Eating disorders in patients with cystic fibrosis.

This study was designed to examine rates of eating disorders and psychopathology in patients with cystic fibrosis (CF). Fifty-eight CF patients and 43 healthy control participants were evaluated using structured psychiatric interviews and rating scales. Two control participants and no CF patients were diagnosed with an eating disorder. Additionally, 11 CF patients were diagnosed with one or more psychiatric disorders. Group means on the rating scales did not show clinically meaningful elevations in either group. These data indicate no evidence for elevated rates of eating disorders in CF patients. Similarly, rates of other psychiatric disorders in the CF group were not greater than the prevalence reported in the general population.

Proteinase-free myeloperoxidase increases airway epithelial permeability in a whole trachea model.

In cystic fibrosis the bronchiectatic conducting airways have large numbers of neutrophils in their walls and in their luminal contents. The neutrophil's primary granule enzyme activities of elastase and peroxidase are increased in the sputum of these patients. It has been postulated that these enzymes--together or individually--act to damage the airway epithelium. However, only peroxidase activity has consistently correlated with the degree of structural and functional airway disease in these patients with leakage of plasma protein into the airway lumen (Regelmann et al., Pediatr Pulmonol, 1995; 19:1-9). The present study was designed to test whether human neutrophil-derived myeloperoxidase can independently produce bronchial epithelial damage without the presence of proteases, as measured by increased permeability of the airway epithelium. Human peripheral blood neutrophils were purified, their primary granules isolated, and their peroxidase purified using affinity and ion exchange column chromatography. Activity of the proteinase-free peroxidase was measured using a chromogenic substrate. The effect of this peroxidase on the permeability of excised rat tracheas was measured using radioactive and fluorescent-labeled non-ionic molecules of varying molecular weight. Rat tracheas exposed to 15 minute treatments with either 130 U of peroxidase or hydrogen peroxide (10(-5) M) did not show a significant increase in the permeability of the epithelium to [3H]inulin, [14C]sucrose, and fluorescein isothiocyanate dextran 20 compared with control tracheas. However, those tracheas exposed to 130 U peroxidase followed by 10(-5) M hydrogen peroxide showed an increased permeability to each of the three test solutes. We conclude that proteinase-free myeloperoxidase, in the presence of non-toxic concentrations of its substrates, hydrogen peroxide and halide, produced increases in permeability to non-ionic molecules in the rat trachea within 15 minutes.

Sputum peroxidase activity correlates with the severity of lung disease in cystic fibrosis.

Patients with cystic fibrosis (CF) of the same age differ significantly in their degree of pulmonary disease. Based on preliminary observations, we postulated that the activity of myeloperoxidase would be significantly increased in patients with greater structural lung damage than in those with less lung damage. Acid extracts of weighed sputum samples were assayed for lactoferrin concentrations by ELISA. Activities of peroxidase, cathespsin G, and elastase (with and without proteinase 3) were determined by kinetic analysis using chromogenic substrates. The patients were divided into quartiles based on their Brasfield chest-radiograph score. Patients in the first quartile (least amount of structural lung abnormality) were compared to those in the fourth quartile. The concentration of lactoferrin, a specific (secondary) granule protein of neutrophils, did not differ between the two patient groups. However, the activities of the neutrophil primary granule proteins, peroxidase, elastase, and elastase plus proteinase 3, were significantly elevated in the group with the most structural lung abnormality. Sputum albumin concentration was used to estimate leakages of plasma proteins into the airways. Peroxidase activity, but not the activity of cathepsin G, of elastase, or of elastase plus proteinase 3, correlated significantly with albumin/g sputum in both quartile groups. To confirm the association of sputum peroxidase activity with differences in lung structure and to test its correlation with lung function, spirometry was performed in a second group of patients during the week prior to the time of sputum sampling. In this second group, increased sputum peroxidase activity was associated with worse Brasfield scores and with decreased percent-predicted forced expiratory volume in 1 sec.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for an ecto-ATPase on the cell wall of Streptococcus sanguis.

