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In order to maintain the rapid advance of biophotonics in the U.S. and enhance our competitiveness worldwide, key measurement tools must be in place. As part of a wide-reaching effort to improve the U.S. technology base, the National Institute of Standards and Technology sponsored a workshop titled "Biophotonic tools for cell and tissue diagnostics." The workshop focused on diagnostic techniques involving the interaction between biological systems and photons. Through invited presentations by industry representatives and panel discussion, near- and far-term measurement needs were evaluated. As a result of this workshop, this document has been prepared on the measurement tools needed for biophotonic cell and tissue diagnostics. This will become a part of the larger measurement road-mapping effort to be presented to the Nation as an assessment of the U.S. Measurement System. The information will be used to highlight measurement needs to the community and to facilitate solutions.

Background: Limited data exist about the molecular types of methicillin-resistant Staphylococcus aureus (MRSA) strains found in children with cystic fibrosis (CF). We sought to characterize MRSA strains from these patients and compare them with MRSA strains from non-CF pediatric patients.Methods: All MRSA isolates were collected prospectively at Children's Medical Center in Dallas, TX, and the University of Chicago Comer Children's Hospital in 2004 to 2005. All CF MRSA isolates underwent susceptibility testing, multilocus sequence typing, Panton-Valentine leukocidin gene detection (pvl+), and staphylococcal chromosome cassette mec (SCCmec) typing.Results: A total of 22 of 34 MRSA isolates (64.7%) from patients with CF belonged to clonal complex (CC) 5 and contained SCCmec II, so-called health-care associated MRSA (HA-MRSA) strains. Nine of 34 MRSA strains (26.5%) were CC 8, and contained SCCmec IV, so-called community-associated MRSA (CA-MRSA) strains. The CA-MRSA strains tended to be isolated from newly colonized CF patients. In contrast, CC8 isolates predominated among the non-CF patients (294 of 331 patients; 88.8%). MRSA isolates from children with CF were more likely to be resistant to clindamycin (65% vs 19%, respectively) and ciprofloxacin (62% vs 17%, respectively) compared with strains from non-CF patients (p < 0.001). There was no difference in the rate of pvl+ isolate recovery from children with CF undergoing a surveillance culture (7 of 23 children) compared with those with pulmonary exacerbation (3 of 11 children; p = 1.0).Conclusions: Both CA-MRSA (CC8) isolates and HA-MRSA (CC5) isolates populate the respiratory tracts of children with CF. HA-MRSA isolates predominated, but CA-MRSA strains predominated among CF patients with newly acquired MRSA strains and among the non-CF patients. The presence of CA-MRSA strains in children with CF was not associated with exacerbation or necrotizing pneumonia. [ABSTRACT FROM AUTHOR]

Background: Lumacaftor/ivacaftor was approved by the Food and Drug Administration (FDA) as a combination treatment for Cystic Fibrosis (CF) patients who are homozygous for the F508del mutation. The objective of this study was to assess the cost-effectiveness of lumacaftor/ivacaftor combination for the treatment of CF homozygous for F508del CF Transmembrane Conductance Regulator (CFTR) mutation., Methods: A Markov-state transition model following a cohort of 12 year-old CF patients homozygous for F508del CFTR mutation in the United States (US) over two, four, six, eight and ten years from a payer's perspective was developed using TreeAge Pro 2016. Markov states included: mild (percentage of predicted forced expiratory volume in 1 s or FEV1 > 70%), moderate (FEV1 40-70%), severe (FEV1 < 40%) disease, post-transplant, and death. Pulmonary exacerbation and lung transplant were included as transition states. All the input parameters were estimated from the literature. A 1-year cycle length and 3% discount rate were applied. To assess uncertainty in long-term treatment effects, several scenarios were modelled: 100% long-term effectiveness (base-case), defined as improvement in FEV1 in the first year followed by no annual FEV1 decline and a constant reduction in pulmonary exacerbations throughout, 75%, 50%, 25% and 0% (worst case) long-term effectiveness, where treatment effects were intermediate from the second year of treatment until the end of the time horizon. Other scenarios included changing the starting age of the cohort to 6 and 25 years. Primary outcome included incremental cost-effectiveness ratio (ICER) in terms of cost per quality adjusted life year (QALY) gained. One-way and probabilistic sensitivity analyses were performed to determine uncertainty., Results: Under the base-case, Lumacaftor/ivacaftor resulted in higher QALYs (7.29 vs 6.84) but at a very high cost ($1,778,920.88) compared to usual care ($116,155.76) over a 10-year period. The ICER for base-case and worst-case scenarios were $3,655,352 / QALY, and $8,480,265/QALY gained, respectively. In the base-case, lumacaftor/ivacaftor was cost-effective at a threshold of $150,000/QALY-gained when annual drug costs were lower than $4153. The results were not substantially affected by the sensitivity analyses., Conclusions: The intervention produces large QALY gains but at an extremely high cost, resulting in an ICER that would not typically be covered by any insurer. Lumacaftor/ivacaftor's status as an orphan drug complicates coverage decisions.

