A sensitive new bioassay for tumor necrosis factor.

Tumor necrosis factor is an important cytokine involved in inflammation and assay of this cytokine in biological fluids may be important in the understanding of several disease processes. This report describes an improved TNF bioassay employing a newly isolated subclone of the cell line NCTC-clone 929 as well as a novel fluorescence indicator system for detecting viability of the target cells. The limit of detection for the TNF hypersensitive cell line with this fluorescence viability assay was 68 +/- 2.5 fg/ml, which is approximately 3 x more sensitive than the parental clone and approximately 10 x more sensitive than that reported by Branch et al. (1991) using the neutral red indicator system. The hypersensitivity of the clone gradually declined over a 45-day period and at regular intervals new cells were cultivated from frozen stocks. Two different serum sources, bovine fetal serum and horse serum, and four different serum concentrations (5, 10, 15, 20%) were evaluated to optimize sensitivity. No difference was found between serum sources but sensitivity was significantly reduced if < 15% serum was used.

Adamantanamine derivatives. Antimicrobial activities of certain Mannich bases.

A series of Mannich condensation products containing the beta-(1-adamantylamino)propiophenone skeleton (type I) and the o-(1-adamantylaminomethyl)phenol skeleton (type II) was synthesized and tested for antimicrobial activity against certain bacteria and fungi by the agar diffusion and tube dilution methods. Type I compounds were more active than type II compounds and had a broad-spectrum effect on certain gram-negative and gram-positive bacteria, acid-fast bacteria, a yeast, and a mold.

Fungal spores: hazardous to health?

Fungi have long been known to affect human well being in various ways, including disease of essential crop plants, decay of stored foods with possible concomitant production of mycotoxins, superficial and systemic infection of human tissues, and disease associated with immune stimulation such as hypersensitivity pneumonitis and toxic pneumonitis. The spores of a large number of important fungi are less than 5 microm aerodynamic diameter, and therefore are able to enter the lungs. They also may contain significant amounts of mycotoxins. Diseases associated with inhalation of fungal spores include toxic pneumonitis, hypersensitivity pneumonitis, tremors, chronic fatigue syndrome, kidney failure, and cancer.

Toxicity of mycotoxins for the rat pulmonary macrophage in vitro.

The presence of mycotoxins in grains is well documented. Workers in grain handling occupations are commonly exposed to grain dust aerosols. Work in our laboratory has shown that T-2 toxin is highly toxic to rat alveolar macrophages in vitro, causing loss of viability, release of radiolabeled chromium, inhibition of macromolecular synthesis, inhibition of phagocytosis, and inhibition of macrophage activation. Similarly, patulin caused a significant release of radiolabeled chromium, decrease in ATP levels, significant inhibition of protein and RNA synthesis, and inhibition of phagocytosis. The data show that both T-2 toxin and patulin are highly toxic to rat alveolar macrophages in vitro. The data further suggest that the presence of these mycotoxins in airborne respirable dust might present a hazard to exposed workers.

Overview of investigations into pulmonary hemorrhage among infants in Cleveland, Ohio.

Idiopathic pulmonary hemorrhage was diagnosed in 37 infants in the Cleveland, Ohio, area between 1993 and 1998. This rare disorder has been related to 12 deaths, including 7 originally thought to be sudden infant death syndrome. Thirty of the infants were African American, all of whom lived in a limited geographic area of eastern metropolitan Cleveland, an area of older housing stock. An investigation led by the Centers for Disease Control and Prevention has found an association with household exposure to a toxigenic mold, Stachybotrys chartarum, and other fungi. The rapidly growing lungs of young infants appear to be especially vulnerable to the toxins made by toxigenic molds. Environmental tobacco smoke was frequently present in the infants' homes and may be a trigger precipitating the acute bleeding. Stachybotrys, although not thought to be a common mold, is known to have a wide geographic distribution. An additional 101 cases of acute, idiopathic pulmonary hemorrhage have been reported in infants in the United States over the past 5 years. In this overview, the investigations are summarized, the clinical profile is described, the toxicity of S. chartarum is discussed, and pathophysiologic concepts are presented.