Certain strains of viridans streptococci bind platelets, which release ATP from dense granules and then aggregate. By hydrolyzing the released ATP to the platelet agonist, ADP, cell wall-associated ATPase activity of Streptococcus sanguis may amplify the aggregation of platelets. To identify and characterize this ecto-ATPase activity, whole cells were incubated with [14C]-ATP. The cell-free nucleotides were separated by thin-layer chromatography and quantified by liquid scintillation counting. Whole-cell activity showed temperature and pH optima in the physiological range. To isolate a soluble fraction with ATPase activity from the cell wall, whole cells were digested under osmotically stable conditions to produce protoplasts. Protoplasts and cells were separated from soluble cell wall materials by centrifugation. ATPase activity in cell fractions was identified by zymograms of native 8% polyacrylamide gels after electrophoresis. The ecto-ATPase preparation, membrane and cytoplasmic ATPase in lysed protoplasts showed different zymograms and sensitivity to inhibition by DCCD, ouabain vanadate, azide and NEM. In electron micrographs of ultrathin sections of cells of S. sanguis, ATPase activity was localized to the cell wall. Since the pattern of localization to the wall changed with the phase of growth, the ecto-ATPase of S. sanguis may be associated with the development and maintenance of the cell wall.

Platelet-interactive products of Streptococcus sanguis protoplasts.

To isolate a more native, platelet-interactive macromolecule (class II antigen) of Streptococcus sanguis, cultured protoplasts were used as a source. Protoplasts were optimally prepared from fresh washed cells by digestion with 80 U of mutanolysin per ml for 75 min at 37 degrees C while osmotically stabilized in 26% (wt/vol) raffinose. Osmotically stabilized forms were surrounded by a 9-nm bilaminar membrane, as shown by transmission electron microscopy. Protoplasts were cultured in chemically defined synthetic medium and osmotically stabilized by ammonium chloride. Spent culture media were harvested daily for 7 days. Each day, soluble proteins were isolated from media, preincubated with platelet-rich plasma, and tested for inhibition of platelet aggregation induced by S. sanguis cells. Products released from S. sanguis protoplasts and reactive with an anti-class II antigen immunoaffinity matrix were able to inhibit S. sanguis-induced platelet aggregation. As resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, anti-class II-reactive protoplast products included silver-stained bands of 67, 79, 115, 216, and 248 kDa. The 115-kDa protein fraction was isolated by gel filtration and ion-exchange chromatography. This form of the class II antigen contained N-formylmethionine at its amino terminus. Rhamnose constituted 18.2% of the total residual dry weight and nearly half of its carbohydrate content. Diester phosphorus constituted 1% of this fraction. After trypsinization of the protoplast products from either preparation, a 65-kDa protein fragment was recovered. This protoplast protein fragment and the S. sanguis cell-derived 65-kDa class II antigen, previously implicated in the induction of platelet aggregation, were shown to be functionally and immunologically identical.

Reduction of sputum Pseudomonas aeruginosa density by antibiotics improves lung function in cystic fibrosis more than do bronchodilators and chest physiotherapy alone.

We evaluated patients with cystic fibrosis (CF) and moderate obstructive lung disease in pulmonary exacerbation in a double-blind placebo-controlled trial to determine the contribution of antibiotic-mediated reduction in sputum bacterial density to clinical improvement. For the first 4 days of study, all patients received bronchodilating aerosols and chest physiotherapy but no antibiotics. During this time, the patients showed significant improvement in mean FVC, FEV1, and maximal midexpiratory flow rate (FEF25-75). In 12 of 13 trials, the patients showed no significant increases in the density of Pseudomonas aeruginosa during these first 4 days. In these 12 trials, the patients were stratified by their initial FVC and randomized to receive either parenteral tobramycin and ticarcillin (n = 7) or placebo (n = 5), in addition to continued aerosol and chest physiotherapy. In the remaining trial, the patient had a significant rise in the density of P. aeruginosa and was assigned to the antibiotic group. During the next 14 days of therapy, the antibiotic group showed significantly (p less than 0.01) greater reductions in log10 colony-forming units (cfu) of P. aeruginosa per gram of sputum and greater increases in FVC, FEV1, and FEF25-75 than did the placebo group. The degree of decrease in log10 cfu P. aeruginosa/g sputum correlated significantly (p less than 0.001) with the degree of improvement in FVC, FEV1, and FEF25-75.(ABSTRACT TRUNCATED AT 250 WORDS)