We describe a laser-driven x-ray plasma source designed for ultrafast x-ray absorption spectroscopy. The source is comprised of a 1 kHz, 20 W, femtosecond pulsed infrared laser and a water target. We present the x-ray spectra as a function of laser energy and pulse duration. Additionally, we investigate the plasma temperature and photon flux as we vary the laser energy. We obtain a 75 μm FWHM x-ray spot size, containing ∼10(6) photons/s, by focusing the produced x-rays with a polycapillary optic. Since the acquisition of x-ray absorption spectra requires the averaging of measurements from >10(7) laser pulses, we also present data on the source stability, including single pulse measurements of the x-ray yield and the x-ray spectral shape. In single pulse measurements, the x-ray flux has a measured standard deviation of 8%, where the laser pointing is the main cause of variability. Further, we show that the variability in x-ray spectral shape from single pulses is low, thus justifying the combining of x-rays obtained from different laser pulses into a single spectrum. Finally, we show a static x-ray absorption spectrum of a ferrioxalate solution as detected by a microcalorimeter array. Altogether, our results demonstrate that this water-jet based plasma source is a suitable candidate for laboratory-based time-resolved x-ray absorption spectroscopy experiments.

An emerging tool in airway biology is the precision-cut lung slice (PCLS). Adoption of the PCLS as a model for assessing airway reactivity has been hampered by the limited time window within which tissues remain viable. Here we demonstrate that the PCLS can be frozen, stored long-term, and then thawed for later experimental use. Compared with the never-frozen murine PCLS, the frozen-thawed PCLS shows metabolic activity that is decreased to an extent comparable to that observed in other cryopreserved tissues but shows no differences in cell viability or in airway caliber responses to the contractile agonist methacholine or the relaxing agonist chloroquine. These results indicate that freezing and long-term storage is a feasible solution to the problem of limited viability of the PCLS in culture.

Purpose of Review: Asthma is a major public health problem that afflicts nearly one in 20 people worldwide. Despite available treatments, asthma symptoms remain poorly controlled in a significant minority of asthma patients, especially those with severe disease. Accordingly, much ongoing effort has been directed at developing new therapeutic strategies; these efforts are described in detail below., Recent Findings: Although mucus hypersecretion is an important component of asthma pathobiology, the primary mechanism of morbidity and mortality in asthma is excessive narrowing of the airway. The key end- effector of excessive airway narrowing is airway smooth muscle (ASM) contraction; overcoming ASM contraction is therefore a prominent therapeutic strategy. Here, we review exciting new advances aimed at ASM relaxation., Summary: Exciting advances in ASM biology have identified new therapeutic targets for the prevention or reversal of bronchoconstriction in asthma.

