Continuous-flow Chamber Research Articles

Differential Extracellular Signaling Via FcγR and FMLP in Functionally Distinct Antigen-Presenting Cell Subsets: Ultraviolet-Induced Epidermal Macrophages Versus Langerhans Cells

Sunburned skin is characterized by expanded numbers of macrophages (ultraviolet [UV]-MPH), and these UV-MPH differ from Langerhans cells (LC) in their abilities to initiate T-cell-mediated immune reactions. UV-MPH and LC may themselves be differentially responsive to the surrounding milieu, which may in turn modulate their immunoregulatory activity. We asked whether immunologic signal responsiveness, as assessed by cytosolic calcium mobilization, differed among normal human LC, UV-MPH, and normal blood monocytes. LC from normal skin and UV-MPH from UV-exposed skin were distinguished from keratinocytes in epidermal cell suspensions by labeling with anti-HLA-DR. Intracellular calcium content was monitored in real time with the calcium indicator, indo-1, after cross-linking Fc gamma RI, Fc gamma RII, CD11b, CD11c, or CD18 molecules, or addition of interleukin-1 alpha, IL-1 beta, interferon-gamma, bradykinin, substance P, or FMLP. Using flow cytometric analysis of cell suspensions, UV-MPH and blood monocytes were triggered by cross-linking Fc gamma RII (flux of 6.05 and 12.2, respectively). UV-MPH could also be triggered by Fc gamma RI crosslinking and FMLP (flux of 6.41 and 15.54, respectively). By contrast, none of these inflammatory stimuli could cause cytosolic calcium mobilization in normal LC (Flux of -0.2 by FcRII, and 0.18 by FMLP). Because LC calcium flux may be dependent upon extracellular attachments, LC were anchored onto fibronectin-coated coverslips and then their Fc gamma RII was crosslinked in a continuous flow chamber. However, image analysis also failed to detect calcium flux. Neither population responded to interleukin-1, interferon-gamma, bradykinin, substance P, or beta 2 integrin crosslinking. These results indicate that blood monocytes and infiltrating macrophages differ substantially from LC in their responses to immune complexes and chemoattractants. Differential responsiveness to the inflammatory milieu may influence the antigen presenting or effector capabilities of these populations.

Bacterivory: a novel dual role for thraustochytrids in the sea

Two thraustochytrids, Thraustochytrium striatum Schneider and Schizochytrium mangrovei Raghukumar, isolated in 1989 in axenic cultures using pine pollen in seawater from mangroves in Goa, India, were observed in continuous-flow chambers. When grown in the presence of bacteria, both species produced amoebae, which moved using pseudopodia and phagocytosed bacterial cells. Several amoebae were produced in ‘sporangia’, or isolated vegetative cells transformed directly into amoebae. These findings were confirmed with several single-cell isolations of the protists. The demonstration of phagotrophic amoebae in these organisms suggests a dual role for thraustochytrids in nature, namely a fungus-like breakdown of complex organic molecules as well as bacterivory. This phenomenon also places the thraustochytrics in a unique relationship with many other protistan groups.

A continuous-flow system for measuring in vitro oxygen and nitrogen metabolism in separated stream communities

SUMMARY. 1. This paper describes a continuous-flow system that we have used extensively for measurement of oxygen exchange and nitrogen transformations in different communities of lowland streams, i.e. the water, the sediment, and the macrophyte—biofilm community. The system, which is set up on the stream bank, consists of several macrophyte and sediment chambers equipped with a double-flow system that ensures an internal water velocity close to that in the stream and which, by continuously renewing the water, mimics diel fluctuation in stream temperature and water chemistry. Water temperature and dissolved oxygen are measured continuously and the data stored on magnetic tape. Water samples are collected in a refrigerator and analysed using standard chemical procedures. 2. The application of the system is illustrated using results obtained during a 2-day summer experiment in a shallow macrophyte—rich stream. The biological processes in the stream were mainly associated with the macrophyte—biofilm community and the sediment, those in the water being negligible. Oxygen release was confined to the macrophyte—biofilm community, the sediment consuming oxygen both by day and by night. Whole-system gross production and dark respiration occurred at similar rates (6–7g O2 m−2 day−1), net balance being about zero. Inorganic nitrogen was consumed both by the sediment and to a greater extent by the macrophytes, the diel average consumption being 1g N m−2 day−1. 3. The sum of the activity in the macrophyte and sediment chambers corresponded to the overall activity of the stream section as determined by upstream/downstream mass balance. This indicates that the results obtained with the continuous-flow chambers realistically describe the oxygen and the nitrogen metabolism of the stream.