Pluronic F-68 reduces the endothelial adherence and improves the rheology of liganded sickle erythrocytes.

A perfluorochemical blood substitute emulsified with Pluronic F-68 has been shown to improve the filterability of deoxygenated sickle red cells. We questioned whether some of the effect was independent of oxygen loading and studied the influence of Fluosol DA (Green Cross, Osaka, Japan) and Pluronic on the rheology and adhesion of sickle red cells saturated with oxygen and carbon monoxide. A 5-vol/vol% concentration of Fluosol or equivalent concentration of Pluronic was equally effective at improving the filtration of washed sickle cells through 5-micron-diameter pores at wall shear stresses approximating 1,000 dyne/cm2. The same concentration of Pluronic reduced the extensional static rigidity of irreversibly sickled cells (ISC) by 25% and also abolished the adherence of gravity-sedimented sickle cells to endothelial monolayers in the presence of saline or plasma. The inhibition of adherence was not reversible by washing and was accomplished with equal ease by isolated treatment of sickle cells or endothelium. Pluronic had no effect on the rheology or adhesion of normal adult red cells. Neither Fluosol nor Pluronic changed sickle or normal cell shape, mean cell volume, mean cell density, or cell density distribution. A lubricating effect of Pluronic on cell surfaces could explain all of the rheological observations and offers another direction of inquiry in the search for therapy for sickle cell disease.

Size and filterability of human and hamster pulmonary macrophages exposed to cigarette smoke.

Pulmonary alveolar macrophages (PAM) obtained from healthy human cigarette smokers generated greater pressure in filtration through small cylindrical pores than PAM from nonsmoking controls. A well standardized hamster smoking model was employed to study the structural basis for the impaired PAM filtration accompanying cigarette smoke exposure. Hamsters also demonstrated increased PAM filtration pressure through cylindrical pores less than half a cell diameter in size after inhalation of cigarette smoke, but not after exposure to vapor phase smoke that had been strained through a particle filter. Cigarette smoker PAM were equally filterable as control through larger diameter channels, and showed comparable improvements in filterability with cytochalasin B treatment. Altered PAM filtration was associated with the formation of characteristic cytoplasmic smoker inclusions, an increase in cell size, and loss of redundant, ruffled surface membrane. The study of smoker and control PAM separated into different sizes on the basis of variations in cell density suggested that discrepancies in cell size were insufficient to explain the filtration disparity. A loss of availability of surface membrane for deformation appeared to be the most important factor responsible for the impaired filtration.

Micropipette aspiration of human platelets after exposure to aggregating agents.

The present study examined the influence of activation on platelet deformability. Aspiration of cells after exposure to thrombin, adenosine 5' -diphosphate, or the calcium ionophore A23187 at concentrations producing shape change and stickiness revealed significant changes from control cells. At the lowest negative pressure, 4 X 10(-2) dynes/cm (-1 cm H2O), there were no differences in lengths of membrane segments aspirated from control and activated platelets. Each subsequent increase in negative pressure up to 35 X 10(-2) dynes/cm (-7.5 cm H2O) resulted in significantly longer aspirated segments on activated cells compared to control cells. Greater negative pressures did not cause further increases in lengths of membrane segments drawn into the pipette. Thus, activation, which results in constriction of the circumferential microtubule, makes more membrane available for aspiration as negative pressure is increased. In both control and activated platelets, the microtubule coils served as a barrier to further lengthening of aspirated membrane segments as negative pressure was increased beyond 35 X 10(-2) dynes (-7.5 cm H2O).

