Epidemiological samplings for long-term HBM-studies during a pandemic situation - Experiences and lessons-learned, the German Environmental Specimen Bank during the COVID-19 pandemic.

For the investigation of diseases and other harmful environmental influences (e.g., chemicals) epidemiological studies rely on high quality human samples, among others. Collecting samples and data in the field can pose an enormous challenge to the study team with regard to health protection and occupational safety, especially in the context of a pandemic where there was great uncertainty about the biological risks associated with SARS-CoV-2. The German Environmental Specimen Bank (German ESB) is a key element of environmental and human biomonitoring in Germany with the aim to document and assess trends of human and environmental exposure to chemicals over time and to provide scientific data for policy decision makers. Starting with a pilot study in 1978 human samples are now collected at four sampling locations annually, while sampling is carried out with a highly standardized mobile laboratory since 2013. Due to the corona pandemic 3 of 4 ESB sampling campaigns had to be cancelled in 2020. However, a continuous sampling is crucial to generate current policy relevant data on chemical exposure. Hence, a protection and hygiene concept has been developed including COVID-19 testing with the goal to protect the health of participants and employees during sampling and to meet legal requirements, while sustaining the standardized procedures of sampling and sample preparation. The concept is based on a flexible approach to allow adjustments to changing government regulations and recommendations in the course of the pandemic. By implementing this concept, all samplings were successfully carried out in 2021 & 2022, with the pandemic still ongoing. This paper provides an example of good practice and valuable insights in how to collect human samples during a pandemic.

Assessing background contamination of sample tubes used in human biomonitoring by non-targeted liquid chromatography-high resolution mass spectrometry.

Controlling and minimising background contamination is crucial for maintaining a high quality of samples in human biomonitoring targeting organic chemicals. We assessed the contamination of three previous types and one newly introduced medical-grade type of sample tubes used for storing human body fluids at the German Environmental Specimen Bank. Aqueous extracts from these tubes were analysed by non-targeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) before and after a dedicated cleaning procedure. After peak detection using MZmine, Bayesian hypothesis testing was used to group peaks into those originating either from instrumental and laboratory background contamination, or actual tube contaminants, based on if their peak height was reduced, increased or not affected by the cleaning procedure. For all four tube types 80-90% of the 2475 peaks (1549 in positive and 926 in negative mode) were assigned to laboratory/instrumental background, which we have to consider as potential sample tube contaminants. Among the tube contaminants, results suggest a considerable difference in the contaminant peak inventory and the absolute level of contamination among the different sample tube types. The cleaning procedure did not affect the largest fraction of peaks (50-70%). For the medical grade tubes, the removal of contaminants by the cleaning procedure was strongest compared to the previous tubes, but in all cases a small fraction increased in intensity after cleaning, probably due to a release of oligomers or additives. The identified laboratory background contaminants were mainly semi-volatile polymer additives such as phthalates and phosphate esters. A few compounds could be assigned solely as tube-specific contaminants, such as N,N-dibutylformamide and several constituents of the oligomeric light stabiliser Tinuvin-622. A cleaning procedure before use is an effective way to standardise the used sample tubes and minimises the background contamination, and therefore increases sample quality and therewith analytical results.

Genome-wide DNA methylation sequencing identifies epigenetic perturbations in the upper airways under long-term exposure to moderate levels of ambient air pollution.