Review of methods applicable to the assessment of mold exposure to children.

This article presents discussion of the assessment of the exposure of children to fungi, substances derived from fungi, and the environmental conditions that may lead to exposure. The principles driving investigations of fungal contamination and subsequent exposure are presented as well as guidelines for conducting these investigations. A comprehensive description of available research sampling and analysis techniques is also presented.

Acute pulmonary hemorrhage in infants associated with exposure to Stachybotrys atra and other fungi.

BACKGROUND: A geographic cluster of 10 cases of pulmonary hemorrhage and hemosiderosis in infants occurred in Cleveland, Ohio, between January 1993 and December 1994. STUDY DESIGN: This community-based case-control study tested the hypothesis that the 10 infants with pulmonary hemorrhage and hemosiderosis were more likely to live in homes where Stachybotrys atra was present than were 30 age- and ZIP code-matched control infants. We investigated the infants' home environments using bioaerosol sampling methods, with specific attention to S atra. Air and surface samples were collected from the room where the infant was reported to have spent the most time. RESULTS: Mean colony counts for all fungi averaged 29 227 colony-forming units (CFU)/m3 in homes of patients and 707 CFU/m3 in homes of controls. The mean concentration of S atra in the air was 43 CFU/m3 in homes of patients and 4 CFU/m3 in homes of controls. Viable S atra was detected in filter cassette samples of the air in the homes of 5 of 9 patients and 4 of 27 controls. The matched odds ratio for a change of 10 units in the mean concentration of S atra in the air was 9.83 (95% confidence interval, 1.08-3 X 10(6)). The mean concentration of S atra on surfaces was 20 X 10(6) CFU/g and 0.007 x 10(6) CFU/g in homes of patients and controls, respectively. CONCLUSION: Infants with pulmonary hemorrhage and hemosiderosis were more likely than controls to live in homes with toxigenic S atra and other fungi in the indoor air.

In vivo pulmonary response to Aspergillus terreus spores.

The fate of intratracheally instilled Aspergillus terreus spores was followed in both rabbits and rats. Phagocytosis of the spores by the pulmonary macrophage was rapid in that approx. 42% of the observed spores were associated with the macrophages immediately after instillation. Direct penetration of the lung architecture by the spores was not observed but spores were seen in the alveolar interstitium at 3 hr after instillation and in the tracheobroncheal lymph nodes at 24 hr. Granulomas formed between 48 hr and 1 week after exposure. In the absence of apparent spore or spore extract toxicity and precipitating antibodies against Aspergillus terreus, the observed reactions preclude the possibility that the lesions were the consequence of hypersensitivity. This model of pulmonary response to fungal spores may be of future value for characterizing further the pathology associated with certain occupational exposures to moldy materials.

Comparison of mutagenic and recombinogenic effects of some adenine analogues in Saccharomyces cerevisiae D7.

2-Aminopurine, 2-amino-N6-hydroxyadenine and N6-hydroxyaminopurine were compared in suspension test with growing and non-growing cells for their mutagenic and recombinogenic (reciprocal and nonreciprocal) activities in Saccharomyces cerevisiae strain D7. Ethyl methanesulfonate was used as a positive control. No increases above spontaneous frequencies were observed when non-growing cells were treated with the base analogues although EMS induced concentration-dependent responses at all 3 genetic end-points. When growing cells were treated, HAP was recombinogenic and mutagenic and AHA was mutagenic, but only weakly recombinogenic. HAP induced comparable numbers of revertants at much lower concentrations than AHA. 2AP failed to induce any detectable response even at concentrations as high as 2400 microgram/ml.

Genotoxic properties of 2,4,7-trinitro-9-fluorenone.

