Online monitoring of volatile organic compounds emitted from human bronchial epithelial cells as markers for oxidative stress.

Particulate air pollution is associated with adverse respiratory effects and is a major factor for premature deaths.In-vitroassays are commonly used for investigating the direct cytotoxicity and inflammatory impacts due to particulate matter (PM) exposure. However, biological tests are often labor-intensive, destructive and limited to endpoints measured offline at single time points, making it impossible to observe the progression of cell response upon exposure. Here we explored the potential of a high-resolution proton transfer reaction mass spectrometer (PTR-MS) to detect the volatile organic compounds (VOCs) emitted by human bronchial epithelial cells (BEAS-2B) upon exposure to PM. Cells were exposed to single components (1,4-naphthoquinone and Cu(II)) known to induce oxidative stress. We also tested filter extracts of aerosols generated in a smog chamber, including fresh and aged wood burning emissions, as well asα-pinene secondary organic aerosol (SOA). We found that 1,4-naphthoquinone was rapidly internalized by the cells. Exposing cells to each of these samples induced the emission of VOCs, which we tentatively assigned to acetonitrile, benzaldehyde and dimethylbenzaldehyde, respectively. Emission rates upon exposure to fresh and aged OA fromα-pinene oxidation and from biomass burning significantly exceeded those observed after exposure to similar doses of Cu(II), a proxy for transition metals with high oxidative potential. Emission rates of biomarkers from cell exposure toα-pinene SOA exhibited a statistically significant, but weak dose dependence. The emission rates of benzaldehyde scaled with cell death, estimated by measuring the apical release of cytosolic lactate dehydrogenase. Particle mass doses delivered to the BEAS-2B cells match those deposited in the human tracheobronchial tract after several hours of inhalation at elevated ambient air pollution. The results presented here show that our method has the potential to determine biomarkers of PM induced pulmonary damage in toxicological and epidemiological research on air pollution.

[Vascular factors in glaucoma]. / Facteurs vasculaires du glaucome.

The exact pathophysiology of glaucoma is not fully understood. Understanding of the vascular pathophysiology of glaucoma requires: knowing the techniques for measuring ocular blood flow and characterizing the topography of vascular disease and the mechanisms involved in this neuropathy. A decreased mean ocular perfusion pressure and a loss of vascular autoregulation are implicated in glaucomatous disease. Early decrease in ocular blood flow has been identified in primary open-angle glaucoma and normal pressure glaucoma, contributing to the progression of optic neuropathy. The vascular damage associated with glaucoma is present in various vascular territories within the eye (from the ophthalmic artery to the retina) and is characterized by a decrease in basal blood flow associated with a dysfunction of vasoregulation.

Aspiration toxicology of hydrocarbons and lamp oils studied by in vitro technology.

Medical literature regularly reports on accidental poisoning in children after aspiration of combustibles such as lamp oils which usually contain hydrocarbons or rape methyl esters (RMEs). We aimed to analyze the toxic potential of alkanes and different combustible classes in vitro with regard to biologic responses and mechanisms mediating toxicity. Two different in vitro models were used, i.e. (i) a captive bubble surfactometer (CBS) to assess direct influence of combustibles on biophysical properties of surfactant film and (ii) cell cultures (BEAS-2B and R3/1 cells, primary macrophages, re-differentiated epithelia) closely mimicking the inner lung surface. Biological endpoints included cell viability, cytotoxicity and inflammatory mediator release. CBS measurements demonstrate that combustibles affect film dynamics, i.e. the surface tension/area characteristics during compression and expansion, in a dose and molecular chain length dependent manner. Cell culture results confirm the dose dependent toxicity. Generally, cytotoxicity and cytokine release are higher in short-chained alkanes and hydrocarbon-based combustibles than in long-chained substances, e.g. highest inducible cytotoxicity in BEAS-2B was for hexane 84.6%, decane 74% and hexadecane 30.8%. Effects of RME-based combustibles differed between the cell models. Our results confirm data from animal experiments and give new insights into the mechanisms underlying the adverse health effects observed.

Effects of photodynamic therapy on subfoveal blood flow in neovascular age-related macular degeneration patients.

