Dorr G. Dearborn

Evaluation of Stachybotrys chartarum in the house of an infant with pulmonary hemorrhage: quantitative assessment before, during, and after remediation.

Stachybotrys chartarum is an indoor mold that has been associated with pulmonary hemorrhage cases in the Cleveland, Ohio, area. This study applied two new quantitative measurements to air samples from a home in which an infant developed PH. Quantitative polymerase chain reaction and a protein synthesis inhibition assay were used to determine the level ofS. chartarum spores and their toxicity in air samples taken before, during, and after a remediation program was implemented to remove the fungus. Initial spore concentrations were between 0.1 and 9.3 spores/m3 of air, and the toxicity of air particulates was correspondingly low. However, the dust in the house contained between 0.4 and 2.1×103 spores/mg (as determined by hemocytometer counts). The remediation program removed all contaminated wallboard, paneling, and carpeting in the water-damaged areas of the home. In addition, a sodium hypochlorite solution was used to spray all surfaces during remediation. Although spore counts and toxicity were high during remediation, air samples taken postremediation showed no detectable levels ofS. chartarum or related toxicity. Nine isolates ofS. chartarum obtained from the home were analyzed for spore toxicity, hemolytic activity, and random amplified polymorphic DNA banding patterns. None of the isolates produced highly toxic spores (>90 μg T2 toxin equivalents per gram wet weight spores) after growth for 10 and 30 days on wet wallboard, but three isolates were hemolytic consistently. DNA banding patterns suggested that at least one of these isolates was related to isolates from homes of infants with previously investigated cases.

Inhibitory effect of cystic fibrosis serum on pseudomonas phagocytosis by rabbit and human alveolar macrophages.

Summary: This report presents experimental observations indicating the presence of an inhibitory activity in cystic fibrosis (CF) serum which impairs phagocytosis of Pseudomonas aeruginosa by rabbit as well as human alveolar macrophages. Of the 49 patient serum samples studied, 40 consistently showed ≥ 60% inhibition, 3 showed no inhibition and 6 were in the range of 20–60% inhibition of Pseudomonas phagocytosis. In parallel studies, the phagocytosis of S. aureus and S. marcescens was found not to be inhibited by CF serum. Mixing of CF serum with normal serum could not overcome the inhibitory effect, indicating the presence of an inhibitory factor rather than the lack of a necessary component. The inhibitory activity is not lost upon exposure of serum to glass, upon freezing the serum once, or upon heating at 56 C for 30 minutes.Speculation: The serum of cystic fibrosis patients selectively inhibits alveolar macrophage function in vitro; the expression of this inhibitory activity in vivo may compromise effective host control of infection. Investigation of the origin, nature and pathophysiologic role of the activity may suggest new approaches to the control of Pseudomonas pulmonary infection.Pultionary infection is a major factor in the morbidity and mortality associated with cystic fibrosis (CF) (6). Pseudomonas, a uhrquitous organism in the environment, is cystic fibrosis (CF) (6). Pseudomonas, a uhrquitous organism in the environment, is usually not pathogenic for healthy individuals. However, individuals with the chronic lung disease of CF are particularly susceptible to opportunistic Pseudomonas aeruginosa infections. The frequency of this organism in CF pulmonary infections is inadequately explained. It is well known that most CF patients have elevated levels of Pseudomonas antibodies in their sera and pulmonary secretions (l2,14). While recently there has been an indication that a lymphocyte unresponsiveness to Pseudomonas may be acquired as the infection progresses (18,19), no other immunologic abnormality has been consistendy observed (5.10). Extrapulmonary infection is extremely rare and sepsis is almost never, seen after the first months of life (22). This unusual susceptibility to Pseudomonas despite apparently normal systemic humoral and cellular immunity. suggests that local pulmonary host defense mechanisms are defective in CF. Several recent studies have indicated that lung defenses can, to a certain extent, function independently of systemic humoral and cell mediated immune systems (9,15,20,21).Lung defenses include mucociliary transport as well as phagocytic cells, lymphocytes, and immunoglobulin secretion. Since mucociliary transport in some CF patients is compromised (5). clearing of the bacteria becomes more dependent on the efficient action of the phagocytic cells. Previous studies in our laboratory (2) and by Biggar, et al. (I) have shown that CF serum impairs phagocytosis of Pseudomonas by rabbit alveolar macrophages. This report presents experimental observations indicating the presence of an inhibitory activity in CF serum which impairs phagocytosis of Pseudomonas by human as well as rabbit alveolar macrophages.

