The effect of aerosol distribution on airway responsiveness to inhaled methacholine in patients with asthma.

It has been demonstrated that airway deposition of inhaled aerosols is more heterogeneous in patients with asthma than in normal subjects. Nevertheless, the influence of abnormal airway deposition on responses to bronchoactive aerosols is poorly understood. We altered bronchopulmonary deposition heterogeneity of methacholine aerosol in nine asymptomatic patients with asthma by controlling inspiratory flow at high (approximately 60 L/min) versus low (approximately 12 L/min) rates on 2 study days and determined the effect on the provocative dose of methacholine causing a 20% fall in FEV1 (PD20) (often used as a measure of airway responsiveness). Deposition uniformity was quantified from gamma-camera scans of the lungs in terms of the distribution of a technetium-labeled aerosol that was inhaled rapidly or slowly before the inhalation of methacholine. Increased deposition in an inner (large, central airways) versus an outer (peripheral airways and alveoli) zone of the right lung (inner/outer ratio, greater than 1) and higher values of skew (an index of deposition asymmetry) and kurtosis (an index of deposition range) indicated enhanced heterogeneity of deposition. Mean (+/- SD) inner/outer ratio was significantly higher during rapid inspiration compared to slow inspiration with 2.91 +/- 0.51 and 1.84 +/- 0.30, respectively (p less than 0.01). Mean skew and kurtosis were also significantly higher after rapid inspiration, with 1.12 +/- 0.35 and 3.86 +/- 1.25, respectively, compared to 0.74 +/- 0.36 and 2.64 +/- 0.77 after slow inhalation (p less than 0.01). Geometric mean PD20 methacholine was significantly reduced when the aerosol was inhaled rapidly, with 5.9 cumulative methacholine units compared to 15.7 units after slow inhalation (p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of quantitative aerosol techniques for use in bronchoprovocation studies

To investigate airway physiology by use of inhaled aerosols, it is frequently necessary to measure the actual amount of material deposited on the airway wall as well as the site of particle deposition. To satisfy these needs, radiolabeled aerosols and gamma camera techniques have been used to measure regional deposition of inhaled particles. To make quantitative measurements of the amount deposited, previous investigators have used a "phantom" technique to indirectly calibrate the gamma camera for the attenuation of gamma rays through the lungs and chest wall. For this calibration, the phantom is a simulated lung containing a known amount of radioactivity. Radioactive counts emitted from the phantom are assumed to be attenuated in the same manner as the intact human lung. The present article describes a technique to determine directly the amount of inhaled aerosol deposited in the lung and simultaneously to calibrate the gamma camera for each individual subject. We used right angle light scattering and a gamma camera to measure individual values of the deposition fraction (DF) of inhaled aerosol deposited in the lung and the coefficient of attenuation (AC) of gamma rays in normal and obstructed lungs of human subjects. Radiolabeled monodisperse aerosols 1 and 2 microns in diameter were used. Knowledge of the activity of the inhaled aerosol (microcurie per liter), the volume inhaled, and the measured DF determined each subject's AC (counts per minute per microcurie). DF varied by an order of magnitude in normal (0.04 to 0.48) and obstructed (0.16 to 0.75) of subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of inspiratory flow rate regulation on nebulizer output and on human airway response to methacholine aerosol

Increased inspiratory flow rate has been demonstrated to decrease pulmonary deposition of inhaled aerosols. To study the effect of inspiratory flow rate regulation on the physiologic response to an active substance administered by aerosol, we compared the effect of high unregulated flow rate (66 to 212 L/min) with regulated low flow rate (20 to 35 L/min) on nebulizer output and on the pulmonary response to methacholine in patients with asthma. Four No. 646 DeVilbiss nebulizers were used in sequence with a nebulization dosimeter to deliver tenfold incremental concentrations of methacholine aerosol (mass median aerodynamic diameter = 1.52 micron; geometric standard deviation = 1.96) ranging from 0.025 to 25 mg/ml. When flow was unregulated, nebulizer output was not greater than when flow was regulated, but coefficients of variation of output were significantly greater (p less than 0.01). The PD20 on the two unregulated days was significantly different (p = 0.01), whereas the PD20 on the two flow regulated days was not significantly different (p greater than 0.05). We conclude that regulation of inspiratory flow rate at rates within the range of tidal breathing significantly decreases variability in nebulizer output and variation of pulmonary responses to methacholine challenge.     