AIM: To assess the short-term changes in choroidal blood flow (ChBF) after photodynamic therapy (PDT) in patients with neovascular age-related macular degeneration (AMD). METHODS: Fourteen patients with exudative AMD were included after a complete ophthalmological examination, fluorescein and indocyanine green angiography and optical coherence tomography. Subfoveal ChBF was assessed using laser Doppler flowmetry (LDF) in both treated (n=14) and nontreated contralateral (n=8) eyes, 1 h and 1 week after PDT. Ocular perfusion pressure was calculated. RESULTS: The detection sensitivity of the LDF measurements at 2-min intervals before PDT in treated eyes was 7.4% for volume, 6.3% for velocity, and 10.4% for ChBF. The initial mean visual acuity was 0.68+/-0.3 logMar. Macular thickness at baseline as measured by OCT3 was at median (interquartile range), 326.5 microm (188-367). At 1 h and 7 days after PDT, a significant increase in velocity (15.8 and 24.4%, respectively) and a significant decrease in volume (11 and 17.9%, respectively) were noted in treated eyes. Choroidal blood flow and ocular perfusion pressure (OPP) remained similar during follow-up. No significant change in flow parameters was reported in untreated eyes. CONCLUSION: The LDF technique provides feasible and reliable measurements of blood flow parameters before and after PDT in a selective population of patients with exudative AMD. The prognostic value of these early blood flow parameter changes also needs to be assessed.

Corneal and retinal temperatures under various ambient conditions: a model and experimental approach.

BACKGROUND: To determine the distribution of temperatures between the cornea and retina under various external conditions, such as illumination and external temperature, using a simplified heat exchange model. MATERIALS AND METHODS: The human eye is modeled as a water sphere surrounded by different heat sources: choroidal and ciliary body blood flow, tears evaporation, metabolic activity, diffuse retinal illumination and external convection. Corneal and retinal temperatures are derived from this model using finite element theory applied to heat transport. Each of five subjects (28 +/- 10 years old) were placed in three different environments: - 20 degrees C, 20 degrees C and 40 degrees C. After 15 mn, their corneal temperatures were measured with an infrared camera. Corneal temperatures are compared with the values obtained from the model, assuming the same blood flow for all environments. RESULTS: 1) Ciliary body and choroid contribute at least 20 times more than retina to the retinal temperature; 2) temperature increase of the retina due to illumination is negligible; 3) a 10 % reduction of choroidal blood flow induces a corneal temperature decrease of 0.2 degrees C at - 20 degrees C and < 0.1 degrees C at 20 degrees C and 40 degrees C. At normal choroidal blood flow, changes in ambient temperature have a negligible effect on retinal temperature. Measured corneal temperatures agreed with the values from the model: 26.4 +/- 0.9 versus 26.8 degrees C, at - 20 degrees C and 36.2 +/- 0.5 versus 36.7 at 40 degrees C. CONCLUSIONS: Our simplified eye model predicts in a satisfactory way measured corneal temperatures under very different conditions. Calculations show that changing external temperatures from - 20 to + 40 degrees C affects the retinal temperature by less than 1.8 degrees C.

Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase.

Here, we describe a method that offers a unique way to engineer plasmids with precision but without digestion using restriction enzymes for the insertion of DNA. The method allows the insertion of PCR fragments in between any two nucleotides within a target plasmid. The only requirement is that the amplified fragments must be embedded between DNA sequences homologous to the site in which the integration is planned. This method is an adaptation of the QuikChange Site-Directed Mutagenesis protocol. It is simpler than the existing cloning strategies and is suitable for multiparallel constructions of new plasmids. We have demonstrated its utility by constructing plasmids in which we have successfully integrated PCR fragments up to 1117 bp.

Size of the ligand complex between the N-terminal domain of the gene III coat protein and the non-infectious phage strongly influences the usefulness of in vitro selective infective phage technology.