This report is primarily concerned with methods for optical calibration of laser power for continuous wave (CW) light sources, predominantly used in photodynamic therapy (PDT). Light power calibration is very important for PDT, however, no clear standard has been established for the calibration procedure nor the requirements of power meters suitable for optical power calibration. The purposes of the report are to provide guidance for establishing calibration procedures for thermopile type power meters and establish calibration uncertainties for most commercially available detectors and readout assemblies. The authors have also provided a review of the use of various power meters for CW and pulsed optical sources, and provided recommended temporal frequencies for optical power meter calibrations and guidance for routine quality assurance procedure.

Rationale: In the normal lung, breathing and deep inspirations potently antagonize bronchoconstriction, but in the asthmatic lung this salutary effect is substantially attenuated or even reversed. To explain these findings, the prevailing hypothesis focuses on contracting airway smooth muscle and posits a nonlinear dynamic interaction between actomyosin binding and the tethering forces imposed by tidally expanding lung parenchyma., Objective: This hypothesis has never been tested directly in bronchial smooth muscle embedded within intraparenchymal airways. Our objective here is to fill that gap., Methods: We designed a novel system to image contracting intraparenchymal human airways situated within near-normal lung architecture and subjected to dynamic parenchymal expansion that simulates breathing., Measurements and Main Results: Reversal of bronchoconstriction depended on the degree to which breathing actually stretched the airway, which in turn depended negatively on severity of constriction and positively on the depth of breathing. Such behavior implies positive feedbacks that engender airway instability., Overall Conclusions: These findings help to explain heterogeneity of airflow obstruction as well as why, in people with asthma, deep inspirations are less effective in reversing bronchoconstriction.

Expression and function of Kv7 (KCNQ) voltage-activated potassium channels in guinea pig and human airway smooth muscle cells (ASMCs) were investigated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), patch-clamp electrophysiology, and precision-cut lung slices. qRT-PCR revealed expression of multiple KCNQ genes in both guinea pig and human ASMCs. Currents with electrophysiological and pharmacological characteristics of Kv7 currents were measured in freshly isolated guinea pig and human ASMCs. In guinea pig ASMCs, Kv7 currents were significantly suppressed by application of the bronchoconstrictor agonists methacholine (100 nM) or histamine (30 μM), but current amplitudes were restored by addition of a Kv7 channel activator, flupirtine (10 μM). Kv7 currents in guinea pig ASMCs were also significantly enhanced by another Kv7.2-7.5 channel activator, retigabine, and by celecoxib and 2,5-dimethyl celecoxib. In precision-cut human lung slices, constriction of airways by histamine was significantly reduced in the presence of flupirtine. Kv7 currents in both guinea pig and human ASMCs were inhibited by the Kv7 channel blocker XE991. In human lung slices, XE991 induced robust airway constriction, which was completely reversed by addition of the calcium channel blocker verapamil. These findings suggest that Kv7 channels in ASMCs play an essential role in the regulation of airway diameter and may be targeted pharmacologically to relieve airway hyperconstriction induced by elevated concentrations of bronchoconstrictor agonists.

Tidal breathing, and especially deep breathing, is known to antagonise bronchoconstriction caused by airway smooth muscle (ASM) contraction; however, this bronchoprotective effect of breathing is impaired in asthma. Force fluctuations applied to contracted ASM in vitro cause it to relengthen, force-fluctuation-induced relengthening (FFIR). Given that breathing generates similar force fluctuations in ASM, FFIR represents a likely mechanism by which breathing antagonises bronchoconstriction. Thus it is of considerable interest to understand what modulates FFIR, and how ASM might be manipulated to exploit this phenomenon. It was demonstrated previously that p38 mitogen-activated protein kinase (MAPK) signalling regulates FFIR in ASM strips. Here, it was hypothesised that the MAPK kinase (MEK) signalling pathway also modulates FFIR. In order to test this hypothesis, changes in FFIR were measured in ASM treated with the MEK inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene). Increasing concentrations of U0126 caused greater FFIR. U0126 reduced extracellular signal-regulated kinase 1/2 phosphorylation without affecting isotonic shortening or 20-kDa myosin light chain and p38 MAPK phosphorylation. However, increasing concentrations of U0126 progressively blunted phosphorylation of high-molecular-weight caldesmon (h-caldesmon), a downstream target of MEK. Thus changes in FFIR exhibited significant negative correlation with h-caldesmon phosphorylation. The present data demonstrate that FFIR is regulated through MEK signalling, and suggest that the role of MEK is mediated, in part, through caldesmon.

