levels in cystic fibrosis (CF) sputum and to assess any relationship to recognized markers of inflammation such as sputum NE, interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), urine TNF receptor 1 (TNFr1), plasma IL-6, and serum C-reactive protein (CRP). Proteinase activities were measured in the sputum of 36 clinically stable CF patients using spectrophotometric and fluorogenic assays. Immunoblots were also used to confirm enzyme activity data. All other parameters were measured by ELISA. Patients had a mean age of 27.2 (8.2) years, FEV. of 1.6 (0.79) L and BMI of 20.7 (2.8). Both cathepsin B and S activities were detected in all samples, with mean concentrations of 18.0 (13.5) microg/ml and 1.6 (0.88) microg/ml, respectively and were found to correlate not only with each other but with NE, TNF-alpha and IL-8 (in all cases . < 0.05). Airway cathepsin B further correlated with circulatory IL-6 and CRP however, no relationship for either cathepsin was observed with urine TNFr1. This data indicates that cathepsin B and S may have important roles in the pathophysiology of CF lung disease and could have potential as markers of inflammation in future studies.

Continuity of adolescent and early adult partner violence victimisation: association with witnessing violent crime in adolescence.

BACKGROUND: Although exposure to peer and family violence is a documented risk factor for adolescent dating violence, less is known about the relationship between violent crime exposure and dating violence victimisation. METHODS: Participants in the National Longitudinal Study of Adolescent Health (n = 4794) aged 13-17 years self-reported witnessing violent crime (someone being shot or stabbed) in the 12 months prior to Wave I interview (1994-95), physical partner violence victimisation within the 18 months prior to Wave II interview (1995-96), and physical and sexual partner violence victimisation within the 18 months prior to Wave III interview (2001). RESULTS: Twelve per cent of respondents reported dating violence victimisation at Wave II. Witnessing violent crime was positively associated with victimisation in crude (OR = 2.11, 95% CI 1.56 to 2.86) and adjusted (AOR = 1.53, 95% CI 1.09 to 2.15) analyses. Of the adolescent partner violence victims (n = 549), 32% reported continued victimisation into early adulthood; after adjusting for gender, age, urbanicity and childhood maltreatment history, witnessing violent crime in adolescence was negatively associated with having non-violent relationships in early adulthood (AOR = 0.40, 95% CI 0.19 to 0.84). In cross-sectional and longitudinal analyses, associations between violent crime exposure and victimisation did not vary by age, gender or race/ethnicity. CONCLUSION: Adolescents exposed to violent crime experience an increased risk of partner violence victimisation in adolescence and continuing victimisation into adulthood. Targeting dating violence prevention and intervention programmes to geographic areas with high levels of violent crime may be an efficient strategy to reach higher risk adolescents. Reducing community violent crime may also have spillover effects on partner violence.

Altered sensory experience induces targeted rewiring of local excitatory connections in mature neocortex.

Experience-dependent plasticity in adulthood is slower than during development. Previous experience can accelerate adult cortical plasticity. However, the contributions of functional synaptic changes and modifications in neuronal structure to the acceleration of adult cortical plasticity remain unclear. If structural remodeling was important then it should be exhibited by neuronal connections that have altered during plasticity. We trimmed rodents' whiskers to induce experience-dependent plasticity and reconstructed pairs of layer 2/3 (L2/3) pyramidal neurons after electrophysiological recording. We reported recently that local excitatory connections strengthen without a change in synapse number in cortex with retained sensory input (spared) (Cheetham et al., 2007). Here, we show that strengthened connections are rewired. The rewiring involves remodeling of the axonal arbor of excitatory connections with only minor changes in postsynaptic dendritic trees. The axonal remodeling resulted in a greater length of presynaptic axon close to postsynaptic dendrites at existing local excitatory connections in spared cortex. In control cortex, the length of axon close to dendrite in unconnected pairs of L2/3 pyramidal neurons was similar to that in synaptically connected pairs of L2/3 pyramidal neurons. This finding suggests that the probability of forming a synapse and, therefore, establishing a connection, is not driven solely by the length of axon close to dendrite. The axonal remodeling that we describe is not associated with altered synapse number, but instead increases the number of sites where synapses could be formed between synaptically connected neurons with minimal structural changes. This enables rapid and cost-efficient rewiring of local excitatory connections when re-exposed to similarly altered sensory experience in adulthood.