A salivary collection method for young children

Salivary assays are a major physiological measure in studies of child development. Traditional collection techniques have generally involved children chewing on sterile dental cotton rolls. However, research suggests that, for an accurate assay, potential contaminants need to be minimized, both from oral stimulants and the collection device. Moreover, the use of cotton requires that additional saliva be collected to compensate for the amount absorbed by the cotton itself. For these reasons I adapted the passive drooling collection protocol for use with young children. To this end, a game was created which involves frog puppets constructed so that a funnel, which serves as the frog's mouth, connects to a cryogenic vial housed in the frog's body. Details on the specifics of this methodology as well as the effectiveness of this approach are discussed.     

The functional benefit of anti-inflammatory aerosols in the lung periphery

BACKGROUND: The target of anti-inflammatory therapy in asthma is thought to be situated, at least partly, in the lung periphery, and inhaled steroid aerosols are being engineered to reach it. However, the potential effect of such aerosols cannot be fully evaluated by conventional lung function tests because these are insensitive to peripheral lung structure. OBJECTIVE: A prospective cohort study was conducted to investigate whether ultrafine steroid aerosols can elicit a response in the lung periphery, using a validated multibreath washout technique that can distinguish acinar from conductive lung zone function. METHODS: In 30 stable patients with asthma with a wide range of disease severity (FEV(1) 27% to 108% predicted), we assessed conductive and acinar airway function abnormality at baseline, with patients on a standard dry powder steroid aerosol and after switching them to an ultrafine steroid aerosol. RESULTS: Only in those patients with abnormal acinar airway function at baseline (n = 16) did acinar heterogeneity show a consistent improvement after switching to an ultrafine steroid aerosol; the improvement was also correlated with baseline acinar heterogeneity (r = -0.67; P = .007). Although all patients with asthma also presented conductive airway abnormality at baseline, no changes were observed in this lung zone with the switch to the ultrafine aerosol (P > .1). CONCLUSION: Among stable patients with asthma, those with acinar lung zone abnormality at baseline have the potential to receive functional benefit from an ultrafine steroid aerosol. Clinical studies comparing the efficacy of steroid aerosols targeted to the deep lung should at least include a measurement of peripheral lung zone function. CLINICAL IMPLICATIONS: A new noninvasive measure of small airways function reveals why, and for which particular patients with asthma, small steroid aerosol particles can be of therapeutic use.     

Antigen challenge and canine tracheal mucus

Tracheal mucus hypersecretion was demonstrated in 7 dogs with cutaneous sensitivity to extracts of Ascaris suum when the dogs were exposed to aerosols of the same antigen. There was a correlation between antigen skin sensitivity and the aerosol dose at which hypersecretion first occurred. In addition to hypersecretion, antigen challenge led to significant alterations in mucus viscoelasticity. The dose-response pattern was similar to that seen with methacholine and histamine aerosol administration in dogs employing the same methodology. The antigen-induced hypersecretion was blocked or substantially reduced by pretreatment with atropine, H1-antagonists, and thiazinamium chloride. These observations suggest that mucus hypersecretion associated with A. suum challenge in dogs involves histamine release and proceeds at least in part via a cholinergic mechanism.     