The selective infective phage (SIP) technology allows a rapid positive selection of interacting pairs of biological molecules that restore to non-infectious phages their ability to infect the bacterial host. After a successful infection, the phage is amplified and the DNA encoding the interacting ligand is isolated from the phage genome and sequenced. In our studies we have evaluated the usefulness of SIP for the identification and cloning of proteins interacting with a biotinylated target binding to a newly designed adapter molecule consisting of streptavidin fused to the C-terminus of the extracellular domain of the phage minor coat protein III. The new adapter was expressed in Escherichia coli and refolded from inclusion bodies. The two different domains joined within the chimaera were found to be biologically functional. We also demonstrated that non-covalent interactions between a non-infectious phage displaying a short peptide, which specifically binds the streptavidin, and the adapter molecule restore phage infectivity. To evaluate the potential of SIP as a general and generic tool for the screening of cDNA libraries that encode the ligands displayed at the surface of the phage and binding to biotinylated targets, we have increased both the size of the displayed ligand on the phage and the size of the biotinylated target bound to the streptavidin domain of the adapter molecule. In our model systems we show that the size of either the ligand or the target is a limiting factor for the technology.

Does lack of Cftr gene lead to developmental abnormalities in the lung?

Lung disease is the major cause of death in cystic fibrosis (CF), but the effect of gene mutation on the morphology of the main structural compartments of the lung is poorly understood. We show here, to our knowledge for the first time, a quantitative comparison of the fine pulmonary structures of cftr mutant versus non-cf mice. Pertinent volumes and surface areas were estimated in 10 homozygous cftrm1HGU mutants and 11 non-cf littermates by unbiased stereology at the light microscopic level. Our data did not reveal any statistical differences between group means for any of the 9 parameters considered. In other words, our data do not supply any significant evidence that the lack of the Cftr gene is accompanied by any developmental abnormalities in the lung, at least as far as the parameters studied are concerned.

Retention of Teflon particles in hamster lungs: a stereological study.

The significance of aerosols in medicine is increased when the distribution of inhaled aerosols in the different respiratory tract compartments and their interaction with lung structures are known. The aim of this study was to investigate the retention of the hydrophobic Teflon spheres used in human beings so as to analyze their regional distribution and to study their interaction with lung structures at the deposition site. Six intubated and anesthetized Syrian Golden hamsters inhaled aerosols of Teflon particles with an aerodynamic diameter of 5.5 microns by continuous negative-pressure ventilation adjusted to slow breathing. Lungs were fixed by intravascular perfusion within 21 minutes after inhalation was started, and tissue samples were taken and processed for light and electron microscopy. The stereological (fractionator) analysis revealed that particle retention was the greatest in alveoli (72.4%), less in intrapulmonary conducting airways (22.9%), and the least in extrapulmonary mainstem bronchi (0.3%) and trachea (4.4%). Particles were found submerged in the aqueous lining layer and in close vicinity to epithelial cells. In intrapulmonary conducting airways, 21.5% of Teflon particles had been phagocytized by macrophages. This study with highly hydrophobic Teflon particles clearly demonstrates that for spheres of this size, surface tension and line tension forces rather than the particles' surface free energy are decisive for the displacement of particles into the aqueous phase by surfactant. It was this displacement that enabled subsequent interaction with macrophages. Refined knowledge of particle retention may help us to better understand the biological response to inhaled particles.

Subfoveal choroidal blood flow in response to light-dark exposure.

PURPOSE: To document the response of subfoveal choroidal blood flow (ChBF) in the human eye induced by light and dark exposures and provide some insight into the mechanism underlying this response. METHODS: In a group of 12 volunteers (age, 25-60 years), ChBF was measured with a confocal laser Doppler flowmeter. Wavelength of the probing laser beam was 785 nm (90 microW at the cornea). ChBF was recorded in room light, in darkness, in room light after dark adaptation, and during strong green light exposure after exposure to room light. After dark adaptation of both eyes, ChBF was also measured in one eye while only the fellow eye was exposed to strong visible light. RESULTS: Although ChBF was stable during room light condition, it decreased significantly by 15% (P < 0.01) during dark adaptation. After 6 minutes of room light following 20 minutes of darkness, ChBF was back to baseline. Strong, diffuse, green light exposure over a field of 40 degrees, as well as the probing laser beam, had no detectable effect on ChBF. No change in ChBF was detected when the fellow eye was illuminated after both eyes had been dark adapted. CONCLUSIONS: The findings did not confirm the presence of an active process of ChBF regulation in response to light exposure in humans. They demonstrate, however, a reversible decrease in ChBF that occurs after a transition from room light to darkness, which could involve a neural mechanism.

Interaction of fungal spores with the lungs: distribution and retention of inhaled puffball (Calvatia excipuliformis) spores.

