Novel Cellular Luminescence Probes for Immunological and Toxicological Assessments

Escherichia coli K-12 (pEGFPluxABCDEAmp) (E. coli-lux), constitutively emitting bioluminescence (BL), was constructed and its BL emitting properties tested in different growth and killing conditions. The BL emission directly correlated with the number of viable E. coli-lux cells, and when subjected to the antimicrobial agent, the diminishment of the BL signal was linked directly to the number of killed bacterial cells. The method provided a very convenient application, especially when compared to conventional plate counting assays. This novel real-time based method was utilized in both immunological and toxicological assessments. The parameters such as the activation phase, the lytic phase and the capacity of the killing of the serum complement system were specified not only in humans but also in other species. E. coli-lux was also successfully used to study the antimicrobial activities of insect haemolymph. The mechanisms of neutrophil activity, like that of a myeloperoxidase (MPO)-H2O2-halide system, were studied using the E. coli-lux approach. The fundamental role of MPO was challenged, since during the actual killing in described circumstances in phagolysosome the MPO system was inactivated and chlorination halted. The toxicological test system, assessing indoor air total toxicity, particularly suitable for suspected mold damages, was designed based on the E. coli-lux method. Susceptibility to the vast number of various toxins, both pure chemicals and dust samples from the buildings and extracts from molds, were investigated. The E. coli-lux application was found to possess high sensitivity and specificity attributes. Alongside the analysis system, the sampling kit for indoor dust was engineered based on the swipe stick and the container. The combination of practical specimen collector and convenient analysis system provided accurate toxic data from the dust sample within hours. Neutrophils are good indicators of the pathophysiological state of the individual, and they can be utilized as a toxicological probe due to their ability to emit chemiluminescence (CL). Neutrophils can either be used as probe cells, directly exposed to the agent studied, or they can act as indicators of the whole biological system exposed to the agent. Human neutrophils were exposed to the same toxins as tested with the E. coli-lux system and measured as luminol amplified CL emission. The influence of the toxins on the individuals was investigated by exposing rats with moniliniformin, the mycotoxin commonly present in Finnish grains. The activity of the rat neutrophils was found to decrease significantly during the 28 days of exposure.Escherichia coli K-12 transformoitiin uudella bakteerilusiferaasigeenikasetin sisältämällä pEGFPluxABCDEAmp plasmidilla. Aktiivinen bakteerilusiferaasioperoni tuottaa konstitutiivista bioluminesenssia emittoivia E. coli soluja, ilman lisättyä substraattia. Uusi konstruktio nimettiin E. coli-lux:si ja sen bioluminesenssiemissio-ominaisuuksia mitattiin erilaisissa inkubaatio-olosuhteissa. Tutkimuksissa havaittiin E. coli-lux:in tuottaman bioluminesenssin olevan suoraan verrannollinen reaktioseoksessa olevien elävien bakteerisolujen lukumäärään ja kun reaktioon lisättiin antimikrobiaalista yhdistettä, valosignaalin laskun havaittiin korreloivan kuolleiden bakteerisolujen lukumäärän kanssa. Konventionaaliseen maljalaskentamenetelmään verrattuna E. coli-lux – sovellus tarjosi erittäin käytännöllisen vaihtoehdon bakteerien elinkyvyn ja tapon mittaamiseen ja sitä hyödynnettiin tässä kirjassa esitellyissä immunologisissa ja toksikologisissa menetelmissä koetinorganismina. Synnynnäisen immuniteetin tutkimuksessa E. coli-lux konstruktiota hyödynnettiin seerumin komplementtisysteemin aktivaatioreaktioteiden kineettisten parametrien kuten aktivaatio- ja lyyttisen faasin nopeuden sekä bakteeritappokapasiteetin määrittämiseen ihmisen, kirjolohen, sian, rotan, sekä lepakon seerumeista. Komplementin komponenteille spesifisten monoklonaalisten vasta-aineiden sekä antiseerumeiden avulla selvitettiin tiettyjen komponenttien vaikutusta tapporeaktioon. Lisäksi E. coli-lux menetelmää käytettiin hyönteisten veren, hemolymfan, antimikrobiaalisten ominaisuuksien selvittämisessä. Polymorfonukleaaristen leukosyyttien, eli neutrofiilien aktiivisuutta, fagosytoosia ja myeloperoksidaasi (MPO) -vetyperoksidi (H2O2) -kloridi-systeemin roolia fagolysosomaalisessa mikrobitapossa tutkittiin in vivo ja in vitro olosuhteissa, käyttämällä koettimena E. coli-lux soluja. MPO-systeemin perinteinen rooli neutrofiilien pääasiallisena bakteeritappomekanismina kyseenalaistettiin, koska olosuhteissa jossa mikrobitappo tapahtuu, MPO:n klorinaatioaktiivisuus havaittiin olemattomaksi. Tulokset viittasivat H2O2:n fagolysosomaalisen konsentraation itsessään olevan riittävän suuri mikrobitappoon. E. coli-lux konstruktiota käytettiin perustana uuden, rakennuksen sisäilman kokonaistoksisuutta mittaavan menetelmän kehitykseen. Testi perustuu kohteista kerättyjen sisäpölynäytteiden analysointiin, jossa E. coli-lux altistetaan pölyistä tehdyille H2O- ja DMSO- uutoksille ja toksisuus havaitaan valoemission heikkenemisenä. Toksisuus ilmoitetaan EC50 arvoina mg pölyä/ml uutosnestettä kohden. Menetelmä pystyy tunnistamaan kosteus- ja homevaurioiden sekä muiden sisäilmaongelmia aiheuttavien myrkkylähteiden toksisuuden. Analyysimenetelmän spesifisyys ja sensitiivisyys on verrannollinen perinteisiin mammaliansoluilla tehtäviin toksisuustesteihin ja lisäksi E. coli-lux systeemi on edullisempi ja nopeampi. E. coli-lux menetelmällä löydetty toksisuus korreloi tilankäyttäjien terveyshaittojen kanssa. Analyysimenetelmän ohella on kehitetty uusi näytteenottomenetelmä, jossa huonepölyä kerätään 1 – 10 mg pyyhkäisemällä useasta eri kohdasta kuitupuikolla ja Neutrofiilit toimivat hyvänä nisäkäsyksilön patofysiologisen tilan indikaattorina ja myös niitä voidaan käyttää koetinsoluina toksikologisessa tutkimuksessa. Neutrofiilien kyky emittoida kemiluminesenssiä fagosytoosin hengitysryöpsähdyksen yhteydessä mahdollistaa solujen toiminnan havainnoinnin levynlukijalumienometrillä. Neutrofiilejä voidaan altistaa suoraan toksiineille ja Ihmisen neutrofiilejä käytettiin E. coli-lux-systeemin toksisuusmittauksissa referenssikoettimina. Neutrofiilit voivat myös toimia koko altistetun yksilön systeemin indikaattoreina ja niitä käytettiin tutkittaessa suomalaisista viljoista eristetyn mykotoksiinin, moniliformiinin, vaikutusta rottien immuunipuolustukseen 28 päivää kestäneessä subkliinisessä kokeessa. Oraaliasuspensiona annosteltu moniliformiini laski merkitsevästi rottien neutrofiilien aktiivisuutta.Siirretty Doriast