Recent Advances in the Diagnosis and Management of Ebola Virus Disease

Outbreaks of Ebola virus, a causative agent of hemorrhagic fever, in West Africa and the Democratic Republic of the Congo have highlighted the need for assays that can rapidly identify individuals infected with Ebola virus. These assays need to accurately detect and rule-out Ebola virus disease (EVD) and, ideally, not require advanced laboratory techniques. Over the course of the most recent outbreaks, field studies have evaluated the performance of rapid antigen and automated molecular tests, many of which were given emergency use authorization by the Food and Drug Administration. Overall, molecular assays demonstrated high sensitivity, while antigen testing had the highest sensitivity when the viral load was high. Specificity was high across the evaluated test options. In addition to the evaluation of diagnostic test methods, newly developed treatment options and the efficacy of ring vaccination to prevent disease transmission have been investigated. Findings from these field studies have demonstrated that many of the newly developed diagnostic assays, as well as experimental treatments and vaccination, are promising tools for controlling EVD.     

Comparison of intermittent and continuous inhalation provocation tests

To improve standardization of inhalation provocation tests, two tests utilizing intermittent and continuous inhalation of methacholine aerosol were compared in five normal and ten asthmatic patients. During the intermittent inhalation test, methacholine aerosols with stepwise incremental concentrations were inhaled during tidal breathing for two minutes with a following 5-minute pause interval. Specific airway conductance (SGaw) and respiratory resistance (Rrs) were measured one-half, one and one-half, and five minutes after the end of each inhalation period. Specific airway conductance and Rrs were measured with the panting and forced oscillation methods, respectively. During the continuous inhalation test, the same stepwise incremental concentrations of methacholine aerosol were inhaled during tidal breathing for two minutes without a pause while Rrs was continuously measured. The cumulative dose of methacholine required to reduce SGaw by 35% of the baseline value (PD35 SGaw) during the intermittent inhalation test was significantly correlated with the cumulative methacholine dose required to reduce Grs (= 1/Rrs) by 35% of the baseline value during the continuous inhalation test (PD35 Grs) (r = .98). There was also a significant correlation between the slopes of the intermittent inhalation test and continuous inhalation test curves (r = .80). The results indicate that methacholine provocation can be measured as reliably using a simple continuous inhalation method as by a more complex intermittent one.     

Identification of soybean dust as an epidemic asthma agent in urban areas by molecular marker and RAST analysis of aerosols

From 1981 to 1987, 26 outbreaks of asthma have occurred in the city of Barcelona, Spain, affecting a total of 687 subjects and causing 1155 emergency room admissions. Assays of urban aerosols collected with high-volume samplers between October 1986 and May 1989 have indicated that soybean dust originating from harbor activities (and not traffic or industrial pollution) is the causal agent for these epidemics. Soybean particulates in the filters have been characterized from the composition of the alcohol fraction, namely, campesterol, stigmasterol, beta-sitosterol, n-triacontan-1-ol, and n-dotriacontan-1-ol, constituting a series of aerosol components correlated with the epidemiologic data. This result has also been confirmed by immunochemical assay of specific soybean allergens. The concentration of these sterols in the air corresponds to a 24-hour average level of soybean dust in the order of 25 micrograms/m3 on epidemic days. These results suggest the advisability of monitoring soybean dust in air particulates of populated areas surrounding soybean storage or processing plants. The techniques presented here afford a simple way for the recognition of soybean dust in aerosols containing high concentrations of organic pollutants of traffic or industrial origin.     

Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexed luminex assay

Several different methods have been developed to quantitate neutralizing antibody responses to human papillomaviruses (HPVs), including in vivo neutralization assays, in vitro pseudoneutralization assays, competitive radioimmunoassays (cRIAs), and enzyme-linked immunosorbent assays. However, each of these techniques possesses one or more limitations that preclude testing large numbers of patient sera for use in natural history studies and large vaccine clinical trials. We describe here a new multiplexed assay, by using the Luminex Laboratory MultiAnalyte Profiling (LabMAP3) assay system, that can simultaneously quantitate neutralizing antibodies to human papillomavirus types 6, 11, 16, and 18 in 50 micro l of serum. The HPV-Luminex competitive immunoassay measures titers of polyclonal antibodies in serum capable of displacing phycoerythrin-labeled detection monoclonal antibodies binding to conformationally sensitive, neutralizing epitopes on the respective virus-like particles. This competitive Luminex immunoassay was found to be as sensitive, accurate, and precise as the currently used cRIAs. An effective HPV vaccine will most likely require several distinct genotypes to protect against multiple cancer causing papillomaviruses. The HPV-Luminex immunoassay should prove to be a useful tool in simultaneously quantitating antibody immune responses to multiple HPV genotypes for natural history infection studies and for monitoring the efficacy of prospective vaccines.     