This paper discusses a procedure for measuring the absolute spectral responsivity of optical-fiber power meters and computation of the calibration uncertainty. The procedure reconciles measurement results associated with a monochromator-based measurement system with those obtained with laser sources coupled with optical fiber. Relative expanded uncertainties based on the methods from the Guide to the Expression of Uncertainty in Measurement and from Supplement 1 to the "Guide to the Expression of Uncertainty in Measurement"-Propagation of Distributions using a Monte Carlo Method are derived and compared. An example is used to illustrate the procedures and calculation of uncertainties.

Breathing is known to functionally antagonize bronchoconstriction caused by airway muscle contraction. During breathing, tidal lung inflation generates force fluctuations that are transmitted to the contracted airway muscle. In vitro, experimental application of force fluctuations to contracted airway smooth muscle strips causes them to relengthen. Such force fluctuation-induced relengthening (FFIR) likely represents the mechanism by which breathing antagonizes bronchoconstriction. Thus, understanding the mechanisms that regulate FFIR of contracted airway muscle could suggest novel therapeutic interventions to increase FFIR, and so to enhance the beneficial effects of breathing in suppressing bronchoconstriction. Here we propose that the connectivity between actin filaments in contracting airway myocytes is a key determinant of FFIR, and suggest that disrupting actin-myosin-actin connectivity by interfering with actin polymerization or with myosin polymerization merits further evaluation as a potential novel approach for preventing prolonged bronchoconstriction in asthma.

Breathing (especially deep breathing) antagonises development and persistence of airflow obstruction during bronchoconstrictor stimulation. Force fluctuations imposed on contracted airway smooth muscle (ASM) in vitro result in its relengthening, a phenomenon called force fluctuation-induced relengthening (FFIR). Because breathing imposes similar force fluctuations on contracted ASM within intact lungs, FFIR represents a likely mechanism by which breathing antagonises bronchoconstriction. While this bronchoprotective effect appears to be impaired in asthma, corticosteroid treatment can restore the ability of deep breaths to reverse artificially induced bronchoconstriction in asthmatic subjects. It has previously been demonstrated that FFIR is physiologically regulated through the p38 mitogen-activated protein kinase (MAPK) signalling pathway. While the beneficial effects of corticosteroids have been attributed to suppression of airway inflammation, the current authors hypothesised that alternatively they might exert their action directly on ASM by augmenting FFIR as a result of inhibiting p38 MAPK signalling. This possibility was tested in the present study by measuring relengthening in contracted canine tracheal smooth muscle (TSM) strips. The results indicate that dexamethasone treatment significantly augmented FFIR of contracted canine TSM. Canine tracheal ASM cells treated with dexamethasone demonstrated increased MAPK phosphatase-1 expression and decreased p38 MAPK activity, as reflected in reduced phosphorylation of the p38 MAPK downstream target, heat shock protein 27. These results suggest that corticosteroids may exert part of their therapeutic effect through direct action on airway smooth muscle, by decreasing p38 mitogen-activated protein kinase activity and thus increasing force fluctuation-induced relengthening.

A high-energy laser attenuator in the range of 250 mJ (20 ns pulse width, 10 Hz repetition rate, 1064 nm wavelength) is described. The optical elements that constitute the attenuator are mirrors with relatively low reflectance, oriented at a 45 degrees angle of incidence. By combining three pairs of mirrors, the incoming radiation is collinear and has the same polarization orientation as the exit. We present damage testing and polarization-dependent reflectance measurements for 1064 nm laser light at 45 degrees angle of incidence for molybdenum, silicon carbide, and copper mirrors. A six element, 74 times (18 dB) attenuator is presented as an example.

