Electrical Impedance Spectroscopy Quantifies Skin Barrier Function in Organotypic In Vitro Epidermis Models.

3 D human epidermal equivalents (HEEs) are a state-of-the-art organotypic culture model in pre-clinical investigative dermatology and regulatory toxicology. Here, we investigated the utility of electrical impedance spectroscopy (EIS) for non-invasive measurement of HEE epidermal barrier function. Our setup comprised a custom-made lid fit with 12 electrode pairs aligned on the standard 24-transwell cell culture system. Serial EIS measurements for seven consecutive days did not impact epidermal morphology and readouts showed comparable trends to HEEs measured only once. We determined two frequency ranges in the resulting impedance spectra: a lower frequency range termed EISdiff correlated with keratinocyte terminal differentiation independent of epidermal thickness and a higher frequency range termed EISSC correlated with stratum corneum thickness. HEEs generated from CRISPR/Cas9 engineered keratinocytes that lack key differentiation genes FLG, TFAP2A, AHR or CLDN1 confirmed that keratinocyte terminal differentiation is the major parameter defining EISdiff. Exposure to pro-inflammatory psoriasis- or atopic dermatitis-associated cytokine cocktails lowered the expression of keratinocyte differentiation markers and reduced EISdiff. This cytokine-associated decrease in EISdiff was normalized after stimulation with therapeutic molecules. In conclusion, EIS provides a non-invasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects and repair.

High resolution mass spectrometric suspect screening, wide-scope target analysis of emerging contaminants and determination of legacy pollutants in adult black-tailed godwit Limosa limosa limosa in the Netherlands - A pilot study.

The Dutch breeding population of the black-tailed godwit Limosa limosa limosa has declined substantially over recent decades; the role of contaminants is unknown. We analysed liver samples from 11 adult birds found dead on their breeding grounds in SW Friesland 2016-2020, six from extensive, herb-rich grasslands, five from intensive grasslands. We carried out LC and GC wide-scope target analysis of more than 2400 substances, LC suspect screening for more than 60,000 substances, target analysis for Cd, Hg, Ni and Pb, organo-phosphate flame retardants (OPFRs), dechlorane plus compounds and selected polybrominated diphenyl ether flame retardants (PBDEs), and bioassay for polybrominated dibenzo-p-dioxins and dibenzofurans (PBDDs/PDBFs) and dioxin-like polychlorinated biphenyls (dl-PCBs). Residues of 29 emerging contaminants (ECs) were determined through wide-scope target analysis. Another 20 were tentatively identified through suspect screening. These contaminants include industrial chemicals (personal care products, surfactants, PAHs and others), plant protection products (PPPs) and pharmaceuticals and their transformation products. Total contaminant load detected by wide-scope target analysis ranged from c. 155 to c. 1400 ng g-1 and was generally lower in birds from extensive grasslands. Heatmaps suggest that birds from intensive grasslands have a greater mix and higher residue concentrations of PPPs, while birds from extensive grasslands have a greater mix and higher residue concentrations of per- and polyfluoroalkyl substances (PFAS). All four metals and two OPFRs were detected. All tested PBDEs were below the respective LODs. Bioassay revealed presence of PBDDs, PBDFs and dl-PCBs. Further research is required to elucidate potential health risks to godwits and contaminant sources.

Influence of dissolution on the uptake of bimetallic nanoparticles Au@Ag-NPs in soil organism Eisenia fetida.

A key aspect in the safety testing of metal nanoparticles (NPs) is the measurement of their dissolution and of the true particle uptake in organisms. Here, based on the tendency of Ag-NP to dissolve and Au-NP to be inert in the environment, we exposed the earthworm Eisenia fetida to Au core-Ag shell NPs (Au@Ag-NPs, Ag-NPs with a Au core) and to both single and combined exposures of non-coated Au-NPs, Ag-NPs, Ag+ and Au+ ions in natural soil. Our hypothesis was that the Ag shell would partially or completely dissolve from the Au@Ag-NPs and that the Au core would thereby behave as a tracer of particulate uptake. Au and Ag concentrations were quantified in all the soils, in soil extract and in organisms by inductively coupled plasma mass spectrometry (ICP-MS). The earthworm exposed to Au@Ag-NPs, and to all the combinations of Ag and Au, were analyzed by single particle inductively coupled plasma time-of-flight mass spectrometry (spICP-TOFMS) to allow the quantification of the metals that were truly part of a bimetallic particle. Results showed that only 5% of the total metal amounts in the earthworm were in the bimetallic particulate form and that the Ag shell increased in thickness, suggesting that biotransformation processes took place at the surface of the NPs. Additionally, the co-exposure to both metal ions led to a different uptake pattern compared to the single metal exposures. The study unequivocally confirmed that dissolution is the primary mechanism driving the uptake of (dissolving) metal NPs in earthworms. Therefore, the assessment of the uptake of metal nanoparticles is conservatively covered by the assessment of the uptake of their ionic counterpart.

Persistent Organic Pollutants in two species of migratory birds from Rothera Point, Adelaide Island, Antarctica.

