Kati Huttunen

Low-level exposure to ambient particulate matter is associated with systemic inflammation in ischemic heart disease patients

Short-term exposure to ambient air pollution is associated with increased cardiovascular mortality and morbidity. This adverse health effect is suggested to be mediated by inflammatory processes. The purpose of this study was to determine if low levels of particulate matter, typical for smaller cities, are associated with acute systemic inflammation. Fifty-two elderly individuals with ischemic heart disease were followed for six months with biweekly clinical visits in the city of Kotka, Finland. Blood samples were collected for the determination of inflammatory markers interleukin (IL)-1β, IL-6, IL-8, IL-12, interferon (IFN)γ, C-reactive protein (CRP), fibrinogen, myeloperoxidase and white blood cell count. Particle number concentration and fine particle (particles with aerodynamic diameters <2.5 μm (PM(2.5))) as well as thoracic particle (particles with aerodynamic diameters <10 μm (PM(10))) mass concentration were measured daily at a fixed outdoor measurement site. Light-absorbance of PM(2.5) filter samples, an indicator of combustion derived particles, was measured with a smoke-stain reflectometer. In addition, personal exposure to PM(2.5) was measured with portable photometers. During the study period, wildfires in Eastern Europe led to a 12-day air pollution episode, which was excluded from the main analyses. Average ambient PM(2.5) concentration was 8.7 μg/m(3). Of the studied pollutants, PM(2.5) and absorbance were most strongly associated with increased levels of inflammatory markers; most notably with C-reactive protein and IL-12 within a few days of exposure. There was also some evidence of an effect of particulate air pollution on fibrinogen and myeloperoxidase. The concentration of IL-12 was considerably (227%) higher during than before the forest fire episode. These findings show that even low levels of particulate air pollution from urban sources are associated with acute systemic inflammation. Also particles from wildfires may exhibit pro-inflammatory effects.

Metabolite profiles of Stachybotrys isolates from water-damaged buildings and their induction of inflammatory mediators and cytotoxicity in macrophages

The metabolite profiles of 20 Stachybotrys spp.isolates from Finnish water-damaged buildings were compared with their biological activities. Effects of purified compounds on cytotoxicity and production of inflammatory mediators such as nitric oxide, IL-6 and TNFα in murine RAW264.7 macrophage cells were studied. The 11isolates belonging to the satratoxin-producing chemotype were highly cytotoxic to the macrophages. The isolates inducing inflammatory mediators all belonged to the atranone-producing chemotype, but pure atranones B, and D did not elicit a response in the bioassay. Altogether, cytotoxicity ofStachybotrys sp. isolates appear to be related to satratoxin production whereas the specific component inducing inflammatory responses in atranone-producing isolates remains obscure.

Inflammatory responses in RAW264.7 macrophages caused by mycobacteria isolated from moldy houses.

Mycobacterial strains (nonpathogenic Mycobacterium terrae, potentially pathogenic Mycobacterium avium-complex and Mycobacterium scrofulaceum), isolated from a moldy building, were studied with respect to their ability to stimulate macrophages (RAW264.7) to produce inflammatory mediators, and to cause cytotoxicity. Reactive oxygen species (ROS) were measured by chemiluminescence, cytokines (TNF-alpha, IL-6, IL-1, IL-10) immunochemically, nitric oxide (NO) by Griess-method, expression of inducible NO-synthase (iNOS) with Western Blot analysis and cytotoxicity with MTT-test. All the strains induced dose- and time-dependent production of NO, IL-6 and TNF-alpha in macrophages, whereas IL-1 or IL-10 production was not detected. The production of ROS and cytotoxicity was increased with the highest doses. Interestingly, different strains had significant differences in their ability to induce these responses, M. terrae being the most potent and M. avium-complex the weakest one. These results indicate that both non- and potentially pathogenic strains of mycobacteria present in moldy buildings are capable of activating inflammatory mechanisms in macrophages.