A continuous flow-chamber to study prey preferences of Golden Perch (Macquaria ambigua Richardson) larvae

This paper describes the use of a continuous-flow chamber for maintaining small and fragile fish larvae and their prey in a healthy state and under suitable conditions over a 24 h period for studies on prey preferences. Survival of golden perch (Macquaria ambigua Richardson) larvae and prey was high and ten replicates on food preferences were carried out simultaneously. The larvae preferred small Daphnia carinata King (780–860 μm) over larger ones (2000–3120 and 3680–5440 μm) and preferred cladocerans (Daphnia carinata King and Moina micrura Kurz) over calanoids (Boeckella de Guerne & Richard). The chamber is also suitable for studies on feeding behaviour and diet requirements of fish larvae.

A Study of the Hydration Properties of Selected Laser Dye Aerosols Including Continuous-Flow Parallel Plate and Alternating-Gradient Thermal Diffusion Cloud Chamber Measurements in the High Supersaturation Regime

The hydration behavior of aerosols, made up of various fluorescent dyes, when exposed to water saturation or supersaturated conditions has been studied. Critical supersaturation spectra are reported. The dyes are found to behave as high molecular weight ionic compounds that obey Kohler theory. Their relevant Kohler parameters are measured. This study makes use of and compares results from the isothermal haze, continuous-flow, and alternating-gradient thermal diffusion cloud chambers. The ability of the continuous-flow thermal diffusion chamber to operate correctly at high supersaturations is shown.

A comparison of the static and flow methods for the detection of ice nuclei

The use of the membrane-filter processing chamber to study ice nuclei concentrations has become wide-spread since its introduction by Bigg et al. in 1961. The technique is convenient because of the simplicity of its operation and because it could be run remote from the place of field study. It has however been found to suffer from a number of drawbacks, namely, the volume effect, the chamber height effect, the vapour depletion effect, etc. Comparison of the results obtained by running a traditional filter processor and a continuous flow chamber under identical temperature and humidity conditions for polluted Manchester air has shown that the latter technique detects more ice nuclei than the former one by a factor of about 14±4. These results confirm that the filter technique suffers from the vapour depletion effect. The present results are in agreement with Bigg et al., Mossop and Thorndike, and King. In the light of our findings the filter technique does not appear to be a standard method. Therefore the ice nuclei data obtained with the filter method should not be extended to clouds in order to study their microphysical properties.

Survival of pathogenic bacteria in various freshwater sediments.

Four human-associated bacteria, Pseudomonas aeruginosa, Salmonella newport, Escherichia coli, and Klebsiella pneumoniae, were tested for survival in five freshwater sediments. Bacterial survival in continuous-flow chambers was monitored over 14-day periods on sediments ranging from organically rich high-clay fractions to organically poor sandy fractions. Bacterial die-off ranged from 1 to 5 orders of magnitude in sediments. E. coli survived as long as or longer than S. newport. P. aeruginosa and K. pneumoniae tended to survive longer than E. coli. Survival of E. coli and S. newport was greater in sediments containing at least 25% clay. Good reproducibility allowed the development of linear models to describe die-off rates.

Measurements of natural deposition and condensation-freezing ice nuclei with a continuous flow chamber

Ice nucleus concentrations in samples of city air have been determined by using a continuous flow diffusion chamber. The chamber has been set-up so that ice nuclei may be activated as they pass through regions of controlled supersaturation and temperature similar to those experienced in natural clouds. It has been found that two distinct modes of nucleation may be discerned by adjusting the flow rate through the chamber to permit a range of nucleus activation and crystal growth times. For short passage times, deposition nuclei may be activated both above and below water saturation. At longer growth times, and above water saturation, condensation-freezing nuclei have time to activate and grow. The total nucleus concentration detected is thus the sum of the two distinct types activated in these experiments. The resulting concentrations are considerably in excess of estimates obtained with a conventional filter technique and help to account for the discrepancies between observed ice nucleus and measured ice crystal concentrations in clouds.

