A comprehensive battery of flow cytometric immunoassays for the in vitro testing of chemical effects in human blood cells

BackgroundThere is a growing need for immunological assays to test toxic and modulatory effects of chemicals. The assays should be easy to use, reproducible and superior to cell line-based assays. We have therefore developed a comprehensive portfolio of assays based on primary human blood cells that are suitable for testing chemical effects.MethodsThe flow cytometry-based assays were designed to target a wide range of human peripheral blood mononuclear cells and whole blood, including T cells, NK cells, B cells, basophils and innate-like T cells such as γδT, MAIT and NKT cells. We have selected a set of activation markers for each immune cell, e.g: CD154 (T cells), CD137, CD107a (NK cells), CD63 (basophils), CD69, CD83 (B cells), CD69, IFN-γ (MAIT cells) and we selected cell specific stimuli: aCD3 antibodies (T cells); E. coli and cytokines IL-12/15/18 (MAIT cells); CpG ODN2006, R848 or aCD40 antibodies (B cells), fMLP or aFcϵR1 (basophils) or K562 cells (NK cells).ResultsBy selecting immune cell-specific markers and cell-specific stimuli, we were able to induce particular immune responses from the targeted immune cells. For example, the response to stimulation with anti-CD3 antibodies was in 36.8% of CD107a+CD8+ cells. Cytokine stimulation induced the production of IFN-γ in 30% of MAIT cells. After stimulation with E. coli, around 50% of MAIT cells produced TNF. About 40% of basophils responded to aFcƐR1 stimulation. Similar activation ranges were achieved in K562-stimulated NK cells.ConclusionOur test portfolio covers the most relevant immune cells present in human blood, providing a solid basis for in vitro toxicity and immunomodulatory testing of chemicals. By using human blood, the natural composition of cells found in the blood can be determined and the effects of chemicals can be detected at the cellular level

The Activation of Mucosal-Associated Invariant T (MAIT) Cells Is Affected by Microbial Diversity and Riboflavin Utilization in vitro

Recent research has demonstrated that MAIT cells are activated by individual bacterial or yeasts species that possess the riboflavin biosynthesis pathway. However, little is known about the MAIT cell activating potential of microbial communities and the contribution of individual community members. Here, we analyze the MAIT cell activating potential of a human intestinal model community (SIHUMIx) as well as intestinal microbiota after bioreactor cultivation. We determined the contribution of individual SIHUMIx community members to the MAIT cell activating potential and investigated whether microbial stress can influence their MAIT cell activating potential. The MAIT cell activating potential of SIHUMIx was directly related to the relative species abundances in the community. We therefore suggest an additive relationship between the species abundances and their MAIT cell activating potential. In diverse microbial communities, we found that a low MAIT cell activating potential was associated with high microbial diversity and a high level of riboflavin demand and vice versa. We suggest that microbial diversity might affect MAIT cell activation via riboflavin utilization within the community. Microbial acid stress significantly reduced the MAIT cell activating potential of SIHUMIx by impairing riboflavin availability through increasing the riboflavin demand.We show that MAIT cells can perceive microbial stress due to changes in riboflavin utilization and that riboflavin availability might also play a central role for the MAIT cell activating potential of diverse microbiota

An in vitro model system for testing chemical effects on microbiome-immune interactions – examples with BPX and PFAS mixtures

IntroductionMore than 350,000 chemicals make up the chemical universe that surrounds us every day. The impact of this vast array of compounds on our health is still poorly understood. Manufacturers are required to carry out toxicological studies, for example on the reproductive or nervous systems, before putting a new substance on the market. However, toxicological safety does not exclude effects resulting from chronic exposure to low doses or effects on other potentially affected organ systems. This is the case for the microbiome-immune interaction, which is not yet included in any safety studies. MethodsA high-throughput in vitro model was used to elucidate the potential effects of environmental chemicals and chemical mixtures on microbiome-immune interactions. Therefore, a simplified human intestinal microbiota (SIHUMIx) consisting of eight bacterial species was cultured in vitro in a bioreactor that partially mimics intestinal conditions. The bacteria were continuously exposed to mixtures of representative and widely distributed environmental chemicals, i.e. bisphenols (BPX) and/or per- and polyfluoroalkyl substances (PFAS) at concentrations of 22 µM and 4 µM, respectively. Furthermore, changes in the immunostimulatory potential of exposed microbes were investigated using a co-culture system with human peripheral blood mononuclear cells (PBMCs). ResultsThe exposure to BPX, PFAS or their mixture did not influence the community structure and the riboflavin production of SIHUMIx in vitro. However, it altered the potential of the consortium to stimulate human immune cells: in particular, activation of CD8+ MAIT cells was affected by the exposure to BPX- and PFAS mixtures-treated bacteria. DiscussionThe present study provides a model to investigate how environmental chemicals can indirectly affect immune cells via exposed microbes. It contributes to the much-needed knowledge on the effects of EDCs on an organ system that has been little explored in this context, especially from the perspective of cumulative exposure