Dampness and mould in schools and respiratory symptoms in children: the HITEA study

Background The adverse respiratory health effects of dampness and mould in the home have been extensively reported, but few studies have evaluated the health effects of such exposures in schools. Objectives To assess the associations between dampness and mould in school buildings and respiratory symptoms among 6–12-year-old pupils in three European countries with different climates. Methods Based on information from self-reports and observations, we selected 29 primary schools with and 27 without moisture damage in Spain, the Netherlands and Finland. Information on respiratory symptoms and potential determinants was obtained using a parent-administered questionnaire among 6–12-year-old pupils. Country-specific associations between moisture damage and respiratory symptoms were evaluated using multivariable multilevel mixed effects logistic regression analysis. Results Data from 9271 children were obtained. Nocturnal dry cough was consistently associated with moisture damage at school in each of the three countries: OR 1.15; 95% CI 1.00 to 1.30 with p for heterogeneity 0.54. Finnish children attending a moisture damaged school more often had wheeze (OR 1.36; CI 1.04 to 1.78), nasal symptoms (OR 1.34; CI 1.05 to 1.71) and respiratory-related school absence (OR 1.50; CI 1.10 to 2.03). No associations with these symptoms were found in the Netherlands or Spain (p for heterogeneity <0.05). Conclusions Moisture damage in schools may have adverse respiratory health effects in pupils. Finnish school children seem to be at higher risk, possibly due to quantitative and/or qualitative differences in exposure.

COMPARISON OF MYCOBACTERIA-INDUCED CYTOTOXICITY AND INFLAMMATORY RESPONSES IN HUMAN AND MOUSE CELL LINES

Environmental mycobacteria, which are ubiquitous in nature, are also detected in moisture-damaged buildings. Their potential role inducing the adverse health effects associated with living in moisture damaged buildings requires clarification. To establish a model for these studies, we evaluated inflammatory responsiveness in different cell lines exposed to environmental mycobacterial species. Four mycobacterial isolates belonging to Mycobacterium avium complex and Mycobacterium terrae, recovered from the indoor air sampled when a moldy building was being demolished, were studied for their cytotoxicity and ability to stimulate the production of inflammatory mediators in mouse RAW264.7 and human 28SC macrophage cell lines, and human A549 lung epithelial cell line. Lipopolysaccharide (LPS) was used as a positive control. Production of cytokines (tumor necrosis factorα, TNF-α; interleukin 6, IL-6; and interleukinβ, IL-1β) was analyzed immunochemically, nitric oxide (NO) by the Griess method, expression of inducible NO synthase with Western blot analysis, and cytotoxicity with the MTT test. Both human and mouse cells produced NO and IL-6 after mycobacterial exposure. Mouse macrophages also showed production of TNF-α induced by both mycobacteria and LPS, whereas the human cell lines failed to produce TNF-α after mycobacterial exposure and the human epithelial cell line also failed to respond to LPS. Similarly, only mouse macrophages produced IL-1β. Mycobacterial exposure was not cytotoxic to human cells and was only slightly cytotoxic to mouse macrophages. The results indicate that environmental mycobacterial isolates from moldy buildings are capable of activating inflammatory mechanisms in both human and murine cells. The human and mouse cell lines, however, differ significantly in the grade and type of the responses.Environmental mycobacteria, which are ubiquitous in nature, are also detected in moisture-damaged buildings. Their potential role inducing the adverse health effects associated with living in moisture damaged buildings requires clarification. To establish a model for these studies, we evaluated inflammatory responsiveness in different cell lines exposed to environmental mycobacterial species. Four mycobacterial isolates belonging to Mycobacterium avium complex and Mycobacterium terrae, recovered from the indoor air sampled when a moldy building was being demolished, were studied for their cytotoxicity and ability to stimulate the production of inflammatory mediators in mouse RAW264.7 and human 28SC macrophage cell lines, and human A549 lung epithelial cell line. Lipopolysaccharide (LPS) was used as a positive control. Production of cytokines (tumor necrosis factor alpha, TNF-alpha; interleukin 6, IL-6; and interleukin beta, IL-1beta) was analyzed immunochemically, nitric oxide (NO) by the Griess method, expression of inducible NO synthase with Western blot analysis, and cytotoxicity with the MTT test. Both human and mouse cells produced NO and IL-6 after mycobacterial exposure. Mouse macrophages also showed production of TNF-alpha induced by both mycobacteria and LPS, whereas the human cell lines failed to produce TNF-alpha after mycobacterial exposure and the human epithelial cell line also failed to respond to LPS. Similarly, only mouse macrophages produced IL-1beta. Mycobacterial exposure was not cytotoxic to human cells and was only slightly cytotoxic to mouse macrophages. The results indicate that environmental mycobacterial isolates from moldy buildings are capable of activating inflammatory mechanisms in both human and murine cells. The human and mouse cell lines, however, differ significantly in the grade and type of the responses.