Simultaneous Quantitation of Antibodies to Neutralizing Epitopes on Virus-Like Particles for Human Papillomavirus Types 6, 11, 16, and 18 by a Multiplexed Luminex Assay

Several different methods have been developed to quantitate neutralizing antibody responses to human papillomaviruses (HPVs), including in vivo neutralization assays, in vitro pseudoneutralization assays, competitive radioimmunoassays (cRIAs), and enzyme-linked immunosorbent assays. However, each of these techniques possesses one or more limitations that preclude testing large numbers of patient sera for use in natural history studies and large vaccine clinical trials. We describe here a new multiplexed assay, by using the Luminex Laboratory MultiAnalyte Profiling (LabMAP3) assay system, that can simultaneously quantitate neutralizing antibodies to human papillomavirus types 6, 11, 16, and 18 in 50 μl of serum. The HPV-Luminex competitive immunoassay measures titers of polyclonal antibodies in serum capable of displacing phycoerythrin-labeled detection monoclonal antibodies binding to conformationally sensitive, neutralizing epitopes on the respective virus-like particles. This competitive Luminex immunoassay was found to be as sensitive, accurate, and precise as the currently used cRIAs. An effective HPV vaccine will most likely require several distinct genotypes to protect against multiple cancer causing papillomaviruses. The HPV-Luminex immunoassay should prove to be a useful tool in simultaneously quantitating antibody immune responses to multiple HPV genotypes for natural history infection studies and for monitoring the efficacy of prospective vaccines.     

Characterization of Respiratory Drug Delivery with Enhanced Condensational Growth using an Individual Path Model of the Entire Tracheobronchial Airways

The objective of this study was to evaluate the delivery of inhaled pharmaceutical aerosols using an enhanced condensational growth (ECG) approach in an airway model extending from the oral cavity to the end of the tracheobronchial (TB) region. The geometry consisted of an elliptical mouth-throat (MT) model, the upper TB airways extending to bifurcation B3, and a subsequent individual path model entering the right lower lobe of the lung. Submicrometer monodisperse aerosols with diameters of 560 and 900 nm were delivered to the mouth inlet under control (25 °C with subsaturated air) or ECG (39 or 42 °C with saturated air) conditions. Flow fields and droplet characteristics were simulated using a computational fluid dynamics model that was previously demonstrated to accurately predict aerosol size growth and deposition. Results indicated that both the control and ECG delivery cases produced very little deposition in the MT and upper TB model (approximately 1%). Under ECG delivery conditions, large size increases of the aerosol droplets were observed resulting in mass median aerodynamic diameters of 2.4–3.3 μm exiting B5. This increase in aerosol size produced an order of magnitude increase in aerosol deposition within the TB airways compared with the controls, with TB deposition efficiencies of approximately 32–46% for ECG conditions. Estimates of downstream pulmonary deposition indicted near full lung retention of the aerosol during ECG delivery. Furthermore, targeting the region of TB deposition by controlling the inlet temperature conditions and initial aerosol size also appeared possible.     