BACKGROUND: The biologic responses to inhaled airborne fungal spores, which are well-known allergen carriers, would be better understood if we had an insight into their pattern of distribution and interaction with lung structures. OBJECTIVES: To investigate the retention characteristics of inhaled basidiospores, which often represent the major portion of the spore load in air-sampling surveys and to analyze their regional distribution within and interaction with the lungs. METHODS: Intubated and anesthetized Syrian Golden hamsters inhaled aerosols of puffball (Calvatia excipuliformis) spores, with an aerodynamic diameter of 3.1 micrometer, either by spontaneous breathing (group A, n = 3) or by continuous negative-pressure ventilation (group B, n = 4). Lungs were fixed by intravascular perfusion of fixative solution within 29 minutes of the initial inhalation, and tissue samples were then processed for light and electron microscopy. RESULTS: Stereological (fractionator) analysis of lung tissue revealed that the greatest number of spores was deposited within the alveoli (67.2% in group A and 89.8% in group B). The intrapulmonary conducting airways retained an intermediate proportion (32.3% in group A and 10.0% in group B), whereas the extrapulmonary mainstem bronchi and trachea held the lowest proportion (0.5% or less). Deposited spores were lodged within the aqueous lining layer and in close proximity to the epithelial cells. Within the intrapulmonary conducting airways, 22. 3% of the spores in group A and 9.0% of those in group B had been engulfed by macrophages. CONCLUSION: This study demonstrates that inhaled 3-micrometer-diameter basidiospores become distributed over a large surface area. It also reveals that such particles are displaced by surfactant (surface forces) into the aqueous lining layer of airways and alveoli, thereby facilitating subsequent phagocytosis by macrophages. This interaction of spores with lung structures may be important for the development of respiratory allergies induced by airborne fungal allergens.

[Effect of light on choroidal blood flow in the fovea centralis]. / Effet de la lumière sur le flux sanguin de la choroïde dans la région de la fovea centralis.

PURPOSE: To investigate whether light and dark exposures induce a response of choroidal blood flow (ChBF) in the foveal region in humans. METHODS: In a group of healthy volunteers (age 25-60 years) ChBF was measured using a new confocal laser Doppler flowmeter (probing laser at 785 nm, power at the cornea = 90 microW). ChBF was recorded at room light, in darkness, at room light following dark adaptation, and during strong light exposure following room light. RESULTS: While ChBF was stable during room light condition, it decreased significantly by 15% (p < 0.01) during darkness. After 6 min of room light following darkness, ChBF was back to baseline. Strong diffuse, green light exposure over a field of 45 degrees had no detectable effect on ChBF. In all the experiments, no significant change of blood pressure was detected. CONCLUSIONS: Our findings did not confirm the presence of an active process of ChBF regulation in response to strong light exposure in humans. They demonstrate, however, a reversible decrease in ChBF occurring after a transition from room light to darkness.

[Optical Doppler velocimetry of red blood cells at different depths in retinal vessels by varying the coherence length of the source: feasibility study]. / Vélocimétrie par effet Doppler optique des globules rouges à différentes profondeurs dans les vaisseaux rétiniens en variant la longueur de cohérence temporelle de la source: etude de faisabilité.

PURPOSE: To report on a novel approach to measure the velocity of red blood cells (RBCs) at different retinal vessel depths. METHOD: The technique is an extension of conventional laser Doppler velocimetry using light sources of various coherence lengths (CL). Light scattered by the moving RBCs interferes with that reflected from the anterior vessel wall only if the optical path difference is shorter than CL. Therefore, using low coherence light sources, localized measurements of RBCs velocity can be performed. RESULTS: Measurements of RBCs velocity at different depths in a main retinal vein (diameter: 152 microns) of a volunteer has been performed using 4 different light sources with CLs of 14 microns, 21 microns, 32 microns and > m. Measured values are in good agreement with theoretically predicted values. CONCLUSION: This new approach permits to measure RBCs velocity at different depths of retinal vessels in the human retina.

[The mucociliary system of the lung--role of surfactants]. / Der mukoziliäre Apparat der Lunge--die Rolle des Surfactant.