Investigating the short- and long-term effects of antibacterial agents using a real-time assay based on bioluminescent E. coli-lux

We have previously established that the E. coli-lux assessment is a convenient tool for rapid measurements of the kinetical features of short-term toxicity caused by various factors. In this study, kinetic measurements of seven specifically acting model antibacterials (i.e., polymyxin B, chloramphenicol, nalidixic acid, kanamycin, deoxynivalenol, erythromycin and tetracycline) and two metals (AgNO3 and CdCl2) against E. coli-lux through a bioluminescence- and optical density-based real-time assay that combined short- and long-term toxicity assessments were performed. Bacteria were exposed to antibacterials and the effects were reported as the half-maximum effective concentration (EC50) after 30 min and 10 h. Regarding the 10-hour endpoints, all reference compounds, except deoxynivalenol, showed dose-response inhibition in the studied concentration range. The analysis of chloramphenicol, kanamycin, erythromycin, tetracycline and nalidixic acid clearly revealed the limitations of short-term inhibition tests. No significant differences were observed between the results obtained from luminescence inhibition and growth inhibition assays. The kinetical data from measurements provide differentiation between bacteriostatic and bactericidal mechanisms of various types of antibacterial agents. The combined assessment of short- and long-term effects reduces the risk of the underestimation of toxicity due to an inaccurate endpoint selection. The cost-efficient and fully automated E. coli-lux assessment technique may offer possibilities for high-throughput screening procedures.</p