Sensitive and reproducible quantitation of mucosal HIV-1 RNA and DNA viral burden in patients with detectable and undetectable plasma viral HIV-1 RNA using endoscopic biopsies

Mucosal tissue is the main portal of entry for HIV-1 infection and, in macaques, has been demonstrated to be a significant compartment for viral replication and CD4+ T lymphocyte depletion. Quantitating tissue viral burden in addition to plasma viral load provides insights into HIV-1 pathogenesis and an additional means to gauge antiretroviral response. The aim of this study was to develop reliable, reproducible, and sensitive assays to quantitate tissue viral burden of HIV-1 RNA and DNA using 1-3 endoscopically acquired, rectosigmoid biopsies. Total DNA and RNA were simultaneously extracted following homogenization from the same tissue samples. Quantitative polymerase chain reaction (PCR) assay in the HIV-1 LTR region was used to detect viral DNA and RT-PCR for viral RNA. It was determined that HIV-1 RNA and DNA can be reproducibly quantified from a single rectosigmoid biopsy with minimal intra-assay or intra-patient variability. These results reflect high recovery of extracted nucleic acids with calculated results accurately reflecting in vivo levels. The techniques outlined differ from currently available approaches by incorporating control standards to identify loss or degradation of RNA and DNA from acquisition through the in vitro assay and permit extraction with high yields of RNA and DNA from the same tissue sample.     

Methacholine responsiveness increases after ultrasonically nebulized water but not after ultrasonically nebulized hypertonic saline in patients with asthma

Airway obstruction can be induced in patients with asthma by the inhalation of ultrasonically nebulized aerosols of nonisotonic solutions. It is the change in osmolarity of the periciliary fluid that is believed to be the stimulus for bronchoconstriction. However, it is not known whether hyperosmolar and hypo-osmolar aerosols induce asthma via the same mechanism. We have previously reported that patients with asthma have a reduction in the dose of provoking agent that induces a 20% fall in FEV1 (PD20) for methacholine after challenge with nebulized water. To determine whether hyperosmolar aerosols also increase sensitivity to methacholine, we studied 13 subjects with asthma on 3 days. On day 1, the PD20 to methacholine was determined. On day 2, a challenge with nebulized 4.5% saline was followed by a challenge with methacholine 40 to 60 minutes later. On day 3, a challenge with nebulized water was followed by a methacholine challenge. Sensitivity to methacholine was significantly increased after water (p less than 0.02) but not after 4.5% saline. Furthermore, there was no relationship between the PD20 to water and to 4.5% saline. When the Spearman's correlation coefficient was used to compare sensitivity to the challenges, there was a significant relationship between the PD20 to 4.5% saline and methacholine (p less than 0.01) but not between the PD20 to water and methacholine. These results suggest that the mechanism of asthma induced by hyperosmolar and hypo-osmolar solutions is different.     

Use of laser flow-type fluorescence aerosol particle counter to evaluate the concentration of microbes in the surface air under high dust content

The paper deals with the use of a laser flow-type fluorescence aerosol particle counter to evaluate the concentrations of microbes in the surface air under high dust content. Various circuits of flow-type optic aerosol recorders are analyzed. Flow spectral luminescence analysis of some particles flow while exciting the fourth harmonics of a pulse laser on yttrium-aluminium garnet with neodymium by ultraviolet radiation is shown to be the most optimum method for indication of individual aerosol particles. Experiments were conducted on the authors' model of a pilot plant based on this method. The model of a laser flow-type optic analyzer was developed for experimental studies that give a clear display of biological aerosols in complex aerosols. The laser flow-type analyzer-based unit developed may provide a fluorescence signal of aerosol particles in the flow of a sample and that light diffusion signal from them at an exciting light wavelength of 266 nm. Experiments with BVC aerosols and soil dust particles were conducted in different regions of Russia. They showed it possible to detect and to rapidly calculate soil microorganisms by laser flow-type fluorescence assay of individual particles when excited by ultraviolet radiation.     

Inhaled methacholine does not influence the cellular composition of bronchoalveolar lavage fluid

A methacholine or a histamine test is often used to quantitate bronchial responsiveness, in studies investigating the influence of various inhaled stimuli on the responsiveness and composition of bronchoalveolar lavage (BAL) fluid. Histamine has been shown to evoke an inflammatory cell response in BAL fluid. In order to test the influence of the methacholine test on the cellular composition of BAL fluid, we investigated 14 healthy, nonsmoking volunteers with a methacholine test 24 h before performing a BAL. The results showed no significant changes in the cellular composition of BAL fluid, as compared with control BAL in the same persons.     