While the link between exposure to high levels of ambient particulate matter (PM) and increased incidences of respiratory and cardiovascular diseases is widely recognized, recent epidemiological studies have shown that low PM concentrations are equally associated with adverse health effects. As DNA methylation is one of the main mechanisms by which cells regulate and stabilize gene expression, changes in the methylome could constitute early indicators of dysregulated signaling pathways. So far, little is known about PM-associated DNA methylation changes in the upper airways, the first point of contact between airborne pollutants and the human body. Here, we focused on cells of the upper respiratory tract and assessed their genome-wide DNA methylation pattern to explore exposure-associated early regulatory changes. Using a mobile epidemiological laboratory, nasal lavage samples were collected from a cohort of 60 adults that lived in districts with records of low (Simmerath) or moderate (Stuttgart) PM10 levels in Germany. PM10 concentrations were verified by particle measurements on the days of the sample collection and genome-wide DNA methylation was determined by enzymatic methyl sequencing at single-base resolution. We identified 231 differentially methylated regions (DMRs) between moderately and lowly PM10 exposed individuals. A high proportion of DMRs overlapped with regulatory elements, and DMR target genes were involved in pathways regulating cellular redox homeostasis and immune response. In addition, we found distinct changes in DNA methylation of the HOXA gene cluster whose methylation levels have previously been linked to air pollution exposure but also to carcinogenesis in several instances. The findings of this study suggest that regulatory changes in upper airway cells occur at PM10 levels below current European thresholds, some of which may be involved in the development of air pollution-related diseases.

A step towards harmonising human biomonitoring study setup on European level: Materials provided and lessons learnt in HBM4EU.

Internal exposure of the human body to potentially harmful chemical substances can be assessed by Human Biomonitoring (HBM). HBM can be used to generate conclusive data that may provide an overview of exposure levels in entire or specific population groups. This knowledge can promote the understanding of potential risks of the substances of interest or help monitoring the success of regulatory measures taken on the political level. Study planning and design are key elements of any epidemiologic study to generate reliable data. In the field of HBM, this has been done using differing approaches on various levels of population coverage so far. Comparison and combined usage of the resulting data would contribute to understanding exposure and its factors on a larger scale, however, the differences between studies make this a challenging and somewhat limited endeavour. This article presents templates for documents that are required to set up an HBM study, thus facilitating the generation of harmonised HBM data as a step towards standardisation of HBM in Europe. They are designed to be modular and adaptable to the specific needs of a single study while emphasising minimum requirements to ensure comparability. It further elaborates on the challenges encountered during the process of creating these documents during the runtime of the European Joint Programme HBM4EU in a multi-national expert team and draws up lessons learnt in the context of knowledge management.

HBM4EU feasibility studies: Lessons learned in combining health and human biomonitoring studies.

BACKGROUND: The European Human Biomonitoring Initiative (HBM4EU) is a joint program evaluating humans' exposure to several environmental substances and their potential health effects. One of the main objectives of HBM4EU is to make use of human biomonitoring (HBM) to assess human exposure to chemicals in Europe to better understand the associated health impacts and to improve chemical risk assessment. In parallel to HBM studies, health examination surveys (HESs), nutrition/dietary surveys, and disease specific health surveys are conducted in many European countries. In HESs, information collected by questionnaire(s) is supplemented with physical examinations and analysis of clinical and biological biomarkers in biological samples. HBM and health examination survey (HES) use similar data collection methods and infrastructures hence the feasibility of combining these two is explored in this paper. METHODS: Within HBM4EU, three feasibility studies (in Finland, Germany, and UK/England) were conducted to evaluate opportunities and obstacles of combining HBM and health studies. In this paper we report lessons learned from these feasibility studies. RESULTS: The Finnish feasibility study called KouBio-KUOPIO study was a new initiative without links to existing studies. The German feasibility study added a HBM module to the first follow-up examination of the LIFE-Adult-Study, a population-based cohort study. The UK feasibility integrates a sustainable HBM module into the Health Survey for England (HSfE), an annual health examination survey. Benefits of combining HBM and HESs include the use of shared infrastructures. Furthermore, participants can receive additional health information from HES, and participation rates tend to be higher due to the potential to obtain personal health information. Preparatory phases including obtaining ethical approval can be time-consuming and complicated. Recruitment of participants and low participation rates are common concerns in survey research and therefore designing user-friendly questionnaires with low participant burden is important. Unexpected events such as the COVID-19 pandemic can cause substantial challenges and delays for such studies. Furthermore, experiences from several countries demonstrated that long-term funding for combined studies can be difficult to obtain. CONCLUSIONS: In the future, incorporating HBM modules into existing HESs can provide a feasible and cost-effective method to conduct HBM studies and obtain a wide range of relevant data to support public health policies and research.