We mutagenized male BTBR mice with N-ethyl-N-nitrosourea and screened 1315 of their G3 offspring for airway hyperresponsiveness. A phenovariant G3 mouse with exaggerated methacholine bronchoconstrictor response was identified and his progeny bred in a nonspecific-pathogen-free (SPF) facility where sentinels tested positive for minute virus of mice and mouse parvovirus and where softwood bedding was used. The mutant phenotype was inherited through G11 as a single autosomal semidominant mutation with marked gender restriction, with males exhibiting almost full penetrance and very few females phenotypically abnormal. Between G11 and G12, facility infection eradication was undertaken and bedding was changed to hardwood. We could no longer detect airway hyperresponsiveness in more than 37 G12 offspring of 26 hyperresponsive G11 males. Also, we could not identify the mutant phenotype among offspring of hyperresponsive G8-G10 sires rederived into an SPF facility despite 21 attempts. These two observations suggest that both genetic and environmental factors were needed for phenotype expression. We suspect that rederivation into an SPF facility or altered exposure to pathogens or other unidentified substances modified environmental interactions with the mutant allele, and so resulted in disappearance of the hyperresponsive phenotype. Our experience suggests that future searches for genes that confer susceptibility for airway hyperresponsiveness might not be able to identify some genes that confer susceptibility if the searches are performed in SPF facilities. Experimenters are advised to arrange for multigeneration constancy of mouse care in order to clone mutant genes. Indeed, we were not able to map the mutation before losing the phenotype.

Superimposition of force fluctuations on contracted tracheal smooth muscle (TSM) has been used to simulate normal breathing. Breathing has been shown to reverse lung resistance of individuals without asthma and animals given methacholine to contract their airways; computed tomography scans also demonstrated bronchial dilation after a deep inhalation in normal volunteers. This reversal of airway resistance and bronchial constriction are absent (or much diminished) in individuals with asthma. Many studies have demonstrated that superimposition of force oscillations on contracted airway smooth muscle results in substantial smooth muscle lengthening. Subsequent studies have shown that this force fluctuation-induced relengthening (FFIR) is a physiologically regulated phenomenon. We hypothesized that actin filament length in the smooth muscle of the airways regulates FFIR of contracted tissues. We based this hypothesis on the observations that bovine TSM strips contracted using acetylcholine (ACh) demonstrated amplitude-dependent FFIR that was sensitive to mitogen-activated protein kinase (p38 MAPK) inhibition- an upstream regulator of actin filament assembly. We demonstrated latrunculin B (sequesters actin monomers thus preventing their assimilation into filaments resulting in shorter filaments) greatly increases FFIR and jasplakinolide (an actin filament stabilizer) prevents the effects of latrunculin B incubation on strips of contracted canine TSM. We suspect that p38 MAPK inhibition and latrunculin B predispose to shorter actin filaments. These studies suggest that actin filament length may be a key determinant of airway smooth muscle relengthening and perhaps breathing-induced reversal of agonist-induced airway constriction.

We have developed a measurement system based on a correlation method to characterize the nonlinearity of a detector's response over a large range of laser pulse energy. The system consists of an excimer-laser source, beam-shaping optics, a beam splitter, a monitor detector, a set of optical filters, and the detector under test. Detector nonlinearities as large as 10% or greater over an entire measurement range at an excimer-laser wavelength of 193 nm are observed. The measurement range of the current system is approximately 300 nJ to 50 mJ of laser pulse energy at the detector under test. The typical expanded measurement uncertainty of nonlinearity is 0.6% (k = 2).