The genotoxic effects of 2,4,7-trinitro-9-fluorenone (TNF) were studied in assays employing procaryotic (Salmonella typhimurium and Escherichia coli) and eucaryotic (Saccharomyces cerevisiae, mouse lymphoma L5178Y and Chinese hamster ovary) cells. The results show that TNF is a potent mutagen for procaryotes. It causes both frame-shift and base-pair substitution mutations, although frame-shift mutations were predominant. In Saccharomyces cerevisiae, this compound appeared to be too toxic to permit detection of genotoxic effects. TNF was also toxic to mouse lymphoma cells and Chinese hamster ovary cells but the toxic effects were reduced by metabolic activation. TNF induced a clear increase in sister-chromatid exchanges in CHO cells and in mutant frequency in mouse lymphoma cells both in the presence and absence of metabolic activation.

Mutagenicity of oil-shale ash.

3 oil-shale ash samples were extracted with solvents and analyzed for mutagenicity with a number of tests systems. In Salmonella typhimurium, the ash extracts were highly mutagenic with the Ames his reversion and the ara-resistant systems. Mutation induction by the ash in Salmonella was independent of metabolic activation and was of the frameshift type. These ash extracts showed a substantial killing effect, but failed to induced ad-3 reversion in Neurospora crassa, gene conversion and mitotic crossing-over in Saccharomyces cerevisiae and TGr mutation in cultured CHO cells.

Chemotaxis of alveolar macrophages and neutrophils in response to microbial products derived from organic dust.

Mechanisms of chemotaxis of alveolar macrophages (AMs) and neutrophils (PMNs) in response to microbial products derived from organic dust were studied using the blindwell chemotaxis chamber technique. Seven different known etiological agents causing respiratory symptoms were used for experiments: cell extract and endotoxin from Pantoea agglomerans (synonyms: Erwinia herbicola, Enterobacter agglomerans), cell extracts from Thermoactinomyces vulgaris and Aspergillus fumigatus, protease from Bacillus thermoproteolyticus rokko and two preparations of glucans. These agents were evaluated for their ability to direct attraction of alveolar macrophages and neutrophils and stimulation of alveolar macrophages to release chemotactic factors for other alveolar macrophages and neutrophils. The microbial products were able to attract both alveolar macrophages and neutrophils directly in a dose-dependent manner, and the exposure of cultured alveolar macrophages to most agents stimulated chemotactic activity for for alveolar macrophages and neutrophils. The generation and release of this activity by alveolar macrophages may provide a mechanism for the initiation and amplification of inflammatory reactions in the lung after inhalation of organic dust. Results of these in vitro studies may be relevant to the pathogenesis of alveolitis in organic dust-induced lung diseases.

Histamine and endotoxin contamination of hay and respirable hay dust.

Three distinct samples collected from a barn in which an outbreak of respiratory problems occurred were examined for possible etiologic agents. No causal relationship could be established from the results of this study; however histamine concentrations as high as 0.5 ng/mg for bulk hay (in the absence of measurable creatinine levels) along with 6138.3 endotoxin units/mg of hay were present in the samples. Both endotoxin and histamine could be recovered from respirable hay dust. The authenticity of the histamine found in the hay was evaluated with high-performance liquid chromatography and radioimmunoassay. Histamine release caused by hay extracts was evaluated with the use of leukocytes from the farmer and a referent. Histamine is known to modulate the immune system, but the role of occupational or environmental exposure to histamine in respiratory disease is unknown.

Partners for child passenger safety: a unique child-specific crash surveillance system.

Insurance claims data were combined with telephone survey and on-site crash investigation data to create the first large scale, child-focused motor vehicle crash surveillance system in the US. Novel data management and transfer techniques were used to create a nearly real-time data collection system. In the first year of this on-going project, known as Partners for Child Passenger Safety, over 1200 children < or = 15 years of age per week were identified in crashes reported to State Farm Insurance Co. from 15 states and Washington, D.C. Partners for Child Passenger Safety is similar in its design and overall objectives to National Automotive Sampling System (NASS), the only other population-based crash surveillance system currently operating in the US.

Cell wall preparations from environmental yeasts: effect on alveolar macrophage function in vitro.