Micropipette aspiration of human platelets: influence of rewarming on deformability of chilled cells.

Recent studies using micropipette elastimetry have shown that the circumferential microtubule supporting the discoid form of resting platelets has a direct influence on the resistance of the cell to deformation. However, the findings did not resolve whether the mere presence of microtubules, their organization into coils, or location under the cell wall was responsible for resistance to aspiration into micropipettes. In the present study platelets were cooled to 2 degrees to 4 degrees C to remove microtubules completely. The chilled cells were then rewarmed to 37 degrees C, and the influence of microtubule reassembly on resistance to deformation in micropipettes measured at intervals up to 1 hour. Chilled platelets without microtubules were aspirated more than twice as far as control platelets at all negative pressures from 1 to 10 cm H2O (tensions 4 to 41 X 10(-2) dynes/cm). At negative tensions beyond 32.5 X 10(-2) dynes/cm (8 cm H2O), the aspirated lengths of control platelets plateaued until the cells finally fragmented. Aspirated segments of chilled platelets continued to increase in length on exposure to greater negative pressure. Twenty minutes after rewarming at 37 degrees C, chilled cells began to return toward normal resistance to aspiration when only 6% had recovered discoid shape. The range of deformability at this time was narrow, indicating that partial recovery was not caused by development of two cell populations, one with and one without microtubules. The return toward normal resistance continued at 30 minutes when 75% of platelets were discoid, and was identical to that in control platelets after 1 hour at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Opsonin-independent phagocytosis of surface-adherent bacteria by human alveolar macrophages.

Opsonin-independent mechanisms of phagocytosis may be important in host defense of certain body sites such as the lung. In this study, one such mechanism, "surface phagocytosis," was investigated by measuring the uptake of unopsonized [3H]-labeled Staphylococcus aureus and Pseudomonas aeruginosa adherent to a plastic surface by human alveolar macrophages (AM) and peripheral blood polymorphonuclear leukocytes (PMN). Efficient uptake of unopsonized bacteria by both cell types was observed. Electron microscopic studies suggested that the manner in which these cell types encounter adherent bacteria is different. While AM appear to gather in organisms at their periphery as they spread out upon the underlying substrate, PMN seem to sweep bacteria up as they move along the plastic surface. Bacterial killing determined by a fluorochrome microassay was decreased by AM compared to PMN. Although the precise mechanism whereby phagocytes recognize unopsonized bacteria adherent to a surface remains to be determined, this aspect of phagocytic cell function may prove to have clinical relevance.

Human phagocyte interactions with the Lyme disease spirochete.

The interaction between human polymorphonuclear leukocytes and monocytes and the Lyme disease spirochete was investigated by incubating phagocytes with microorganisms adherent to plastic or glass surfaces. Both cell populations readily phagocytized and killed spirochetes, and antibodies facilitated but were not essential for phagocytosis.

Micropipette aspiration of human platelets: influence of microtubules and actin filaments on deformability.

The deformability of human platelets has been evaluated by micropipette aspiration. Control discoid platelets were about ten times as resistant to deformation in the micropipette as red blood cells. Under a constant negative pressure of 10 cm H2O, control platelets developed extension lengths of 0.74 +/- 0.1 micron. Prior treatment with vincristine, colchicine, or low temperature, all of which remove platelet microtubules, was associated with marked increases in lengths of aspirated segments. Taxol or heavy water, which stabilize microtubules, prevented the increased deformability caused by agents that dissolve microtubules. Cytochalasin B, an agent that inhibits assembly of actin microfilaments, also caused an increase in lengths of aspirated segments that could not be prevented by taxol. Vincristine and cytochalasin B, together, caused a greater increase in deformability than either agent alone. These results indicate important roles for microtubules and microfilaments in platelet deformability.