Ice nucleus measurement with a continuous flow chamber

Ice nucleus measurement with a continuous flow chamber

Ice nucleus measurement with a continuous flow chamber

AbstractA continuous flow vapour diffusion chamber has been designed and constructed so that ice nuclei can be activated over ranges of temperature and supersaturation similar to those found in natural clouds. A conventional filter‐processing chamber was also built for carrying out comparative measurements. Simultaneous use of the two chambers under similar conditions of temperature and supersaturation has shown that the filter method leads to an underestimate of the concentration of ice nuclei in an air sample. The ratio of the two simultaneous measurements is strongly dependent on the concentration of Aitken nuclei in the air sample and is also dependent on the chamber supersaturation. Some tentative evidence of the presence of condensation‐freezing nuclei active above water saturation is presented.

Evaluation of Plasmodium berghei for Endotoxin by the Limulus Lysate Assay

The presence of endotoxin in the malaria parasite has long been suspected but has not been directly demonstrated by the Limulus amoebocyte lysate (LAL) assay or other assays. Tests for endotoxins have been done on plasma of infected individuals but not on the parasites themselves (Glew and Levin, 1975, Proc. Soc. Exp. Bio. Med. 148: 508-510). Recently, Tubbs (1980, Trans. R. Soc. Trop. Med. Hyg. 74: 121-123) detected endotoxin activity in the plasma of mice infected with Plasmodium berghei and in patients infected with P. falciparum using the LAL assay, and suggested that the endotoxin was derived from either the parasites or the endogenous bacteria in the gut. By assaying intact plasmodia washed free of contaminating host cells and plasma, it is possible to determine whether the plasmodia are the source of the endotoxin. We obtained free P. berghei using ultrasonic energy and tested the plasmodia for endotoxin activity using the LAL assay. The results obtained are the subject of this report. Groups of mice and rats were infected by the intraperitoneal injection of approximately 106 P. berghei parasites, and later the blood was collected aseptically by cardiac puncture when parasitemia reached 40%. The blood was transferred to pyrogen-free, sterile, heparinized tubes and washed three times in sterile, pyrogen-free 0.9% NaCl. Blood from uninfected mice was treated in an identical manner and served as a control. The blood specimens were then sonicated as previously described (Prior and Kreier, 1972, Exp. Parasitol. 32: 239-243). The continuous-flow chamber and sonicator probe tip were rendered pyrogen-free by heating at 160 C overnight. All equipment and solutions were tested for endotoxin contamination with the LAL test before use. Following sonication the specimens were centrifuged to harvest the plasmodia, and then adjusted to 25% packedcell volume. The specimens were lysed by three cycles of freeze-thawing and assayed quantitatively for the presence of endotoxin using the microdilution LAL assay (Prior and Spagna, 1979, J. Clin. Microbiol. 10: 394395). The minimum sensitivity of the Limulus lysate utilized in the microdilution assay (courtesy of Mallinckrodt Inc., St. Louis, Missouri) was 0.06 ng/ml of LPS using the E. coli reference standard (lot EC-2, Bureau of Biologics, Food and Drug Administration). Aliquots of free parasite and control samples were seeded with known quantities of the E. coli endotoxin and assayed. Because certain heat-labile protein substances may give positive LAL test results, portions of the samples were heated at 100 C for 5 min and then tested for endotoxin activity. Parasites and control samples were also cultured on blood agar aerobically and anaerobically to detect any possible bacterial contamination. The results obtained in four experiments using the LAL assay on the lysed parasite suspensions and controls are shown in Table I. A reaction suggestive of endotoxin was ob-

Analysis of a continuous flow parallel plate thermal diffusion cloud chamber

In order to make reliable measurements of water droplet growth in a thermal diffusion cloud chamber, it is essential to have a detailed picture of all transient behavior and edge effects associated with the temperature and diffusion fields within the chamber. In the application of a continuous flow chamber where test nuclei are injected into a carrier air stream, additional care must be taken to account for the transition from turbulent to laminar flow of the carrier air. Here we present an analysis of the transient behavior and edge effects within a continuous flow parallel plate thermal diffusion cloud chamber including: (a) the transition to the steady state of the temperature field, (b) the transition to the steady state of the water vapor density field, (c) the evolution of the boundary layer along the edge of the chamber and (d) the development of the laminar parabolic flow profile from an initial “plug flow” state at the inlet to the chamber.