Sequence homologies between nucleotide binding regions of CFTR and G‐proteins suggest structural and functional similarities

Sequence homology between the α‐subunits of G‐proteins and other GTP‐binding proteins and certain regions within the nucleotide binding domains (NBDs) of cystic fibrosis transmembrane conductance regulator (CFTR) indicates that these protein structures may be similar. A sequence allignment of the NBDs of CFTR and NBDs from other membrane transporters, forms the basis of a structural model. This model predicts that one of the conserved sequences GGQR, within which a number of CF mutations occur, forms part of the nucleotide binding pocket and serves as an ON/OFF conformational switch as observed in GTP binding proteins. Furthermore, based on subtle sequence differences between the first and second NBDs of CFTR and from structure‐activity data, we suggest that the nucleotide binding site environments of the two NBDs are different.

Ultrastructure and function of alveolar macrophages from cystic fibrosis patients

Summary: Alveolar macrophages were isolated from three cystic fibrosis patients, and the structure and function of these cells were compared to that of normal alveolar macrophages. The cystic fibrosis (CF) and normal alveolar macrophages were able to phagocytize Pseudomonas in the presence of normal serum, but cells from both sources had decreased phagocytosis of Pseudomonas in the presence of CF seram. Phagocytosis of Staphylococcus was not inhibited. Ultrastructural studies showed CF macrophages to be morphologically normal, however, in contrast to CF polymorphonuclear cells, they had not been heavily engaged in phagocytosis. The similarities between CF and normal macrophages suggest that the chronic pulmonary infection of CF may be due to an extrinsic factor in an altered lung environment rather than to any intrinsic cellular defect of the alveolar macrophage.Speculation: Functional and morphologic observations indicate that cystic fibrosis alveolar macrophages are not providing an adequate phagocytic defense against Pseudomonas. This defective phagocytosis does not appear to arise from an intrinsic problem with the macrophages, but rather appears to be due to extrinsic factors, i.e.. an altered lung environment together with a substance(s) present in cystic fibrosis serum which selectively inhibit Pseudomonas phagocytosis.

Housing and allergens: a pooled analysis of nine US studies.

BACKGROUND Housing conditions can contribute to allergen exposures that are linked to asthma, but little is known about which of those conditions are most likely to predict high levels of allergens in settled house dust. METHODS We pooled allergen, housing condition, occupant behavior, demographic, and other data from nine asthma studies (n=950 homes in 6 US cities). Dust mite (Der f 1 or Der p 1), cockroach (Bla g 1 or Bla g 2), mouse (Mus m 1), cat (Fel d 1) and dog (Can f 1) allergens were measured in settled dust from kitchens or bedrooms, and concentrations were categorized according to previously published asthma symptom thresholds. We calculated odds ratios (OR) using logistic regression to identify those housing conditions and occupant behaviors that were associated with clinically significant allergen levels, after adjusting for numerous confounding variables. RESULTS The adjusted results show that high cockroach allergen was associated with cracks or holes in walls (OR=2.1), high dust mite allergen was associated with mold odor (OR=2.5), housing built before 1951 (OR=2.1), and single-family home with slab on grade (OR=1.9); and mouse allergen was associated with rodent control or signs of rodents (OR=3.62) and inversely associated with presence of a cat (OR=0.20). Water leaks and below average housekeeping had unadjusted high odds ratios for high cockroach allergen. CONCLUSION We have identified a number of housing conditions that are consistently associated with increased allergen dust concentrations. This study indicates that screening for housing-based asthma triggers should include presence of cats, dogs, cockroaches, or rodents; water leaks; mold or mold odor; holes or cracks in walls; and below average housekeeping. Single family houses that have basements or crawl spaces or are built before 1951 are also important predictors for increased allergens in housing.

Localization of Satratoxin-G in Stachybotrys chartarum Spores and Spore-Impacted Mouse Lung Using Immunocytochemistry

Satratoxin-G (SG) is the major macrocyclic trichothecene mycotoxin produced by Stachybotrys chartarum (atra) and has been implicated as a cause of a number of animal and human health problems including pulmonary hemorrhage in infants. However, there is little understanding where this toxin is localized in the spores and mycelial fragments of this species or in the lung impacted by SG-sequestered spores. The purpose of this study was to evaluate the distribution of SG in S. chartarum spores and mycelium in culture, and spore-impacted mouse lung in vivo, using immunocytochemistry. SG was localized predominately in S. chartarum spores with moderate labelling of the phialide-apex walls. Labelling was primarily along the outer plasmalemma surface and in the inner wall layer. Only modest labelling was observed in hyphae. Toxin localization at these sites supports the position that spores contain the highest satratoxin concentrations and that the toxin is constitutively produced. In impacted mouse lung, highest SG labelling was detected in lysosomes, along the inside of the nuclear membrane in nuclear heterochromatin and RER within alveolar macrophages. Alveolar type II cells also showed modest labelling of the nuclear heterochromatin and RER. There was no evidence that the toxin accumulated in the neutrophils, fibroblasts, or other cells associated with the granulomas surrounding spores or mycelial fragments. These observations indicate that SG displays a high degree of cellular specificity with respect to its uptake in mouse lung. They further indicate that the alveolar macrophages play an important role in the sequestration and immobilization of low concentrations of the toxin.