Cell-free released components of Streptococcus sanguis inhibit human platelet aggregation.

To study the role of surface components in the selective binding and aggregation of platelet-rich plasma (PRP) by strains of viridans streptococci, we treated the binding, aggregation strain Streptococcus sanguis I 2017-78 by sonication or trypsinization. Morphologically identifiable electron-dense fibrils were released from the cell wall, apparently from an inner electron-dense layer, under conditions that left cells intact. These controlled conditions were determined to cause submaximal loss in adhesion to platelet ghosts and PRP aggregation by treated, washed S. sanguis. Soluble components were recovered from the controlled sonic or L-(tosylamido 2-phenyl)ethyl chloromethyl ketone-trypsin treatments. Each showed dose-response inhibition of aggregation when preincubated with PRP before challenge with fresh, untreated S. sanguis. The time to onset of PRP aggregation was inhibited by 50% with 0.2 mg of TPCK-trypsin peptides or 1.0 mg of the sonicate per ml per 2 X 10(8) platelets. Components of both preparations were immunologically cross-reactive, but lipoteichoic acid was not a major antigen of either. By weight, the TPCK-trypsin peptides were virtually all protein; the sonicate residues identified were about 50% protein and 7% hexose. Each was a complex mixture of components as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 8 TPCK-trypsin peptides and 16 sonicate components were so identified. In contrast, at least four or five components from either preparation were recognized as surface determinants by a rabbit antiserum to whole homologous microbes. Platelet-binding ligands of S. sanguis could be among these determinants.

Phagocytosis by polymorphonuclear leukocytes of Staphylococcus aureus and Pseudomonas aeruginosa adherent to plastic, agar, or glass.

Phagocytic cells may encounter bacteria in vivo that are stationary or adherent to a surface. In this study, recently developed in vitro techniques were adapted to evaluate the interaction of polymorphonuclear leukocytes (PMN) with adherent Staphylococcus aureus and Pseudomonas aeruginosa. By measuring the uptake of radiolabeled bacteria, we found that normal human PMN readily phagocytize these organisms when they are attached to plastic or when they are grown on the surface of nutrient agar. Bacteria adherent to glass elicited a chemiluminescent response, and such organisms were phagocytized and killed by PMN. Opsonization of S. aureus and P. aeruginosa was not required for surface phagocytosis, chemiluminescence, or killing. These new methods should allow evaluation of certain biological and clinical aspects of surface phagocytosis in host defense.

Aggregation of human platelets and adhesion of Streptococcus sanguis.

Platelet vegetations or thrombi are common findings in subacute bacterial endocarditis. We investigated the hypothesis that human platelets selectively bind or adhere strains of Streptococcus sanguis and Streptococcus mutans and aggregate, as a result, into an in vitro thrombus. Earlier ultrastructural studies suggested that aggregation of platelets over time by Staphylococcus aureus was preceded in order by adhesion and platelet activation. We uncoupled the adhesion step from activation and aggregation in our studies by incubating streptococci with platelet ghosts in a simple, quantitative assay. Adhesion was shown to be mediated by protease-sensitive components on the streptococci and platelet ghosts rather than cell surface carbohydrates or dextrans, plasma components, or divalent cations. The same streptococci were also studied by standard aggregometry techniques. Platelet-rich plasma was activated and aggregated by certain isolates of S. sanguis. Platelet ghosts bound the same strains selectively under Ca2+- and plasma-depleted conditions. Fresh platelets could activate after washing, but Ca2+ had to be restored. Aggregation required fresh platelets in Ca2+-restored plasma and was inducible by washed streptococcal cell walls. These reactions in the binding and aggregometry assays were confirmed by transmission electron microscopy. Surface microfibrils on intact S. sanguis were identified. These appendages appeared to bind S. sanguis to platelets. The selectivity of adhesion of the various S. sanguis strains to platelet ghosts or Ca2+- and plasma-depleted fresh washed platelets was similar for all donors. Thus, the platelet binding site was expressed widely in the population and was unlikely to be an artifact of membrane aging or preparation. Since selective adhesion of S. sanguis to platelets was apparently required for aggregation, it is suggested that functionally defined receptors for ligands on certain strains of S. sanguis may be present on human platelets. Some differences in the selectivity and rate of the aggregation response were noted among platelet donors, although the meaning of the variability requires further study. Nonetheless, these interactions may contribute to platelet accretion in the initiation and development of vegetative lesions in the subacute bacterial endocarditis.