What is required to combine human biomonitoring and health surveys?

Obtaining holistic information about health and health determinants at the population level should also include data on environmental risk factors of health. So far, only a few countries have combined, at the national level, health and human biomonitoring (HBM) surveys to collect extensive information on health, lifestyles, biological health determinants and environmental exposures. This paper will provide guidelines on how to combine health and HBM surveys and what is the added value of doing so. Health and HBM surveys utilize similar infrastructure and data collection methods including questionnaires, collection and analysis of biological samples, and objective health measurements. There are many overlapping or comparable steps in these two survey types. At the European level, detailed protocols for conducting a health examination survey or HBM study exists separately but there is no protocol for a combined survey available by now. Our recommendations for combined health and HBM surveys focus on a cross-sectional survey on general population aged 6-79 years. To avoid unnecessary participant burden, for the selection of included measurements basic principle would be to ensure that results of the measurements have a public health relevance and clear interpretation. Combining health and HBM surveys into one survey would produce an extensive database for research to support policy decisions in many fields such as public health and chemical regulations. Combined surveys are cost-effective as only one infrastructure is needed to collect information and recruit participants.

Long-term monitoring of mercury in young German adults: Time trend analyses from the German Environmental Specimen Bank, 1995-2018.

As highlighted in the Minamata Convention, Mercury (Hg) in its various forms poses a substantial risk to human health and the environment. The health relevance of Hg is also recognized by the European Human Biomonitoring Initiative (HBM4EU), which classifies Hg as a priority substance, since considerable knowledge and data gaps on Hg exposure levels and their changes over time still exist in Europe. The German Environmental Specimen Bank (German ESB) provides valuable policy relevant data and long-term trends of substance exposure on a national level for international comparison and evaluation. In this study we analysed data of the German ESB on Hg exposure of young adults aged 20 to 29 including data on urinary Hg levels from 1995 to 2018 and whole blood Hg levels from 2001 to 2010. Results show a clear decrease in both, about 86% in urine total daily Hg excretion from 1995 (0.76 µg/L) to 2018 (0.11 µg/L) (n = 10,069) and about 57% in blood concentrations of Hg from 2001 (1.76 µg/L) to 2010 (0.77 µg/L) (n = 4085). Over the investigated timeframe only a few values exceeded the toxicologically derived health based guidance value HBM I for blood and urine, with these exceedances decreasing over time in line with the general trend. The factors mostly influencing Hg excretion identified in this study are dental amalgam as well as fish and seafood consumption. Besides other factors (e.g. age and sex), also airborne Hg exposure appears to be a low but evident influencing factor in Germany. Although a considerable decrease in internal Hg exposure is recognized in the last decades, the current low-level exposure may cause adverse health effects especially to vulnerable groups such as pregnant women and children. To further elucidate and evaluate current exposure sources and to reduce human exposure to Hg, continuous environmental and human biomonitoring is needed.

Long-term time trend of lead exposure in young German adults - Evaluation of more than 35 Years of data of the German Environmental Specimen Bank.