Inhaled sodium fluoride decreases airway responsiveness to acetylcholine analogs in vivo

The study was conducted to characterize the action of NaF, which had relaxing property in carbachol precontracted isolated bovine bronchus, on airway responsiveness challenged by acetylcholine receptor agonists in rats and asthmatic humans.Tracheal flow rate and airway resistance were measured in anaesthetized rats. NaF was delivered either before carbachol challenge or together with carbachol. Patients with mild asthma were challenged with methacholine aerosol, and NaF was delivered when FEV1 fell by more than 20%. The results indicated that: (1) in rats NaF significantly inhibited carbachol-induced bronchial constriction when inhaled prior to carbachol challenge as airway resistances in the NaF and NaF+verapamil groups were significantly lower than those in the control group; (2) NaF significantly reversed carbachol or methacholine-induced bronchial constriction in asthmatic patients. In conclusion, NaF, delivered in form of aerosol, reduced bronchial responsiveness to carbachol in rats and had a bronchodilating effect on rat and human airways precontracted by inhalation of acetylcholine analogs.     

Comparison of methods for identifying resistant herpes simplex virus and measuring antiviral susceptibility.

BACKGROUND: A number of in vitro assays are used to determine susceptibility of HSV to antiviral agents, but results from these in vitro assays do not necessarily correlate with treatment outcome. OBJECTIVES: A method with improved capability for identifying an isolate as acyclovir (ACV) or penciclovir (PCV) resistant when resistance is borderline could greatly improve the management of HSV disease. STUDY DESIGN: A comparative evaluation of four in vitro assays, plaque reduction (PRA), DNA hybridization, plating efficiency (PEA) and plaque autoradiography (PAR) was performed to accurately identify and measure resistance of a TK-altered clinical HSV isolate (HSV-1 N4) from a patient who was non-responsive to ACV treatment. Two established criteria for the prediction of antiviral resistance, IC(50)> or =2.0 microg/ml or an IC(50) greater than 10x above a sensitive virus IC(50), as well as testing in human (MRC-5) and nonhuman (Vero and CV-1 monkey kidney) cell lines were evaluated. RESULTS: The PRA and DNA hybridization assays accurately identified HSV-1 N4 as ACV(r) in human cells when using the 10x above sensitive virus IC(50) resistance criterion. Moreover, the PEA and PAR assays failed to classify HSV-1 N4 as drug resistant and indicate that these technologies alone are inadequate for identifying resistant virus. CONCLUSIONS: The data presented herein indicate that the PRA and DNA hybridization assays most accurately identified an otherwise borderline-resistant isolate as drug resistant: (i) when a sensitive virus is used within each individual assay as a control, (ii) when ACV and PCV susceptibility is evaluated in human cells, and (iii) when the 10x above sensitive IC(50) criterion is used to classify a virus as drug-resistant. Testing of additional clinical samples is warranted to further confirm these findings.     

Pituitary blood flow

The direction of pituitary blood flow, the amount of pituitary blood flow, its regional control, and the role of the median eminence microcirculation are the subjects of this review. Present concepts of pituitary blood flow are focused almost entirely on its direction and arouse from studies of pituitary vascular anatomy performed almost 50 years ago. The development of new anatomic techniques has led to a reappraisal of pituitary angioarchitecture, stimulated physiological studies to clarify the pattern of blood flow within the entire gland, and led to a reappraisal of accepted concepts of directional pituitary blood flow. The availability of techniques to accurately measure organ blood flow has permitted study of pituitary blood flow; and, when combined with knowledge of pituitary anatomy, the application of these techniques promises to provide a means to develop insight into control of the mechanisms by which chemical messengers are delivered to the pituitary to control its function. New anatomic techniques promise to develop new understanding of the three-dimensional arrangement of median eminence microvasculature and yield new concepts of blood flow regulation within the median eminence that can be tested by physiological means.