Prevalence of Hoarseness in Primary Health Care and Hospitals-Associations With Different Work Tasks and Environmental Factors Among Nurses

OBJECTIVESThe voice is one of the most essential tools necessary for nurses achieve high care satisfaction and safety. Research on hoarseness has mainly focused on professional speakers, like teachers. The aim of this study was to determine the current prevalence of hoarseness among six subgroups of nurses (registered nurses, primary care nurses, pediatric nurses, laboratory nurses, dental nurses, and midwives) and also to identify potential environmental risk factors in their working environment.METHODSThe health data was collected in collaboration with two health care professional trade unions. The findings are based on 15,553 returned health questionnaires which were statistically analyzed.RESULTSThe 1-year prevalence of hoarseness among all participating nurses was 30.2 % in this data, but the variation between different occupational subgroups was significant; ranging from 25 % for laboratory nurses to 38 % for midwives. These findings were in line with perceived environmental problems and the differences between the sub-groups were not explained by the participants suffering from asthma, sinusitis, rhinitis, or common respiratory infections; there were no explanatory differences in the prevalence's of the above-mentioned diseases.CONCLUSIONSThe variation in prevalence of hoarseness between the different occupational subgroups was significant and the prevalence was found to be in line with perceived environmental problems. The most common problems were stuffiness, dry air, and inadequate ventilation. In addition, both a perceived "sewer odor" and "smell of mold or cellar" in one's own working environment were also found to be significant risk factors for hoarseness. Based on this study, the current indoor air problems in Finnish health care facilities can be verified and are a source of risk to nurses' voices.</p

The prevalence of hoarseness among health care professionals: time trends and effect of remediation in working conditions in 2007-2018

Purpose The purpose of this study was to investigate time trends in the prevalence of hoarseness among health care professionals in primary health care units (PHC) and in hospitals from 2007 to 2018. Moreover, purpose was to discover potential indoor environmental quality (IEQ) risk factors as well as to determine the effect of the remediation of the indoor air problems on the prevalence of hoarseness. Methods The health status was collected from all employees in these units/hospitals (N = 1564/1199) with questionnaires and the follow-ups were carried out as an open cohort. Based on building condition inspections, buildings were classified to be an "exposed" or "reference" buildings by third-party experts. The before and after remediation results were compared to reference buildings. Results During follow-up, hoarseness has not increased in those PHC units with good IEQ. In the pilot study, the prevalence of hoarseness in non-exposed reference building was 5.9%, and it stayed approximately at the same level throughout the follow-up. Whereas in buildings with an IEQ problem the prevalence of hoarseness varied between 16.2 and 36.1% and it decreased to 11.4% after the remediations. In a large hospital with severe IEQ problems, the prevalence of hoarseness was 39.1%, and in hospital buildings with a milder exposure 23.3%. The most important risk factors for hoarseness were asthma, allergic rhinitis and IEQ problems. Conclusion A good indoor environment and the remediation of damaged buildings seem to promote a better condition of the voice in health care workers.</p

The Association Between Work-related Stress, Indoor Air Quality and Voice Problems Among Teachers - Is There a Trend?