Lead is a ubiquitous pollutant with well-known effects on human health. As there is no lower toxicological threshold for lead in blood and since data gaps on lead exposure still exist in many European countries, HBM data on lead is of high importance. To address this, the European Human Biomonitoring Initiative HBM4EU classified lead as a priority substance. The German Environmental Specimen Bank (German ESB) has monitored lead exposure since more than 35 years. Using data from the early 1980s to 2019 we reveal and discuss long-term trends in blood lead levels (BLLs) and current internal exposure of young adults in Germany. BLLs in young adults decreased substantially in the investigated period. As results from the ESB sampling site Muenster demonstrate, the geometric mean of BLLs of young adults decreased from 1981 (78,7 µg/L) to 2019 (10.4 µg/L) by about 87%. Trends in human exposure closely correlate with air lead levels (ALLs) provided by the European Monitoring and Evaluation Programme (EMEP). Hence, the decrease of BLLs largely reflects the drop in air lead pollution. Known associations of sex, smoking, alcohol consumption, and housing situation with BLLs are confirmed with data of the German ESB. Although internal lead exposure in Germany decreased substantially, the situation might be different in other European countries. Since 2010, BLLs of young adults in Germany levelled out at approximately 10 µg/L. The toxicity of lead even at low levels is known to cause adverse health effects especially in children following exposure of the child or the mother during pregnancy. To identify current exposure sources and to minimize future lead exposure, continuous monitoring of lead intake and exposure levels is needed.

Towards Harmonized Biobanking for Biomonitoring: A Comparison of Human Biomonitoring-Related and Clinical Biorepositories.

Human biomonitoring (HBM) depends on high-quality human samples to identify status and trends in exposure and ensure comparability of results. In this context, much effort has been put into the development of standardized processes and quality assurance for sampling and chemical analysis, while effects of sample storage and shipment on sample quality have been less thoroughly addressed. To characterize the currently applied storage and shipment procedures within the consortium of the European Human Biomonitoring Initiative (HBM4EU), which aims at harmonization of HBM in Europe, a requirement analysis based on data from an online survey was conducted. In addition, the online survey was addressed to professionals in clinical biobanking represented by members of the European, Middle Eastern and African Society for Biopreservation and Biobanking (ESBB) to identify the current state-of-the-art in terms of sample storage and shipment. Results of this survey conducted in these two networks were compared to detect processes with potential for optimization and harmonization. In general, many similarities exist in sample storage and shipment procedures applied by ESBB members and HBM4EU partners and many requirements for ensuring sample quality are already met also by HBM4EU partners. Nevertheless, a need for improvement was identified for individual steps in sample storage, shipment, and related data management with potential impact on sample and data quality for HBM purposes. Based on these findings, recommendations for crucial first steps to further strengthen sample quality, and thus foster advancement in HBM on a pan-European level are given.

Trends in characteristics of 24-h urine samples and their relevance for human biomonitoring studies - 20 years of experience in the German Environmental Specimen Bank.

To document trends in human exposure to environmental pollutants, the German Environmental Specimen Bank (ESB) has been routinely collecting and archiving 24-h urine samples from young adults at four sampling sites in Germany on an annual basis. For the purpose of normalizing measured analyte concentrations, urinary creatinine (UC), specific gravity (SG), conductivity (CON), and total urine volume (UVtot) of 24-h urine samples have also been recorded. These parameters are however susceptible to variation over time, as well as within/among participants and normalization against them can thus affect the interpretation of data regarding exposure to environmental pollutants. To evaluate the influence of normalization against these parameters, we first sought to determine variations of these parameters with regard to differences between sexes and trends over time. We analysed data from 8619 urine samples collected from 1997 to 2016. We observed an inverse relation between UVtot and UC, SG, and CON. We also found differences between sexes for UC, SG and CON, but not UVtot. UC, SG, and CON showed significant decreasing trends over time in both sexes. In contrast, a significant increase of over 30% in UVtot, independent of participant age and BMI, was revealed. This increase in UVtot and the concomitant sample dilution is likely to have an impact on measured analyte concentrations in 24-h urine samples. Hence, normalization of urinary concentrations is warranted when interpreting time trends of human exposure. Next, urinary calcium (Ca2+) concentrations of ESB participants were used to demonstrate the effects of normalization against each of the four urine parameters. From 1997 to 2016, measured Ca2+ concentrations showed a statistically significant but scientifically implausible decrease. Normalization of Ca2+ concentrations against UVtot (by calculating the total daily excretion), UC, or CON, but not SG, eliminated this decrease. Consistent with previous work, Ca2+ concentrations in urine and total daily Ca2+ excretion were higher for males than females. Normalization against UC, SG, or CON, however, attenuated this difference. Thus, to avoid misinterpretation in trend analysis and sex-specific excretion in 24-h urine samples, the calculation of the total daily excretion is recommended.