We hypothesized that differences in actin filament length could influence force fluctuation-induced relengthening (FFIR) of contracted airway smooth muscle and tested this hypothesis as follows. One-hundred micromolar ACh-stimulated canine tracheal smooth muscle (TSM) strips set at optimal reference length (Lref) were allowed to shorten against 32% maximal isometric force (Fmax) steady preload, after which force oscillations of +/-16% Fmax were superimposed. Strips relengthened during force oscillations. We measured hysteresivity and calculated FFIR as the difference between muscle length before and after 20-min imposed force oscillations. Strips were relaxed by ACh removal and treated for 1 h with 30 nM latrunculin B (sequesters G-actin and promotes depolymerization) or 500 nM jasplakinolide (stabilizes actin filaments and opposes depolymerization). A second isotonic contraction protocol was then performed; FFIR and hysteresivity were again measured. Latrunculin B increased FFIR by 92.2 +/- 27.6% Lref and hysteresivity by 31.8 +/- 13.5% vs. pretreatment values. In contrast, jasplakinolide had little influence on relengthening by itself; neither FFIR nor hysteresivity was significantly affected. However, when jasplakinolide-treated tissues were then incubated with latrunculin B in the continued presence of jasplakinolide for 1 more h and a third contraction protocol performed, latrunculin B no longer substantially enhanced TSM relengthening. In TSM treated with latrunculin B + jasplakinolide, FFIR increased by only 3.03 +/- 5.2% Lref and hysteresivity by 4.14 +/- 4.9% compared with its first (pre-jasplakinolide or latrunculin B) value. These results suggest that actin filament length, in part, determines the relengthening of contracted airway smooth muscle.

The regulation of mucin secretion by airway goblet cells is poorly understood and the receptor-based regulatory mechanisms have not been described in human airways. In the present study, we report that extracellular triphosphate nucleotides regulate the rate of granule release from goblet cells in both normal and cystic fibrosis (CF) airway epithelial explants. Explants isolated from nasal and tracheobronchial tissues were mounted in perfusion chambers and the secretory activity was assessed by videomicroscopic determination of degranulation in single goblet cells and by ELISA determination of mucins secreted into the mucosal perfusate. Baseline degranulation was measured at 0.05 degranulation events (DE)/min. In normal goblet cells, mucosal ATP (10(-4) M, n = 17) induced a biphasic secretory response comprising 29.1 +/- 4.9 DE during the first 5 min, with an initial rate of 118.2 +/- 10.2 DE/min. Mucosal UTP (10(-4) M, n = 9) induced a similar response to ATP (initial rate: 89.2 +/- 23.9 DE/min, 17.9 +/- 5.1 DE in 5 min), but mucosal 2-MeSATP was not an effective agonist (initial rate: 1.5 +/- 1.4 DE/min, 2.3 +/- 0.5 DE in 5 min). Determination of mucins by ELISA confirmed that both ATP and UTP induced similar secretory responses but that 2-MeSATP was not effective. In CF explants, mucosal UTP (10(-4) M, n = 6) induced similar responses to those observed in normal tissues (initial rate: 82.5 +/- 27.5 DE/min, 18.8 +/- 4.1 DE in 5 min). We conclude that human nasal and tracheobronchial goblet cells are stimulated by mucosal nucleotides, probably via a 5'-nucleotide receptor, and that this response is unaffected by CF.

Mucin secretion by goblet cells was determined by quantifying degranulation events (DE) in isolated, superficial epithelium from canine trachea. The epithelium was isolated and explanted to a novel transparent, permeable support, and the goblet cells were visualized by video microscopy. Baseline degranulation events were quantified at 0.05 DE/min. Luminal ATP (10(-4) M, n = 10) stimulated a biphasic secretory response; a burst, maximum rate = 87.9 +/- 25.3, was followed by a plateau, rate = 1.9 +/- 0.3 DE/min. Serosal ATP elicited a complex set of responses: 9 cells failed to respond, 13 exhibited a trivial response, and 31 responded vigorously but with highly variable patterns of degranulation. Nonhydrolyzable 5'-adenylylimidodiphosphate caused degranulation from both sides of the epithelium. Luminal ADP and adenosine were ineffective. Serosal ADP and adenosine elicited a range of responses that was similar in diversity and magnitude to the ATP response. Our conclusions were as follows: 1) goblet cells in the superficial epithelium of the airway can be studied at the single-cell level in explants; 2) nucleotides stimulate goblet cells to secrete mucin; and 3) the goblet cell expresses different nucleotide receptors on its apical and basolateral membranes.