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals: a PARC (Partnership for the Assessment of Risk from Chemicals) project

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC)

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals — a PARC (Partnership for the Assessment of Risk from Chemicals) project

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC)

Comparing construction technologies of single family housing with regard of minimizing embodied energy and embodied carbon

This article concerns the Life Cycle Assessment method of evaluation and the ways in which it can be applied as a tool facilitating the design of buildings to reduce embodied energy and embodied carbon. Three variants of a building were examined with the same functional ground plan and usable floor area of 142.6 m2. Each variant of the building was designed using different construction technologies: bricklaying technology utilizing autoclaved aerated concrete popular in Poland, wooden frame insulated with mineral wool, and the Straw-bale technology. Using digital models (Building Information Model) the building’s energy characteristics was simulated and the embodied energy and embodied carbon of the production stage (also called cradle-to-gate) were calculated. The performed calculations were used to compare the cumulative energy and embodied carbon of each variant for a 40 year long life cycle

Comparing construction technologies of single family housing with regard of minimizing embodied energy and embodied carbon

This article concerns the Life Cycle Assessment method of evaluation and the ways in which it can be applied as a tool facilitating the design of buildings to reduce embodied energy and embodied carbon. Three variants of a building were examined with the same functional ground plan and usable floor area of 142.6 m2. Each variant of the building was designed using different construction technologies: bricklaying technology utilizing autoclaved aerated concrete popular in Poland, wooden frame insulated with mineral wool, and the Straw-bale technology. Using digital models (Building Information Model) the building’s energy characteristics was simulated and the embodied energy and embodied carbon of the production stage (also called cradle-to-gate) were calculated. The performed calculations were used to compare the cumulative energy and embodied carbon of each variant for a 40 year long life cycle

Peripheral Isothermal System of Heat Gain Storage for Thermal Stability in Low-Energy Buildings

The problem of heat storage in low- or ultra-low-energy houses is becoming a crucial issue. The general purpose of this study was to determine the potential for utilising heat gain recovery in a building. The proposed solution is based on an auxiliary latent heat storage tank using paraffin RT24. The tank is connected to an integrated heat recovery system that supplies heat from the internal loop of a mechanical ventilation system. The storage capacity of the tank was determined using the proposed parameter “excess of heat gains” of the thermal zone, and was obtained by measurement. The detailed construction of the tank, the phase change material properties and the quantity were proposed. The data that was collected allowed for the calculation of the temporary charging level as well as the overall seasonal energy stored in the tank. It was shown that during the heating season, the temperature could rise above the set-up value of 20 °C by as much as 8 K at maximum. Although the analysed building was characterised by heavy construction and high thermal mass, the additional heat could be effectively stored and utilised to cover the energy demand of the zone at the level of 88 MJ/a and 208 MJ/a, depending on the airflow rate between the rooms and the heat exchanger, for 140 and 420 m3/h, respectively. The expected energy effect for a low thermal mass construction, e.g., a timber frame was much higher and the results obtained by using the numerical simulation were 116 MJ/a for 140 m3/h, and 273 MJ/a for 420 m3/h, respectively

Life Cycle Assessment as a Major Support Tool within Multi-Criteria Design Process of Single Dwellings Located in Poland

Life cycle assessment is an environmental method which estimates either a process or a building material within the cradle-to-grave cycle. Presently, it is one of a few tools that include all factors which may influence the environment. The authors used this tool to prove effects connected with potential efficient energy levels and a reduction in CO2 emissions within a building’s life cycle. For the purpose of our analyses, several types of single-family building were chosen and they were subjected to analysis in the fixed location of Warsaw. The research scope included a numerical analysis of the buildings concerning the level of embodied energies and the emission of greenhouse gases. The performed analysis proved that, within a 50-year cycle, the difference between the embodied energy from the best and worst building choices can amount to 14.87%, whereas a reduction in embodied carbon emissions can reach 20.65%. Each change in the building’s form and the type of building materials used, regardless of the usable area, influence the environmental impact. Therefore, this paper concludes that LCA, as a management tool, should be used cyclically as part of each phase of the design process. A multi-criteria method for selecting architectural solutions was proposed which considered minimum cumulative primary energy, minimum cumulative carbon emission and minimum cost of constructing a building