Organic dust toxic syndrome (ODTS) is associated with inhalation of high concentrations of organic materials and is a noninfectious illness characterized by fever, malaise, myalgia, and neutrophilic inflammation of the lower respiratory tract. Studies in our laboratory of fungi in fresh lumber have demonstrated that yeasts may predominate and have raised the issue of potential exposure of sawmill workers to yeasts. Zymosan, a cell wall preparation from Saccharomyces cerevisiae, is a potent stimulator of alveolar macrophages (AM). In the present study, preparations from the cell walls of Pichia fabianii, Candida sake, Trichosporon capitatum, Rhodotorula glutinis, and Cryptococcus laurentii were compared with zymosan and beta-1,3-glucan for their ability to stimulate AM and activate complement. All species activated complement. P. fabianii, C. sake, T. capitatum, R. glutinis, C. laurentii, as well as zymosan and glucan, stimulated superoxide anion and leukotriene B4 production in a dose-dependent fashion, but R. glutinis and C. laurentii were much less active. Zymosan, glucan, P. fabianii, and R. glutinis treatment of AM resulted in increased phagocytosis of labeled sheep RBCs, whereas there was no effect with C. sake or C. laurentii and T. capitatum significantly inhibiting phagocytosis. These results suggest that exposure to high concentrations of yeast could provoke pulmonary inflammation resulting in an episode of ODTS.

Inflammatory potential of grain dust.

A comparative analysis of toxicity and inflammatory potential of spring wheat, oat, corn, and flax seed was performed. The extracts were examined by quantification of gram-negative bacterial endotoxin and protein contents, chemotaxis for purified human polymorphonuclear leukocytes, and several alveolar macrophage functions including phagocytosis, release of lactate dehydrogenase, macromolecular synthesis (protein and RNA), and interleukin-1 production. The results suggested that the extracts of all four grain dusts inhibited protein synthesis and decreased phagocytosis of alveolar macrophages at 7.5-25 mg/ml. Protein synthesis was inhibited to 40.2% of control and phagocytosis to 47.9% of control. Interleukin-1 production was stimulated, as indicated by a stimulation index range from 3.8-fold to 14.3-fold over controls. The activity of these grain dust extracts did not correlate with the endotoxin content of the samples. The data demonstrate that the extracts of all four dusts produced ill effects in rat alveolar macrophages in vitro and suggested a potential hazard to agricultural workers exposed to grain dust.

Microbiological analyses and inflammatory effects of settled dusts from rice and hay.

Fourteen samples of settled dust from two factories processing rice and wheat straw near Shanghai, China, were examined by dilution plating for total bacteria, gram-negative bacteria, thermophilic actinomycetes, and fungi. They were also examined for aflatoxin, endotoxin, and potential to stimulate production of human interleukin 1 beta (IL-1 beta) and to consume complement. The concentrations of total microorganisms were consistently greater than 10(7) CFU/g and ranged from 10(7) to 10(9) CFU/g. In general, the level of microbial contamination was greater in the hay dust samples than in the rice dust samples, with bacteria being the most numerous microorganisms observed followed by molds, thermophilic actinomycetes, and yeasts. The predominant fungi were species of Aspergillus, Cladosporium, Penicillium, Trichosporon, and Cryptococcus. No significant levels of aflatoxin were observed and the isolates of A. flavus examined lack significant aflatoxigenic potential. The levels of microorganisms in these samples, the types of organisms found, and the inflammatory mediators such as endotoxin suggest that workers exposed to these dusts may be at risk for respiratory illness.

Pulmonary disease associated with cafeteria flooding.

An illness among office workers, consisting of cough, fever, chills, muscle aches, and chest tightness was associated with water leaks from a cafeteria. Mean single breath carbon-monoxide diffusing capacity (DLCO) of cases differed significantly from that of non-cases. There was a significant decrease in the percent of predicted DLCO with increasing number of symptoms. Testing for precipitating antibodies to microbial agents found in the building revealed no differences between cases and non-cases. DLCO is an appropriate cross-sectional instrument for field investigations of building-associated respiratory disease.