Many pollution particles enter the organism via the lung. In the lung, on a surface of 140 m2, the blood is separated from the air by a tissue barrier of only 1/1000 mm. The conducting airways (trachea, bronchi, bronchioli) are a very effective aerodynamic filter for inhaled particles. The mucociliary transport system functions like a self-cleaning mechanism within the filter. Inhaled particles and particles deposited in the lungs play a crucial aetiological and therapeutic role. The discussion in health policy on the relationship between the increase in air pollution and lung damage is of great importance at the present time. Epidemiological studies of recent years have shown very clearly that there is a correlation between morbidity and mortality as a consequence of respiratory and cardiogenic problems and the concentration of PM10 particles in ambient air. So far, however, this correlation has not been explained. The intrathoracic airways are coated by a respiratory epithelium. This has an irregular coating of viscous liquid, consisting of a low viscous sol phase and a high viscous gel phase. It seems, however, that those phases are not clearly distinguishable. The gel phase is moved towards the pharynx by the metachronal ciliary beat transporting the particles out of the lungs. Furthermore, at the air-liquid interface, there exists a continuous surfactant film which reduces the surface tension as is the case in the alveoli. When particles are deposited on the airway wall, that is, on the surfactant film, they are wetted by surface forces and displaced into the liquid phases. Thus, the surfaces of the particles are probably changed by the surfactant or by surfactant components. Many of these particles are transported in the liquid (gel phase) towards the pharynx (mucociliary transport), whereas some of them remain in close association with the epithelium (sol phase). Such particles remain in the airways for days or even weeks. They are either phagocytised by macrophages and carried off via the airways or taken up by dendritic cells and transported into the tissue from where they reach the lymph nodes via lymph drainage and are presented to the T-lymphocytes. The displacement of particles into the liquid phases, caused by the surfactant, can be considered as the initial step in a complex cascade of defence processes in the lungs. The surface of the particles is probably modified by surfactant or surfactant components. These modified particles may be directed to that clearance pathway which is most beneficial for our health, that is, out of the lungs or into the lymphatic glands, where an immune reaction can be triggered. We therefore consider surfactant to be a primary immune barrier.

Response of choroidal blood flow in the foveal region to hyperoxia and hyperoxia-hypercapnia.

PURPOSE: Arterial carbon dioxide tension and arterial oxygen tension are important determinants of retinal and cerebral blood flow. In the present study the hypothesis that changes in arterial blood gases also influence choroidal blood flow was tested. METHODS: The effect of breathing different mixtures of oxygen (O(2)) and carbon dioxide (CO(2)) on choroidal blood flow in the foveal region was investigated in healthy subjects. The study was performed in a randomized, double-masked four way cross-over design in 16 subjects. Using a compact laser Doppler flowmeter, red blood cell velocity (ChBVel), volume (ChBVol), and flow (ChBF) in the choroidal vasculature were measured during the breathing of various mixtures of O(2)and CO(2) (hyperoxia-hypercapnia): 100% O(2), 97%O(2)+3%CO(2), 95%O(2)+5%CO(2) (carbogen) and 92%O(2)+8%CO( 2). Arterial oxygen tension (pO(2)) and carbon dioxide tension (pCO(2)) were measured from arterialized blood samples from the earlobe. RESULTS: Breathing 100% O(2) had no significant effect on ChBVel (-3.7%), ChBVol (+1.7%) and ChBF (-4.3%). Addition of 3% CO(2) to O(2) also produced no significant change on these blood flow parameters. In contrast, carbogen significantly increased ChBVel (10.0 +/- 4.4%, 95% CI, p < 0.001) and ChBF (12.5 +/- 11.7%, p = 0.002). The effect of 92% O(2) + 8% CO(2) was more pronounced since it significantly increased ChBVel and ChBF by 15.5 +/- 7.5% (p < 0.001) and 16.2 +/- 11.0% (p < 0.001), respectively. None of the gas mixtures induced a significant change in ChBVol. The increase in ChBF was approximately 1.5% per 1 mmHg increase in pCO(2). CONCLUSIONS: This study demonstrates that, in healthy subjects, pCO(2) is an important determinant of foveal choroidal blood flow, whereas pO(2) has little impact on it.

[Measuring choroid blood flow with a new confocal laser Doppler device]. / Mesure du flux sanguin choroïdien au moyen d'un nouvel instrument laser Doppler confocal.