Pulmonary effects of Stachybotrys chartarum in animal studies.

Publisher Summary Stachybotrys chartarum is one of the several environmental fungi that can produce very potent toxic compounds. Animal models provide physicians and environmental scientists with useful tools for assessing risks associated with the respiratory effects of air pollutants. The animal studies to date support the view that pulmonary exposure to the spores of S. chartarum leads to hemorrhagic inflammation and impairment of growth. This has been demonstrated by increases in bronchoalveolar lavage (BAL) fluid of inflammatory cells, proinflammatory mediators, hemoglobin, and proteins along with changes in pulmonary surfactant. Although the earlier experiments were conducted with relatively high doses, recent findings indicate that lower doses, which appear to be closer to the concentrations encountered in indoor air, can elicit similar symptoms. The results of animal studies based on different experimental designs are difficult to compare because of many variables—including spore toxicity, viability, and content of fungal proteins in addition to species, strains, age of animals, and route of administration.

Cytosolic free calcium concentration and intracellular calcium distribution in lymphocytes from cystic fibrosis patients

Lymphocytes prepared from normal individuals and patients with cystic fibrosis (CF) were compared with regard to intracellular Ca2+ concentration, distribution, and handling. No difference between control and CF was found in the concentration of cytosolic free Ca2+ (98 +/- 5 vs 102 +/- 7 nM), and no difference was observed in the kinetics with which control and CF cells restored cytoplasmic Ca2+ toward normal following a perturbation induced by cold-exposure. However, total intracellular Ca2+ is about 25% higher in CF lymphocytes than in control. Of this excess Ca2+, about 50% appears to be sequestered in mitochondria. This suggests that some difference in Ca2+ handling does exist, but the significance of this in cystic fibrosis remains to be determined.

Immunocytochemical localization of stachylysin in Stachybotrys chartarum spores and spore-impacted mouse and rat lung tissue

Stachylysin is a proteinaceous hemolytic agent that is produced by Stachybotrys chartarum. Stachylysin was found, using immunohistochemical and immunocytochemical methods, to be localized in S. chartarum spores/mycelia primarily in the inner wall suggesting that it is constitutively produced. Spores instilled in mouse or rat lung tissues resulted in granuloma formation, which showed the highest stachylysin concentration in the inner wall of the spore and near the spore, with less at distance indicating that it had diffused out from the spore. The in vitro high stachylysin producing strain (58-06) was also highest in vivo, based on immunohistochemistical staining. More stachylysin was observed in the mouse lung tissue at 72 h than at 24 h indicating that production/release is a relatively slow process. The localization of stachylysin in macrophage phagolysosomes suggests that these cells may be involved with hemolysin inactivation. This would be consistent with what is known about asp-hemolysin produced by Aspergillus fumigatus.

Erythrocyte cytosolic free Ca2+ and plasma membrane Ca2+-ATPase activity in cystic fibrosis

The properties of the Ca2+, Mg2+-ATPase of erythrocyte membranes from patients with cystic fibrosis (CF) were extensively compared to that of healthy controls. Following removal of an endogenous membrane inhibitor of the ATPase, activation of the enzyme by Ca2+, calmodulin, limited tryptic digestion or oleic acid, as well as inhibition by trifluoperazine, were studied. The only properties found to be significantly different (CF cells vs controls) were calmodulin-stimulated peak activity (90 vs 101, P less than 0.02) and trypsin-activated peak activity (92 vs 102, P less than 0.02). No significant difference could be measured in the steady-state Ca2+-dependent phosphorylation of CF and control erythrocyte membranes indicating similar numbers of enzyme molecules per cell. The functional state of Ca2+ homeostasis in intact erythrocytes was investigated by measuring the resting cytosolic free Ca2+ levels using quin-2. Both CF and control erythrocytes maintained cytosolic free Ca2+ between 20 to 30 nM. Addition of 50 uM trifluoperazine resulted in an increase in erythrocyte cytosolic free Ca2+ to about 50 nM in both CF and control cells. Estimates of erythrocyte membrane permeability using the steady-state uptake of 45Ca into intact erythrocytes revealed no differences between CF and control cells. These results confirm that there is a small decrease in the calmodulin-stimulated activity of the erythrocyte Ca2+, Mg2+-ATPase in CF. However, this deficit is apparently not large enough to impair the ability of the CF erythrocyte to maintain normal resting levels of cytosolic free Ca2+.