National study of physical and sexual assault among women with disabilities.

OBJECTIVE: To examine the association between the level of disability impairment and physical and sexual assault in a sample of US women at least 18 years of age. DESIGN, SETTING AND PARTICIPANTS: Retrospective longitudinal study of 6273 non-institutionalized US women from 8000 women participating in the 1995-1996 National Violence Against Women (NVAW) Survey. MAIN OUTCOME MEASURE: Women's experiences of physical and sexual assault in the 12 months before the NVAW interview. RESULTS: Most women reported having no disability (n = 5008, 79.8%) and/or not experiencing an assault in the year before their interview (n = 6018, 95.9%). Less than 5% (n = 280) reported having a disability that severely limited daily activities, and 15.7% (n = 985) reported having a disability that moderately limited activities. Less than 4% (n = 218) of the women reported a physical-only assault, and less than 1% (n = 37) reported being sexually assaulted. Women with severe disability impairments were four times more likely to be sexually assaulted than women with no reported disabilities (RR = 4.0, 95% CI 1.5 to 10.6). Little difference in the risk of sexual assault was found between women with moderate disability impairments and those reporting no disabilities (RR = 1.0, 95% CI 0.3 to 2.8). Women with severe (RR = 1.6, 95% CI 0.9 to 3.0) and moderate (RR = 1.2, 95% CI 0.8 to 1.9) disability impairments were at greater risk, although not quite significantly so, of physical-only assault than were women without a disability. CONCLUSION: The findings suggest that women with disabilities that severely limit activities of daily living are at increased risk of sexual assault.

Tuberculosis antigen-specific immune responses can be detected using enzyme-linked immunospot technology in human immunodeficiency virus (HIV)-1 patients with advanced disease.

There are limited data on the efficacy of T cell-based assays to detect tuberculosis (TB) antigen-specific responses in immune-deficient human immunodeficiency virus (HIV) patients. The aim of this study is to determine whether TB antigen-specific immune responses can be detected in patients with HIV-1 infection, especially in those with advanced disease (CD4 T cell count < 300 cells/microl). An enzyme-linked immunospot (ELISPOT) assay, which detects interferon (IFN)-gamma secreted by T cells exposed to TB antigens, was used to assess specific immune responses in a prospective study of 201 HIV-1-infected patients with risk factors for TB infection, attending a single HIV unit. The performance of the ELISPOT assay to detect TB antigen-specific immune responses is independent of CD4 T cell counts in HIV-1 patients. The sensitivity and specificity of this assay for the diagnosis of active tuberculosis does not differ significantly from values obtained in immunocompetent subjects. The negative predictive value of the TB ELISPOT test is 98.2%. A positive predictive value of 86% for the diagnosis of active tuberculosis was found when the combined number of early secretory antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) IFN-gamma spots to CD4 T cell count ratio was > 1.5. TB antigen-specific immune responses can be detected in HIV patients with low CD4 T cell counts using ELISPOT technology in a routine diagnostic laboratory and is a useful test to exclude TB infection in immune-deficient HIV-1 patients. A combination of TB antigen-specific IFN-gamma responses and CD4 T cell counts has the potential to distinguish active tuberculosis from latent infection.

The emerging role of serine proteases in apoptosis.

Unregulated apoptosis can be due to a disruption in the balance and control of both intra- and inter-cellular proteolytic activities leading to various disease states. Many proteases involved in apoptotic processes are yet to be identified; however, several are already well characterized. Caspases traditionally held the predominant role as prime mediators of execution. However, latterly, evidence has accumulated that non-caspases, including calpains, cathepsins, granzymes and the proteasome have roles in mediating and promoting cell death. Increasingly, research is implicating serine proteases within apoptotic processing, particularly in the generation of nuclear events such as condensation, fragmentation and DNA degradation observed in late-stage apoptosis. Serine proteases therefore are emerging as providing additional or alternative therapeutic targets.

Sensory experience alters cortical connectivity and synaptic function site specifically.