IntroductionHoarseness and other voice problems are common in occupations where the person has to speak, sing or shout in a work environment containing dust, noise, gaseous or particulate irritants. In recent years, stress has often been associated with voice problems.ObjectiveThe aim of this study was to examine trends over a period of time of the prevalence of voice problems and reported stress in Finnish school buildings.Study DesignSchool buildings from different parts of the country were studied for ten years using a similar questionnaire (N = 1721). Five schools participated before and after the remediation of an indoor air problem in the school buildings (n = 315).ResultsIn the pilot study (2007–2008), the reported work-related stress was on a very low level (3%) and the prevalence of hoarseness was 10%. After the economic crisis in 2008, the stress was observed to have increased. During the follow-up (2008–2017), the reported stress had continued to increase and in the latest surveys it was 21% and the prevalence of hoarseness 34%. Indoor air factors correlated significantly with hoarseness even when controlling for age, gender, owning pets and job satisfaction in a logistic regression model. In the school buildings where the remediation of the building was completed, the prevalence of hoarseness decreased but the level of stress increased. In general, the proportion of respondents reporting stress was lower than the proportion of respondents with hoarseness. Additionally, the reported stress also correlated with factors other than health in the education branch, especially economic resources and major changes in the core curriculum.ConclusionHoarseness and work-related stress have, to a large extent, different risk factors. Over time, the proportion of individuals with stress has been at a lower level than the proportion of respondents with voice problems; hence, we conclude that it is quite unlikely that stress would be a causative risk factor for hoarseness. We recommend that irritant dust and gases should be reduced from the work environment of teachers to enable recovery from hoarseness and other voice problems. Coping with work-related stress should be alleviated with other measures.</p

Groundwater radon exposure and risk of lung cancer: A population-based study in Finland

Naturally occurring radioactive elements can be found in groundwater and exposure to such elements is associated with an increased risk of lung cancer. In this study, we aimed to observe the association between exposure to these radioactive elements in groundwater and the risk of lung cancer in selected regions in Finland. This is a population-based study from 1955 to 2019 in Finland. The exposed municipalities with their corresponding hospital districts were selected based on radon measurements at groundwater treatment plants. Lung cancer cases were obtained from the Finnish cancer registry. The 5-year incidence rates for lung cancer were calculated and a comparison was made between each of the hospital districts with radon exposure. More than 93,000 cases of lung cancer were reported in the radon-exposed regions over the examined period of 64 years. The highest number of cases was recorded in the Helsinki University hospital district and the least in the Southern Savo hospital district. Similarly, the lung cancer incidence rate was highest in Lapland and lowest in the Southern Savo hospital district. The number of daily smokers in the working-age population appears to have decreased in all the hospital districts from 2013 to 2018. A statistically significant increased risk of lung cancer was observed in the high radon-exposed hospital districts compared to those with lower exposure. Groundwater radon exposure is observed to be associated with an increased risk of lung cancer

Transferring from moisture damaged school building to clean facilities - The avoidance of mold exposure induces a decline in symptoms and improvement in lung function among personnel

Working in a moisture-damaged building can cause different symptoms and effects on lung functions. Moving to a clean environment, it is believed to reduce symptoms and alleviate potential adverse health effects. This case study monitors the health effects of personnel in one school building before and after all school activities were transferred from a moisture-damaged school building to clean premises. The whole school staff was invited to attend this follow-up study. All participants (N = 45) were interviewed, and pulmonary functions were measured by spirometry and exhaled nitric oxide testing (FENO) before transferring the school activities to a new building and the control measurements were performed twice; three months and six months after the transfer. After transferring to temporary facilities, 82% of participants felt that their symptoms were improved or resolved and the pulmonary functions were improved; 50% of those who had decreased pulmonary functions at the beginning, their pulmonary function values returned to normal after three months. Over the next six months, the perceived symptoms continued to reduce so that 93% of the respondents felt fully asymptomatic with respect to indoor air, and the spirometry results improved further. Transferring workers from the damaged building to healthy environment provided beneficial health effects on pulmonary functions and to perceived symptoms even in a relatively short time period. Based on this study, and from the perspective of promoting and protecting the health and well-being of personnel, transferring school activities from a moisture damaged building to clean facilities brought considerable advantages, despite the possible cost and difficulties of finding replacement facilities.</p

