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.

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.

[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.

Evaluation of antibodies fused to minor coat protein III and major coat protein VIII of bacteriophage M13.

A gene coding for an anti-(2-phenyl-5-oxazolone) single-chain Fv antibody (Ab) fragment (anti-phOx scFv) was cloned in-frame into phagemid vectors upstream from genes encoding (i) the wild-type (wt) minor coat protein (cp) III of the filamentous bacteriophage M13 of Escherichia coli, (ii) a truncated version of cpIII (amino acid (aa) positions 198-406), (iii) the wt major cpVIII, or (iv) a hybrid of interleukin-1 beta (IL-1 beta; aa 10-152) and wt cpVIII. Recombinant (re-) phage obtained by phagemid rescue were examined for the efficiency of displaying these various anti-phOx scFv::cp hybrids with commercially available anti-M13 enzyme-linked immunosorbent assays (ELISA), by immunoblotting with an anti-c-myc Ab, and by selection experiments. We found that the highest ELISA signals were obtained with the cpIII constructs and also that more immunoreactive material was detected by blotting than with Ab::cpVIII fusions. Consequently, more scFv::cpIII than scFv::cpVIII phage could be recovered in micropanning experiments with the antigen phOx as target.

The hypervariable region in the genes coding for entomopathogenic crystal proteins of Bacillus thuringiensis: nucleotide sequence of the kurhd1 gene of subsp. kurstaki HD1.

One of the genes for the entomophatogenic crystal protein of Bacillus thuringiensis (subsp. kurstaki strain HD1) has been cloned in Escherichia coli, and its nucleotide sequence determined completely. The gene is contained within a 4360-bp-long HpaI-PstI DNA restriction fragment and codes for a polypeptide of 1,155 amino acid residues. The protoxin protein has a predicted Mr of 130,625. The E. coli-derived protoxin gene product is biologically active against Heliothis virescens larvae in a biotest assay. Extensive computer comparisons with other published B. thuringiensis subsp. kurstaki strains HD1, HD73, and B. thuringiensis subsp. sotto gene sequences reveal hypervariable regions in the first half of the protoxin coding sequence. These regions are responsible for the biological activity of the protein product of the cloned gene, and may explain the different biological activities of these different protoxins.

High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies.

We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h. The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix. This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98%. The final yield was 32 mg/l (10(9) cells/l). Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion. The dissociation constant of the scFv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M. Marks et al. (1991) J. Mol. Biol. 222, 581-597: Marks et al. (1992) Bio/Technology 10, 779-783). This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E. coli expression.

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.

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.

Ultrastructure of the aqueous lining layer in hamster airways: is there a two-phase system?

For particle retention and clearance, the structure and surface properties of the airway lining layer are important. Due to difficulties of its preservation, structural analysis has been hampered, and, hence, the existence of two distinct and continuous phases and how much osmiophilic material is available are unclear. It was the objective of this study to investigate the ultrastructure of the aqueous lining layer in the intrathoracic conducting airways of hamsters. By means of transmission electron microscopy, we investigated the ultrastructure of the airway lining layer in hamsters whose lungs have been fixed by the application of fixative dissolved in nonpolar fluorocarbon, either by instillation via the trachea or injection into the gas exchange parenchyma, together with intravascular perfusion of aqueous fixatives. The results were compared to lungs fixed by intravascular perfusion only. In twelve hamsters, the airway lining layer was found to consist of an aqueous phase and was coated by an osmiophilic film that follows fairly closely the upper-extending contours of cilia protruding from epithelial cells. Substantially less osmiophilic material was preserved in extrapulmonary airways and when nonaqueous fixative was injected. We found that the aqueous lining layer of the intrathoracic airways in hamsters essentially surrounds and covers the cilia, the microvilli, and any other structures like macrophages or deposited particles contained in it and is coated by an osmiophilic film of variable thickness. In healthy animals, a gel phase is expected to be very thin, not clearly separated from the periciliary fluid, and located just beneath the osmiophilic film.

Surfactant and inhaled particles in the conducting airways: structural, stereological, and biophysical aspects.