Neocortical circuitry can alter throughout life with experience. However, the contributions of changes in synaptic strength and modifications in neuronal wiring to experience-dependent plasticity in mature animals remain unclear. We trimmed whiskers of rats and made electrophysiological recordings after whisker cortical maps have developed. Measurements of miniature EPSPs suggested that synaptic inputs to layer 2/3 pyramidal neurons were altered at the junction of deprived and spared cortex in primary somatosensory cortex. Whole-cell recordings were made from pairs of synaptically connected pyramidal neurons to investigate possible changes in local excitatory connections between layer 2/3 pyramidal neurons. The neurons were filled with fluorescent dyes during recording and reconstructed in three dimensions using confocal microscopy and image deconvolution to identify putative synapses. We show that sensory deprivation induces a striking reduction in connectivity between layer 2/3 pyramidal neurons in deprived cortex without large-scale, compensatory increases in the strength of remaining local excitatory connections. A markedly different situation occurs in spared cortex. Connection strength is potentiated, but local excitatory connectivity and synapse number per connection are unchanged. Our data suggest that alterations in local excitatory circuitry enhance the expansion of spared representations into deprived cortex. Moreover, our findings offer one explanation for how the responses of spared and deprived cortex to sensory deprivation can be dissociated in developed animals.

Deaths from violence in North Carolina, 2004: how deaths differ in females and males.

OBJECTIVE: To identify gender differences in violent deaths in terms of incidence, circumstances, and methods of death. DESIGN: Analysis of surveillance data. SETTING: North Carolina, a state of 8.6 million residents on the eastern seaboard of the US. SUBJECTS: 1674 North Carolina residents who died from violence in the state during 2004. METHODS: Information on violent deaths was collected by the North Carolina Violent Death Reporting System using data from death certificates, medical examiner reports, and law enforcement agency incidence reports. RESULTS: Suicide and homicide rates were lower for females than males. For suicides, females were more likely than males to have a diagnosis of depression (55% v 36%), a current mental health problem (66% v 42%), or a history of suicide attempts (25% v 13%). Firearms were the sole method of suicide in 65% of males and 42% of females. Poisonings were more common in female than male suicides (37% v 12%). Male and female homicide victims were most likely to die from a handgun or a sharp instrument. Fifty seven percent of female homicides involved intimate partner violence, compared with 13% of male homicides. Among female homicides involving intimate partner violence, 78% occurred in the woman's home. White females had a higher rate of suicide than African-American females, but African-American females had a higher rate of homicide than white females. CONCLUSIONS: The incidence, circumstances, and methods of fatal violence differ greatly between females and males. These differences should be taken into account in the development of violence prevention efforts.

Neural cell adhesion molecule-associated polysialic acid inhibits NR2B-containing N-methyl-D-aspartate receptors and prevents glutamate-induced cell death.

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of the nervous system and N-methyl-D-aspartate(NMDA)receptor-dependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by approximately 30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 microm glutamate but not by 30 microm glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.

Functionally diverse complement of large conductance calcium- and voltage-activated potassium channel (BK) alpha-subunits generated from a single site of splicing.

The pore-forming alpha-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that undergoes extensive alternative pre-mRNA splicing. However, the extent to which differential exon usage at a single site of splicing may confer functionally distinct properties on BK channels is largely unknown. Here we demonstrated that alternative splicing at site of splicing C2 in the mouse BK channel C terminus generates five distinct splice variants: ZERO, e20, e21(STREX), e22, and a novel variant deltae23. Splice variants display distinct patterns of tissue distribution with e21(STREX) expressed at the highest levels in adult endocrine tissues and e22 at embryonic stages of mouse development. deltae23 is not functionally expressed at the cell surface and acts as a dominant negative of cell surface expression by trapping other BK channel splice variant alpha-subunits in the endoplasmic reticulum and perinuclear compartments. Splice variants display a range of biophysical properties. e21(STREX) and e22 variants display a significant left shift (>20 mV at 1 microM [Ca2+]i) in half-maximal voltage of activation compared with ZERO and e20 as well as considerably slower rates of deactivation. Splice variants are differentially sensitive to phosphorylation by endogenous cAMP-dependent protein kinase; ZERO, e20, and e22 variants are all activated, whereas e21 (STREX) is the only variant that is inhibited. Thus alternative pre-mRNA splicing from a single site of splicing provides a mechanism to generate a physiologically diverse complement of BK channel alpha-subunits that differ dramatically in their tissue distribution, trafficking, and regulation.