Hoarseness Among Young Children in Day-Care Centers

BackgroundChronic respiratory symptoms among toddlers are assumed to be due to allergies and common respiratory infections. Because symptoms and respiratory disease in this age group often continue on to school age and later life, it is important to know the possible risk factors for prevention of the chronic hoarseness.AimWe aimed to determine the current prevalence of hoarseness and other chronic respiratory symptoms among toddlers and young children. Another aim was to examine the risk factors for hoarseness in the building environments of day-care centers (DCC).MaterialAn electronic symptom survey was sent to all parents of children in day-care centers of a large city in southern Finland. In all, 3721 individuals completed the questionnaire (38%), 53.4% were the parents of boys and 46.6% girls.ResultsThe prevalence of hoarseness was 5.6%. The boy's parents reported hoarseness more often than the girls, but no significant difference was observed. Risk factors for hoarseness in a built environment in this age group were noise, visible dust and dirt, mold and a cellar like odor, a sewer smell, other unpleasant smells, stuffiness of the indoor air, a too high or too low temperatures, a cold floor, insufficient ventilation, the age of the DCC building, and wood as the bearing construction of the building. The lifestyle factors that correlated with the prevalence of hoarseness were the amount of time spent outdoors; however, passive smoking, the number of siblings and pets at home did not correlate with hoarseness. Hoarseness was significantly correlated with other chronic respiratory symptoms such as rhinitis, coughs, eye irritation, tiredness, headaches, and stomach problems and also with the regular or periodic use of medication. Hoarseness was also significantly correlated with asthma and allergic rhinitis and also with repeated infections, such as a common cold, cold with a fever, laryngitis, otitis media and acute bronchitis, but not with tonsillitis or pneumonia.When potential confounders had been controlled for with a logistic regression model, the following risk factors in the built environment remained statistically significant: noise, high room temperature, insufficient ventilation and the stuffiness of the indoor air, a solvent odor, wood as the bearing construction and the age of the building.ConclusionsWe conclude that in day-care centers, buildings should be maintained, cleaned and ventilated properly. Concrete and brick used in the construction were protective compared with wood. The acoustic environment should be planned to reduce noise indoors and solvent based chemicals should be avoided. Neither having pets at home or the number of siblings were risk factors, but they were also not found to be protective in this material. All measures that reduce the occurrence of respiratory infections probably also reduce chronic voice problems.</p

Indoor-related microbe damage induces complement system activation in building users

In this comparative study, serum complement system antimicrobial activity was measured from 159 serum samples, taken from individuals from microbe-damaged (70 samples) and from reference buildings (89 samples). Antimicrobial activity was assessed using a probe-based bacterial Escherichia coli-lux bioluminescence system and comparison was made at a group level between the experimental and reference group. The complement activity was higher in users of microbe-damaged buildings compared with the reference group and the significant (P < 0.001) increase in activity was found in the classical reaction pathway. This study strengthens our notion that exposure to indoor-related microbe damage increases the risk for systemic subclinical inflammation and creates a health risk for building users

Indoor exposure to Streptomyces albus and Aspergillus versicolor elevates the levels of spore-specific IgG, IgG1 and IgG3 serum antibodies in building users - A new ELISA-based assay for exposure assessment

Moisture-indicative microbes in buildings are associated with a variety of symptoms, ranging from mild irritation to severe clinical illnesses. These symptoms are caused principally by dried, dormant and dead microbe material like spores, mycelium and microbe metabolites, leading to the activation of the immune system and formation of the antigen-specific immunoglobulins. This activation presumably takes place through the respiratory track and is a normal immune reaction against pathogenic invaders. During continuous exposure, a prolonged state of inflammation will follow, and this forms a considerable health risk for a building's occupant. A new ELISA system utilizing spores from two species Streptomyces albus and Aspergillus versicolor as an antigen was developed to reveal the related immunological processes. In 159 persons, microbial exposure was observed to increase the levels of spore-specific IgG, IgG1 and IgG3 serum antibody levels of individuals residing in microbe-dense buildings compared with the control reference buildings. No differences were detected in the levels of S. albus- and A. versicolor-specific serum IgA or IgM levels.</p