Indoor air particles and bioaerosols before and after renovation of moisture-damaged buildings: The effect on biological activity and microbial flora

Many building-related health problems coincide with moisture damage and mold growth within a building. Their elimination is assumed to improve indoor air quality. The aim of this study was to follow the success of remediation in two individual buildings by analyzing the microbial flora and immunotoxicological activity of filter samples. We compare results from samples collected from indoor air in the moisture-damaged buildings before and after renovation and results from matched reference buildings and outdoor air. The microbial characteristics of the samples were studied by analyzing ergosterol content and determining the composition of fungal flora with quantitative polymerase chain reaction (QPCR). In addition, the concentrations of particles were monitored with optical particle counter (OPC). The immunotoxicological activity of collected particle samples was tested by exposing mouse macrophages (RAW264.7) for 24 h to particle suspension extracted from the filters, and measuring the viability of the exposed cells (MTT-test) and production of inflammatory mediators (nitric oxide, IL-6 and TNF*) in cell culture medium by enzyme-linked immunoassay (ELISA). The results show that for Location 1 the renovation decreased the immunotoxicological activity of the particles collected from damaged building, whereas no difference was detected in the corresponding samples collected from the reference building. Interestingly, only slight differences were seen in the concentration of fungi. In the Location 2, a decrease was seen in the concentration of fungi after the renovation, whereas no effect on the immunotoxicological responses was detected. In this case, the immunotoxicological responses to the indoor air samples were almost identical to those caused by the samples from outdoor air. This indicates that the effects of remediation on the indoor air quality may not necessarily be readily measurable either with microbial or toxicological parameters. This may be associated with different spectrum of harmful agents in different mold and moisture-damaged buildings.

Source-specific fine particulate air pollution and systemic inflammation in ischaemic heart disease patients.

Objective To compare short-term effects of fine particles (PM2.5; aerodynamic diameter <2.5 µm) from different sources on the blood levels of markers of systemic inflammation. Methods We followed a panel of 52 ischaemic heart disease patients from 15 November 2005 to 21 April 2006 with clinic visits in every second week in the city of Kotka, Finland, and determined nine inflammatory markers from blood samples. In addition, we monitored outdoor air pollution at a fixed site during the study period and conducted a source apportionment of PM2.5 using the Environmental Protection Agencys model EPA PMF 3.0. We then analysed associations between levels of source-specific PM2.5 and markers of systemic inflammation using linear mixed models. Results We identified five source categories: regional and long-range transport (LRT), traffic, biomass combustion, sea salt, and pulp industry. We found most evidence for the relation of air pollution and inflammation in LRT, traffic and biomass combustion; the most relevant inflammation markers were C-reactive protein, interleukin-12 and myeloperoxidase. Sea salt was not positively associated with any of the inflammatory markers. Conclusions Results suggest that PM2.5 from several sources, such as biomass combustion and traffic, are promoters of systemic inflammation, a risk factor for cardiovascular diseases.

The Proportions of Streptomyces californicus and Stachybotrys chartarum in Simultaneous Exposure Affect Inflammatory Responses in Mouse RAW264.7 Macrophages

Abstract Adverse health outcomes associated with moisture-damaged buildings originate from an exposure consisting of complex interactions between various microbial species and other indoor pollutants. The concentrations and proportions of microbial components in such environments can vary greatly with the growth conditions. In this study, we aimed to evaluate the effects of simultaneous exposure with modified proportions of actinobacteria Streptomyces californicus and fungi Stachybotrys chartarum on inflammatory responses (cytokines macrophage inflammatory protein 2 [MIP2], interleukin 6 [IL-6] and tumor necrosis factor α [TNFα]; nitric oxide) and cytotoxicity (MTT-test and DNA content analysis) in mouse RAW264.7 macrophage cell line. Five different proportions of microbial spores were studied (Str. californicus: S. chartarum 10:1; 5:1; 1:1; 1:5; 1:10). RAW264.7 cells were coexposed to the total dose of 3 × 105 spores/ml for 24 h and also both of these microbial spores on their own at the respective doses. At least the 1.5-fold synergistic increase in cytokine production of RAW264.7 macrophages was detected when coexposure contained an equal amount or more fungal spores (S. chartarum) than bacterial spores (Str. californicus) compared to the sum response caused by these microbial spores separately. On the contrary, NO production after coexposure was nearly 40% less than the sum response induced by the microbial spores separately, when coexposure contains 5 times more bacterial than fungal spores. In addition, coexposure slightly changed the cytotoxic potency of the spores. The present results revealed that mutual proportions of fungal and bacterial spores in simultaneous exposure affect the nature of their interactions leading to increased or suppressed production of inflammatory mediators in RAW264.7 macrophages.