We have investigated the displacement into the sol phase of inhaled particles deposited in the intrapulmonary conducting airways. Hamsters inhaled an aerosol of monodisperse polystyrene particles of 6 microns diameter. Their lungs were fixed by intravascular perfusion, and light and electron microscopy was used to study the epithelial coating. The surfactant film at the wall-air interface was investigated by measuring its surface tension. The number of particles retained was determined stereologically. In addition we investigated the displacement of spherical particles in vitro on a DPPC monolayer in a Langmuir-Wilhelmy surface balance and determined the surface tension in vivo in the horse trachea by video bronchoscopy, applying the droplet spreading method. We found that particles deposited onto a surfactant film were pulled into the aqueous subphase, and we concluded that surface forces due to the airway surfactant likely displace deposited particles into the periciliary fluid (sol phase). Comparing lungs fixed immediately after inhalation with lungs fixed 24 hr after inhalation revealed that 86% of the particles retained in the intrapulmonary conducting airways immediately after inhalation had been cleared within 24 hr. One-third of the particles of the lungs fixed immediately after inhalation was phagocytized. The combination of structural and stereological analyses with in vitro and in vivo measurements has led to new insights into the role of airway surfactant with respect to the fate of inhaled particles, which may have important consequences regarding the effects of hazardous particles. These studies may also help to evaluate the deposition pattern and clearance of therapeutic particles, with important implications for their therapeutic use.

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.

Diffuse luminance flicker increases retinal vessel diameter in humans.

PURPOSE: To determine retinal vessel diameter variations in response to neuronal activity induced by diffuse luminance flicker. METHODS: The diameter of retinal arteries and veins was measured in 9 normal subjects by computer analysis of fundus pictures taken in monochromatic light under normal conditions of illumination and after 1 min of sinusoidally varying diffuse luminance flicker at 10 Hz. RESULTS: The diameter immediately after flicker was significantly larger than the pre-stimulus diameter by 4.2 +/- 2.2% for the retinal arteries and 2.7 +/- 1.7% for the retinal veins (mean +/- SD). Six is after cessation of the flicker, arterial diameter was not significantly different from that of pre-flicker value. CONCLUSIONS: Diffuse luminance flicker induces an increase in retinal vessel diameter. This increase most probably reflects an increase in retinal blood flow previously evidenced in humans by the blue field simulation technique. The technique needs to be optimized in terms of flicker parameters, to determine whether flicker-evoked retinal diameter changes could represent a useful clinical measure of the capability of the retinal vascular system to vasodilate.

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.

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.

Airway surfactant, a primary defense barrier: mechanical and immunological aspects.

Epidemiologic studies have shown strong associations between mortality and morbidity from respiratory and cardiac causes and exposure to fine (PM10), but not coarse, particulates. A plausible mechanistic explanation for these associations is lacking. It has been shown that particles may be retained for an extended period of time in the airways, and that their clearance is inversely proportional to particle size. Such particles are localized in close association with the airway epithelium, and if they consist of low surface energy material, will be coated with an osmiophilic layer, consistent with surfactant. Particles are displaced into this position by surface and line tension forces exerted by the surfactant film at the air-aqueous interface. Particle displacement due to line tension is much greater for smaller particles in the micrometer range. The surface forces acting on the particles leave deep indentations on the epithelial cells. During the displacement process they may come into contact with airway macrophages in the mucous layer and/or dendritic cells situated in the airway epithelium. The smallest particles may even penetrate the mucosa to enter the interstitial compartment. In addition to altering the physical properties of particles, surfactant coatings reduce particle toxicity and enhance phagocytosis by opsonization. We speculate that surfactant acts as a primary defense barrier and plays a role in antigen presentation and elimination at the air-mucus interface of the airways.

[Forgotten expertise about chiropractic]. / Die vergessenen Gutachten über die Chiropraktik.

Two forgotten experts on chiropractic are revived to expose the lack of scientific foundation of osteopathy, chiropractic and manual medicine. It is reemphasized that for the majority of patients with back complaints a precise and tangible pathologic finding explaining the pain can not be established. Likewise the correlation between radiologic, tomographic and patho-anatomic alterations with the patients complaints leaves much to be desired, particularly for the spinal column. Back ailments plagued humanity already in ancient times, their crippling effects seem, however, to be of iatrogenic origin originating in the 20th century. To explain the success stories of manual healers it is important to distinguish between medical science and the actions of the healer.

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.