UNLABELLED: A new instrument for the measurement of choroidal blood flow in the fovea is presented. It is based on the laser Doppler method and a confocal optical system with an indirect detection of the Doppler shifted light. METHOD: The intensity of the laser beam (785 nm) at the cornea is 90 microW. Measurements were obtained from a normal population of 21 subjects under resting conditions without dilating the pupil. RESULTS: The reproducibility of the choroidal blood flow, based on 5 measurements of 10 s each in 5 randomly selected subjects, is 9%. The minimum detectable change for a statistical significance of p < or = 0.05, based on a population of 21 subjects and 10 s measurements, is 9%. CONCLUSION: This new compact instrument appears to be suitable for the investigation of the physiology and pharmacology of choroidal blood flow and the effect of age-related macular degeneration.

A new methodology for controlled particle inhalation by small rodents.

In order to investigate the deposition, retention, and clearance mechanisms implicated in particle inhalation under standardized conditions, we developed a continuous negative-pressure ventilation system, whereby the breathing pattern in small rodents could be controlled during exposure to aerosols. Using an on-line open-flow set-up, 19 anesthetized, intubated, and paralyzed Syrian golden hamsters, individually contained within a whole-body box, were artificially ventilated under the said continuous negative-pressure conditions, 1 of 5 different combinations of breathing frequency and tidal volume being established. The animals were then exposed to aerosols containing 6-micron diameter polystyrene spheres, and the deposition of particles in the conducting airways was monitored photometrically. During exposure, the level of respiration (mean lung inflation) was stabilized by means of a negative-pressure vent. Breathing frequency and tidal volume, as well as the compliance of the system, remained virtually unchanged during the course of a single experiment, and in each case, a reproducible deposition of particles was achieved. Our findings indicate that tidal volume, but not breathing frequency, has a marked influence on the particle deposition ratio. Breathing frequency exerts opposing and counterbalancing effects on this latter parameter by enhancing the impaction of particles on the one hand, and by decreasing sedimentation on the other.

Identification of the human melanoma-associated chondroitin sulfate proteoglycan antigen epitope recognized by the antitumor monoclonal antibody 763.74 from a peptide phage library.

To identify the epitope of the melanoma-associated chondroitin sulfate proteoglycan (MCSP) recognized by the monoclonal antibody (mAb) 763.74, we first expressed random DNA fragments obtained from the complete coding sequence of the MCSP core glycoproteins in phages and selected without success for binders to the murine mAb 763.74. We then used a library of random heptapeptides displayed at the surface of the filamentous M13 phage as fusion protein to the NH2-terminal portion of the minor coat protein III. After three rounds of selection on the bound mAb, several phages displaying related binding peptides were identified, yielding the consensus sequence Val-His-Leu-Asn-Tyr-Glu-His. Competitive ELISA experiments showed that this peptide can be specifically prevented from binding to mAb 763.74 by an anti-idiotypic MK2-23 mouse:human chimeric mAb and by A375 melanoma cells expressing the antigen MCSP. We screened the amino acid sequence of the MCSP molecule for a region of homology to the consensus sequence and found that the amino acid sequence Val-His-Ile-Asn-Ala-His spanning positions 289 and 294 has high homology. Synthetic linear peptides corresponding to the consensus sequence as well as to the MCSP-derived epitope inhibit the binding of mAb 763.74 to the phages displaying the consensus amino acid sequence. Finally, the biotinylated consensus peptide absorbed to streptavidin-microtiter plates can be used for the detection of mAb 763.74 in human serum. These results show clearly that the MCSP epitope defined by mAb 763.74 has been identified.

Compact laser Doppler choroidal flowmeter.

A compact instrument is described that allows the measurement of the laser Doppler flow parameters, i.e., the velocity, the volume, and flow of blood in the foveal region of the human choroidal vascular system. This new device uses the optical principle of confocality for the delivery of the laser light to the site of measurement and heterodyne detection of the Doppler frequency shifted scattered light. Power of the incident light (785 nm) at the cornea is 90 µW. Measurements were obtained in both eyes of a group of 21 normal volunteers without pupil dilatation. We determined the intrasubject reproducibility and the minimum statistically significant detectable changes in the flow parameters for a group of 21 eyes (one in each subject). Linear correlations were also established between the flow parameters in the right and left eyes. © 1999 Society of Photo-Optical Instrumentation Engineers.