Damage to microsomal membrane by lipid peroxidation.

AbstractAlterations of microsomal membrane integrity were examined during lipid peroxidation. Loss of membrane‐bound NADPH cytochromec reductase activity and protein release into the aqueous phase were related to disruption of the lipophilic region of the membrane. Formation of fluorescent products in the lipid phase of the membrane occurred only in the presence of peroxidation products. Changes in the membrane lipid phase during peroxidation included a decrease in the reactive amino groups of the phospholipids and a decrease in detectable phosphatidyl ethanolamine. In an ultrafiltration continuous flow chamber, peroxidized microsomal membranes were hydrolyzed to a lesser degree by solubilized lysosomal cathepsins and to a greater degree by lysosomal nucleases than were nonperoxidized membranes.

A radiologic and microradiographic study of experimental pulmonary emphysema in dogs.

Anumber of methods for producing pulmonary emphysema in laboratory animals have been studied (3, 4, 7, 9, 11, 18). Their value as models for human emphysema has been criticized mainly on the ground that they fail to simulate adequately the human form of the disease (18). This criticism is not entirely valid since human pulmonary emphysema probably has multiple etiologies leading to a large variety of pathologic, physiologic, and clinical manifestations (17). No animal model based upon a simple etiologic agent can compete in complexity with its human analog. The simple model, however, can provide insight into the mechanism for the production of specific pathophysiologic changes. This report is a study of a model for the disease based upon an inflammatory etiology described by Rossing (6, 19). He found that repeated subacute exposure of dogs to the pulmonary irritant gas phosgene produced a disease resembling human emphysema. Acute exposure to the gas resulted in pulmonary edema, but subacute exposure resulted in terminal bronchiolitis and alveolitis. After repeated exposure, the dogs developed an increased lower airway resistance and an abnormality of pulmonary gas mixing and at necropsy showed pathologic changes consistent with emphysema. This technic for producing an emphysema-like disease in dogs has been used to study gross radiologic changes and fine structural changes in the lungs. Methods Twenty adult beagles weighing between 6 and 11 kg were exposed weekly to a concentration of 0.8 mg/L of phosgene for thirty minutes. A continuous flow chamber with a volume of 280 L similar to the one described by Rossing was used (18). The animals were not anesthetized during exposure. Prior to the initial exposure of the series and one week after the final exposure of the series, a breath-by-breath nitrogen washout, inspiratory and expiratory chest roentgenography, and pulmonary wedge arteriography were performed. During the course of the exposures, chest films on nonanesthetized animals were obtained at frequent intervals to monitor the progression of the acute phase of the disease. For the initial and final studies, the dogs were anesthetized with 30 mg/kg Na pentobarbital. An endotracheal tube with a balloon cuff was inserted, and the animals were placed on a respiratory pump at a tidal volume of 250 to 300 ml and at a rate sufficient just to suppress normal respiration. The breath-by-breath nitrogen washout was done with a rapid nitrogen analyzer. End-breath nitrogen content was used to plot the washout curves (7, 19). The pump was stopped momentarily in full inspiration and expiration to obtain postero-anterior and lateral chest films for measurement of the thoracic index (4). This was defined as the anterior-posterior diameter/lateral diameter.

The Respiratory C14 Pattern of Acetate-1-C14 Oxidation in the Bovine

Summary The construction and use of a large continuous-flow ion chamber is described. This equipment makes possible the continuous measurement of tracer levels of C 14 in the expired air of dairy cattle. The initial studies of the oxidation of carboxyl-labeled acetate in lactating cows indicate a rapid equilibrium of injected labeled acetate with the body pool of acetate and the rapid metabolic turnover of this pool. The manner in which the labeled acetate is oxidized indicates that acetate enters some pathways not directly leading to oxidation.