Discovering time-trends of the German populations exposure to contaminants by analysis of human samples of the German Environmental Specimen Bank (ESB).

The German Environmental Specimen Bank (ESB) is a monitoring instrument of the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety. The permanent biobank facility is run since 1981 containing environmental and human samples from Germany. All samples are collected according to standard operating procedures (SOP). An standardized annual collection of human samples at four different regional sites of the country has been established since 1997. Routine sampling is done once a year, recruiting healthy non occupationally exposed students aged 20-29 years, in an equal gender distribution. The number of participants recruited is approximately 120 students per site and year. Directly after the annual sampling process, the human samples are analyzed for selected environmental chemicals. The time-trends of lead in blood, mercury and pentachlorophenol in 24 h-urine and polychlorinated biphenyls in plasma demonstrated a decrease of exposure during the last two decades by about 40-90 percent. In parallel retrospective studies using cryo-archived samples revealed increasing time trends of emerging chemicals used as substitutes for regulated toxicants. The data demonstrates the great relevance of the ESB for the health related environmental monitoring and shows the importance of human biomonitoring as a tool in information based policy making.

Combining chemical and biological endpoints, a major challenge for twenty-first century's environmental specimen banks.

Environmental specimen banks (ESBs) are not a new phenomenon, but in the last decades, the steep rate in the establishment of new ESBs is a sign to address new research approaches for scientists. In this way, environmental biobanking is becoming a well-organized and effective vehicle to collect samples of high quality making them available for future researchers. The endpoints promoted in the ESBs are mainly based on chemical approaches, but the necessity to add biological endpoint is fundamental (e.g., assessment of the environmental health status). Moreover, advances and development of high sensitive, high-throughput techniques along with ecotoxicological approaches based on biomarkers are stimulating a new demand for stored specimens and associated data. Like in chemically targeted environmental specimen banking, the banked samples for the assessment of biological effects also require guidance informed by knowledge of their practices and challenges, along with policies for the correct advancement of research goals and appropriate and effective biobank governance.

A new approach to standardize multicenter studies: mobile lab technology for the German Environmental Specimen Bank.

Technical progress has simplified tasks in lab diagnosis and improved quality of test results. Errors occurring during the pre-analytical phase have more negative impact on the quality of test results than errors encountered during the total analytical process. Different infrastructures of sampling sites can highly influence the quality of samples and therewith of analytical results. Annually the German Environmental Specimen Bank (ESB) collects, characterizes, and stores blood, plasma, and urine samples of 120-150 volunteers each on four different sampling sites in Germany. Overarching goal is to investigate the exposure to environmental pollutants of non-occupational exposed young adults combining human biomonitoring with questionnaire data. We investigated the requirements of the study and the possibility to realize a highly standardized sampling procedure on a mobile platform in order to increase the required quality of the pre-analytical phase. The results lead to the development of a mobile epidemiologic laboratory (epiLab) in the project "Labor der Zukunft" (future's lab technology). This laboratory includes a 14.7 m(2) reception area to record medical history and exposure-relevant behavior, a 21.1 m(2) examination room to record dental fillings and for blood withdrawal, a 15.5 m(2) biological safety level 2 laboratory to process and analyze samples on site including a 2.8 m(2) personnel lock and a 3.6 m2 cryofacility to immediately freeze samples. Frozen samples can be transferred to their final destination within the vehicle without breaking the cold chain. To our knowledge, we herewith describe for the first time the implementation of a biological safety laboratory (BSL) 2 lab and an epidemiologic unit on a single mobile platform. Since 2013 we have been collecting up to 15.000 individual human samples annually under highly standardized conditions using the mobile laboratory. Characterized and free of alterations they are kept ready for retrospective analyses in their final archive, the German ESB.