Effects of small latex particle uptake on the surface connected canalicular system of blood platelets: a freeze-fracture and cytochemical study.

Recent investigations have suggested that channels of the surface connected open canalicular system are the major source of membrane contributing to increased surface area of platelets following activation or uptake of particulates. However, study of large latex particle phagocytosis suggested that channels filled with spherules become dilated and lose connections with the outer surface, but do not become extruded onto the platelet membrane. The present study has employed electron dense tracers, freeze-fracture and transmission electron microscopy to follow the uptake of small latex spherules by blood platelets. Small particles were rapidly incorporated into the numerous channels without causing significant changes in surface or internal morphology. Intramembranous particles on both E and P faces in replicas of freeze-fractured platelets were unaffected by ingestion of latex spherules. Prolonged exposure to small latex particles caused platelets to lose their discoid form. The irregular spheres contained two or three dilated vacuoles filled with latex and a reduced number of single channels. Results of this and previous studies suggest that channels of the open canalicular system are internalized in platelets after phagocytosis, and are not extruded onto the platelet surface.

Effects of large latex particle uptake of the surface connected canalicular system of blood platelets: a freeze-fracture and cytochemical study.

The complex structural organization of the platelet's surface-connected or open canalicular system (OCS) and close physical relationships to the dense tubular system have been described in previous cytochemical and freeze-fracture ultrastructural studies. Despite observations that suggest that channels of the OCS are seldom, if ever, single, tubelike invaginations of the surface, others have indicated that the OCS is a readily available source of membrane for evagination onto the exposed surface of the platelet after activation or during phagocytosis. In the presence investigation we have utilized freeze-fracture for the first time to evaluate the uptake of large (0.312 micron, SD +/- 0.0022) latex particles by platelets. Results of the study leave no doubt that channels of the OCS serve as the major route for latex ingestion in te unstirred system employed. Prolonged exposure to latex and uptake of many spherules cause marked changes in platelet surface contour and internal organization. The OSC is transformed from a spiderweb of intercommunicating channels into one or two large vacuoles filled with latex. Conversion of the OCS into large sacs is associated with disk-to-sphere transformation and a decrease in the number of openings of the OCS on the platelet surface. Thus, the OCS appears to be effectively interiorized by the process of phagocytosis, rather than evaginated as others have suggested.

Platelets increase neutrophil adherence in vitro to nylon fiber.

The effect of platelets on the adherence of neutrophils to nylon fiber was assessed in whole blood samples and purified neutrophil suspensions in the presence or absence of plasma. In whole blood samples, increasing numbers of platelets were associated (r = 0.47, p less than 0.02) with increasing adherence of neutrophils. Addition of platelets in plasma to purified neutrophil suspensions increased (p less than 0.05) neutrophil adherence from 76.2% +/- 1.4 to 88.0% +/- 2.0. Similarly, addition of washed platelets without plasma also increased (p less than 0.05) neutrophil adherence from 67.9% +/- 5.8 (without added platelets) to 94.2% +/- 1.6 (with 300,000 platelets/mm3 added). In contrast, no augmentation of neutrophil adherence occurred if platelets had their aggregation response suppressed by pretreating platelet donors with aspirin. SEM supported these findings by showing platelets in close association with neutrophils adhering to nylon fiber. These findings emphasize the importance of platelet numbers and reactivity on the adherence of neutrophils.