In Vitro Systems for Toxicity Evaluation of Microbial Volatile Organic Compounds on Humans: Current Status and Trends.

Microbial volatile organic compounds (mVOC) are metabolic products and by-products of bacteria and fungi. They play an important role in the biosphere: They are responsible for inter- and intra-species communication and can positively or negatively affect growth in plants. But they can also cause discomfort and disease symptoms in humans. Although a link between mVOCs and respiratory health symptoms in humans has been demonstrated by numerous studies, standardized test systems for evaluating the toxicity of mVOCs are currently not available. Also, mVOCs are not considered systematically at regulatory level. We therefore performed a literature survey of existing in vitro exposure systems and lung models in order to summarize the state-of-the-art and discuss their suitability for understanding the potential toxic effects of mVOCs on human health. We present a review of submerged cultivation, air-liquid-interface (ALI), spheroids and organoids as well as multi-organ approaches and compare their advantages and disadvantages. Furthermore, we discuss the limitations of mVOC fingerprinting. However, given the most recent developments in the field, we expect that there will soon be adequate models of the human respiratory tract and its response to mVOCs.

Application of impedance measurement to investigate in vitro inhalation toxicity of bacteria.

BACKGROUND: Workers of agriculture and intensive life stock farming are exposed to highly contaminated workplaces. Bioaerosol exposures are suspected to trigger respiratory health effects of the workers. So far, risk evaluation of bioaerosols has been assessed through the infectivity of comprising biological agents that is classified in Europe by four risk groups according to the criteria of Directive 2000/54EC of the European Parliament. However, this directive additionally requires the risk assessment of allergenic and toxigenic effects without further elaboration. The aim of our study was to establish an in vitro screening system that is able to measure inhalative toxic effects of bacteria and their metabolites. METHODS: In this study, we analyzed three bacterial toxins and five culture supernatants of selected bacteria with known toxicity as model agents exposed to the lung epithelial cell line NuLi-1. We used electrical cell-substrate impedance sensing (ECIS) method to monitor real-time cell changes and the viability test Prestoblue™. RESULTS: We confirmed concentration dependent cytotoxic effects of the selected toxins in NuLi-1 cells over a period of up to 48 h. Each toxin resulted in a different but specific impedance profile over time according to their mode of action, whereas viability assay showed the metabolic activity of the cells at a chosen time point without revealing any information on their mode of action. Furthermore, dose-response-relationships were monitored. Tested model bacteria (Streptoccous pneumoniae, Acinetobacter radioresistens, Aerococcus viridans, Aeromonas hydrophila) reacted according to their expected toxicity except one bacterium (Enterococcus faecalis). The established assays revealed the concentration dependent onset and intensity of bacterial cytotoxicity and the viability of the cells at 24 h and 48 h exposure. CONCLUSION: Impedance measurement and the viability assay Prestoblue™ in combination are suitable as sensitive screening methods to analyze toxic potential of bacteria and can therefor support the risk assessment of workplaces in terms of the directive 2000/54/EC.

Serological evidence of asymptomatic infections during Escherichia coli O104:H4 outbreak in Germany in 2011.

INTRODUCTION: The largest known outbreak caused by a rare hybrid strain of Shiga toxin-producing E.coli (STEC) and enteroaggregative E. coli (EAEC) (E.coli O104:H4) of serotype O104:H4 occurred in Germany in 2011. Fenugreek sprouts acted as a transmission vehicle and were widely consumed in the outbreak area at the time of the epidemic. In total 3,842 people developed a clinical illness caused by this strain; however the rates of asymptomatic infections remain unclear. We aimed to develop a serological assay for detection of E.coli O104 LPS specific antibodies and to establish the post-outbreak levels of seropositivity among people with documented exposure to contaminated sprouts. RESULTS AND DISCUSSION: Developed serological assays (ELISA with 84% sensitivity, 63% specificity and Western Blot with 100% sensitivity, 82.5% specificity) identified 33% (16/49) level of asymptomatic infection. Relatively small sample size and a significant time- lapse between the onset of symptoms and serum samples collection (appr. 8 weeks) might explain the assay variability. No association was found between clinical or demographic characteristics and assay positivity. Larger studies are needed to understand the complexity of human immune response and factors influencing development of clinical symptoms. Development of intra-outbreak research plans will substantially aid the conduct of more thorough scientific investigation during an outbreak period.

The PAK1 autoregulatory domain is required for interaction with NIK in Helicobacter pylori-induced NF-kappaB activation.

Helicobacter pylori, the etiological agent of various human gastric diseases, induces the transcription factor nuclear factor kappaB (NF-kappaB) and proinflammatory cytokines/chemokines. We have characterised the direct interaction between p21-activated kinase 1 (PAK1) and NF-kappaB-inducing kinase (NIK) in H. pylori-infected epithelial cells. The dimerisation (DI) motif, which is part of the NH2-terminal autoregulatory domain of PAK1, is critical for this interaction, whereas NIK forms complexes with PAK1 through its carboxy-terminal IkappaB kinase alpha (IKKalpha) binding site. Since the identified interaction sites are also crucial for the binding of activator (Rac/Cdc42 in the case of PAK1) or effector molecules (IKKalpha in the case of NIK), sequential stepwise signalling is suggested. Furthermore, we show that mitogen-activated protein kinase kinase kinases (MAP3K), like TPL2 (tumour progression locus 2) and transforming growth factor beta-activated kinase 1 (TAK1), have no impact on H. pylori-induced activation of NF-kappaB. These results identify the roles of PAK1 and NIK in a unique pathway involved in H. pylori-induced NF-kappaB activation, which is crucial for the induction of the innate immune response.