The structures of mouse and human L1 elements reflect their insertion mechanism.

L1 is an abundant, interspersed repeated DNA element of mammalian genomes. It has achieved its high copy number via retrotransposition. Like other non-LTR retrotransposons, L1 insertion into chromosomal DNA apparently occurs by target-site primed reverse transcription, or TPRT. L1 retrotransposition often generates elements with 5' truncations that are flanked by a duplication of the genomic target site (TSD). It is typically assumed that the 5' truncated elements are the consequence of poor processivity of the L1 reverse transcriptase. However, we find that the majority of young L1 elements from both the human and mouse genomes are truncated at sequences that can basepair with the target site. Thus, to whatever extent truncation is a consequence of poor processivity, we suggest that truncation is likely to occur when target site sequence can basepair with L1 sequence. This finding supports a model for insertion that occurs by two sequential TPRT reactions, the second of which relies upon the homology between the target site and L1. Because perfect heteroduplex formation is not required for all insertions, a dynamic relationship between the primer, template and enzyme during reverse transcription is inferred. 5' truncation may be a successful evolutionary strategy that is exploited by L1 as a means to escape host suppression of transposition.

Reversible depression of transcription during hibernation.

Mammalian hibernators downregulate processes of energy production and consumption while maintaining cellular homeostasis. Energetic costs of transcription must be balanced with demands for gene products. Data from nuclear run-on assays indicate transcriptional initiation is reduced two fold in torpid golden-mantled ground squirrels ( Spermophilus lateralis) as compared to euthermic animals between bouts of torpor. In addition, elongation rates across the temperature range experienced by hibernators indicate a virtual arrest of transcription at the low body temperatures of torpor. Finally, there is no seasonal compensation or species-specific adaptation for increased elongational capacity in the cold. Thus, it appears that hibernators are not specifically adapted to continue transcription during torpor. Taken together, these data indicate that transcription arrests during torpor because of a moderate depression of initiation and a more severe inhibition of elongation, largely due to temperature effects. Restoration of euthermic body temperatures during the interbout arousals reverses this transcriptional depression and permits gene expression.

Cytokine concentrations and neutrophil elastase activity in bronchoalveolar lavage and induced sputum from patients with cystic fibrosis, mild asthma and healthy volunteers.

BACKGROUND: Induced sputum (IS) has been proposed as a non-invasive alternative to bronchoalveolar lavage (BAL) for the assessment and monitoring of airways inflammation. The aim of this study was to compare both methods in patients with cystic fibrosis (CF). The possible differences between subjects with CF, mild asthma and healthy volunteers (HV) was also assessed. METHOD: In a single centre, randomised, two way crossover study, 11 patients with CF, 9 mild asthmatics (MA) and 11 HV underwent BAL and hypertonic saline induction on consecutive days. Free neutrophil elastase (NE), neutrophil elastase/alpha(1)-anti-trypsin complex (NE-AAT), tumour necrosis factor receptor (p55) and interleukin-8 (IL-8) were measured in cell free supernatants. RESULTS: Three CF patients reported serious adverse events following BAL. NE was usually undetectable in both IS or BAL samples and NE-AAT concentrations did not differ consistently between the two sampling methods. IL-8 and p55 levels in the CF patients tended to be higher in IS samples compared with BAL samples (median 19,860 vs. 3,855 pg/ml and 2.55 vs. 0.29 ng/ml, respectively). There was a significant difference in mean p55 concentrations between CF, MA and HV in IS samples (P=0.003) but not in BAL samples (P=0.36). The difference in mean IL-8 concentrations in IS samples between subject groups was statistically different (P=0.023). CONCLUSIONS: IS samples can be safely obtained from CF patients. Analysis of IS samples can help to characterize the inflammatory process in the airways of CF patients. The serious adverse events following BAL in 3 CF patients highlight an inherent risk associated with this procedure.