Neuro-muscular differentiation of adult porcine skin derived stem cell-like cells.

BACKGROUND: Due to the genetic relationship to humans, porcine stem cells are a very important model system to investigate cell differentiation, associated cell signaling pathways, and cell fate. Porcine skin derived stem cells have been isolated from mid-gestation porcine fetus recently. To our knowledge, stem cells from the skin of the adult porcine organism have not been isolated until now. Hence, to our knowledge, we here describe the isolation, expansion, characterization and differentiation of multipotent porcine skin derived stem cell-like cells (pSSCs) from the adult porcine organism for the first time. METHODOLOGY/PRINCIPAL FINDINGS: pSSCs had a spindle shaped morphology similar to mesenchymal stem cells (MSCs). They could be maintained proliferatively active in vitro for more than 120 days and were able to form colonies from single cells. pSSCs expressed Sox2 and Oct3/4, both transcription factors essential to the pluripotent and self-renewing phenotypes of embryonic stem cells, which recently gained attention due to their function in inducing pluripotent stem cells. Furthermore, the expression of the progenitor marker nestin, the somatic stem cell markers Bcrp1/ABCG2, Bmi1, and Stat3 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in undifferentiated pSSCs. Flow cytometry revealed the expression of the MSC related proteins CD9, CD29, CD44 and CD105, but not CD90. After neuronal differentiation cells with a characteristic morphology of neuronal and smooth muscle-like cells were present in the cultures. Subsequent immunochemistry and flow cytometry revealed the down-regulation of nestin and the up-regulation of the neuron specific protein beta-III-tubulin and the astrocyte marker GFAP. Also, alpha-SMA expressing cells increased during differentiation suggesting the neuro-muscular differentiation of these skin derived cells. pSSCs could also be induced to differentiate into adipocyte-like cells when cultured under specific conditions. CONCLUSIONS/SIGNIFICANCE: Adult porcine skin harbors a population of stem cell-like cells (pSSCs) that can be isolated via enzymatic digestion. These pSSCs show characteristic features of MSCs originated in other tissues and express the embryonic stem cell marker Oct3/4, Sox2, and Stat3. Furthermore, pSSCs have the potential to differentiate into cells from two different germ lines, the ectoderm (neurons, astrocytes) and the mesoderm (smooth muscle cells, adipocytes).

Cryobanking of viable biomaterials: implementation of new strategies for conservation purposes.

Cryobanking, the freezing of biological specimens to maintain their integrity for a variety of anticipated and unanticipated uses, offers unique opportunities to advance the basic knowledge of biological systems and their evolution. Notably, cryobanking provides a crucial opportunity to support conservation efforts for endangered species. Historically, cryobanking has been developed mostly in response to human economic and medical needs - these needs must now be extended to biodiversity conservation. Reproduction technologies utilizing cryobanked gametes, embryos and somatic cells are already vital components of endangered species recovery efforts. Advances in modern biological research (e.g. stem cell research, genomics and proteomics) are already drawing heavily on cryobanked specimens, and future needs are anticipated to be immense. The challenges of developing and applying cryobanking for a broader diversity of species were addressed at an international conference held at Trier University (Germany) in June 2008. However, the magnitude of the potential benefits of cryobanking stood in stark contrast to the lack of substantial resources available for this area of strategic interest for biological science - and society at large. The meeting at Trier established a foundation for a strong global incentive to cryobank threatened species. The establishment of an Amphibian Ark cryobanking programme offers the first opportunity for global cooperation to achieve the cryobanking of the threatened species from an entire vertebrate class.