Maturation of cytokine-producing capacity from birth to 1 yr of age

Little is known about the immunologic maturation in the early stages of life. The aim of this study was to investigate maturation of immune system from birth to 1 yr of age and to compare immune functions between mothers and their children. Also the effect of atopy to the immune responses of children was examined. Cord blood samples (n = 228) and peripheral blood samples of children (n = 200) and their mothers (n = 208) 1 yr after birth were collected. Whole blood samples were stimulated for 24 and 48 h with Staphylococcal enterotoxin B (SEB), lipopolysaccharide (LPS) and the combination of phorbol ester and ionomycin (P/I). Production of TNF‐α, IFN‐γ, IL‐5, IL‐8 and IL‐10 was determined using ELISA. Significant mother‐to‐child correlation was detected in cytokine‐producing capacity at the age of 1 yr. TNF‐α (P/I, SEB and LPS stimulation), IFN‐γ (P/I and SEB), IL‐5 (P/I and SEB) and IL‐10 (P/I, SEB and LPS) producing capacity increased from birth to 1 yr of age. In general, stimulated cytokine responses were higher in mothers’ than in children’s blood samples, except in the case of P/I and LPS‐stimulated IL‐8, which were highest at birth. Maternal inhalation atopy was associated with increased cord blood IL‐5 (24 and 48 h) and IL‐10 (48 h) production following P/I stimulation. Also children of food atopic mothers expressed elevated cord blood IL‐10 (48 h, P/I) responses and decreased IFN‐γ/IL‐5 ratio (24 h, P/I). In addition, the production of IFN‐γ (24 and 48 h, P/I) and the IFN‐γ/IL‐5 ratio (24 h and 48 h, P/I) at the age of 1 yr was lower among children with food atopic mothers. In conclusion, our results suggest that both adaptive and innate immune responses increase from birth to 1 yr of age, but are still weak in comparison to adult responses. Cytokine responses of children begin to correlate with those of their mothers during the first year of life. Although only few associations were observed between atopy and cytokine‐producing capacity, our results suggest that children of atopic mothers express Th2‐polarized cytokine pattern.

Chemical and microbial components of urban air PM cause seasonal variation of toxicological activity

The chemical and microbial composition of urban air particulate matter (PM) displays seasonal variation that may affect its harmfulness on human health. We studied the in vitro inflammatory and cellular metabolic activity/cytotoxicity of urban air particulate samples collected in four size-ranges (PM10-2.5, PM2.5-1, PM1-0.2, PM0.2) during four seasons in relatively clean urban environment in Helsinki, Finland. The composition of the same samples were analyzed, including ions, elements, PAH compounds and endotoxins. In addition, microbial contribution on the detected responses was studied by inhibiting the endotoxin-induced responses with Polymyxin B both in the PM samples and by two different bacterial strains representing Gram-positive and -negative bacteria. Macrophage cell line (RAW 264.7) was exposed to the size segregated particulate samples as well as to microbe samples for 24h and markers of inflammation and cytotoxicity were analyzed. The toxicological responses were dependent on the dose as well as size range of the particles, PM10-2.5 being the most potent and smaller size ranges having significantly smaller responses. Samples collected during spring and autumn had in most cases the highest inflammatory activity. Soil components and other non-exhaust particulate emissions from road traffic correlated with inflammatory responses in coarse particles. Instead, PAH-compounds and K(+) had negative associations with the particle-induced inflammatory responses in fine particles, suggesting the role of incomplete biomass combustion. Endotoxin content was the highest in PM10-2.5 samples and correspondingly, the largest decrease in the responses by Polymyxin B was seen with the very same samples. We found also that inhibitory effect of Polymyxin B was not completely specific for Gram-negative bacteria. Thus, in addition to endotoxin, also other microbial components may have a significant effect on the toxicological responses by ambient particulate matter.