Carcasses of South Polar Skuas (Catharacta maccormicki) and Kelp gulls (Larus dominicanus) were opportunistically collected around of Rothera Research station (67°35'8″S and 68°7'59″W) during the 2016/2017 austral summer. Samples of their tissues (muscle, liver and subcutaneous fat) were analysed for Persistent Organic Pollutants (POPs). Organochlorine pesticides (OCPs) showed the highest concentrations, notably for pp'-DDE and HCB. The Polychlorinated biphenyls (PCBs)-profiles demonstrated a clear dominance of hexa- and hepta-CBs, while concentrations of polybrominated diphenyl ethers (PBDEs) remained low. The concentrations of some POPs (e.g. HCB) were lower than in past studies on similar species, however others were within the previous range (PCBs) or even higher than previous reported values (DDE). Although no major interspecific differences in the absolute concentrations of POPs were detected, their profiles varied, being likely related to feeding and migration patterns of each species. The current study provides important baseline data for future monitoring of POPs in Antarctica.

Tracking pan-continental trends in environmental contamination using sentinel raptors-what types of samples should we use?

Biomonitoring using birds of prey as sentinel species has been mooted as a way to evaluate the success of European Union directives that are designed to protect people and the environment across Europe from industrial contaminants and pesticides. No such pan-European evaluation currently exists. Coordination of such large scale monitoring would require harmonisation across multiple countries of the types of samples collected and analysed-matrices vary in the ease with which they can be collected and the information they provide. We report the first ever pan-European assessment of which raptor samples are collected across Europe and review their suitability for biomonitoring. Currently, some 182 monitoring programmes across 33 European countries collect a variety of raptor samples, and we discuss the relative merits of each for monitoring current priority and emerging compounds. Of the matrices collected, blood and liver are used most extensively for quantifying trends in recent and longer-term contaminant exposure, respectively. These matrices are potentially the most effective for pan-European biomonitoring but are not so widely and frequently collected as others. We found that failed eggs and feathers are the most widely collected samples. Because of this ubiquity, they may provide the best opportunities for widescale biomonitoring, although neither is suitable for all compounds. We advocate piloting pan-European monitoring of selected priority compounds using these matrices and developing read-across approaches to accommodate any effects that trophic pathway and species differences in accumulation may have on our ability to track environmental trends in contaminants.

An overview of existing raptor contaminant monitoring activities in Europe.

Biomonitoring using raptors as sentinels can provide early warning of the potential impacts of contaminants on humans and the environment and also a means of tracking the success of associated mitigation measures. Examples include detection of heavy metal-induced immune system impairment, PCB-induced altered reproductive impacts, and toxicity associated with lead in shot game. Authorisation of such releases and implementation of mitigation is now increasingly delivered through EU-wide directives but there is little established pan-European monitoring to quantify outcomes. We investigated the potential for EU-wide coordinated contaminant monitoring using raptors as sentinels. We did this using a questionnaire to ascertain the current scale of national activity across 44 European countries. According to this survey, there have been 52 different contaminant monitoring schemes with raptors over the last 50years. There were active schemes in 15 (predominantly western European) countries and 23 schemes have been running for >20years; most monitoring was conducted for >5years. Legacy persistent organic compounds (specifically organochlorine insecticides and PCBs), and metals/metalloids were monitored in most of the 15 countries. Fungicides, flame retardants and anticoagulant rodenticides were also relatively frequently monitored (each in at least 6 countries). Common buzzard (Buteo buteo), common kestrel (Falco tinnunculus), golden eagle (Aquila chrysaetos), white-tailed sea eagle (Haliaeetus albicilla), peregrine falcon (Falco peregrinus), tawny owl (Strix aluco) and barn owl (Tyto alba) were most commonly monitored (each in 6-10 countries). Feathers and eggs were most widely analysed although many schemes also analysed body tissues. Our study reveals an existing capability across multiple European countries for contaminant monitoring using raptors. However, coordination between existing schemes and expansion of monitoring into Eastern Europe is needed. This would enable assessment of the appropriateness of the EU-regulation of substances that are hazardous to humans and the environment, the effectiveness of EU level mitigation policies, and identify pan-European spatial and temporal trends in current and emerging contaminants of concern.

Perspectives on One Health: a survey of national Delegates to the World Organisation for Animal Health, 2012.

In 2012, the World Organisation for Animal Health (OIE) surveyed all 178 national Delegates to better understand the perceptions and priorities of Veterinary Services with respect to One Health efforts and to guide future work in that area. For the purposes of this survey, the concept or practice of One Health was defined as 'the intersectoral collaborative approach to preventing, detecting, and controlling diseases among animals and humans, including the collaboration among the institutions and systems that support their prevention, detection and control'. A total of 120 OlE Delegates, representing countries from all five OIE regions, responded to the survey. Delegates identified zoonoses, rabies and influenza as high-priority disease areas and disease reporting and food safety as high-priority programme areas for One Health approaches. Veterinary Services participated in joint programmes, involving these priority disease and programme areas, with national intersectoral partners. Delegates reported barriers to implementing One Health approaches, including a lack of resources, the complexity of collaboration, inadequate capacity within their Veterinary Services, and a lack of adequate legislation, policy, guidance and political will for One Health activities. Delegates supported OIE efforts to enhance One Health activities, and requested that the OIE and partners provide technical information and advice and conduct training and capacity-building seminars for One Health. Veterinary Services cannot effectively apply One Health approaches at the national level without effective partnerships across sectors. The OlE can serve as a model for countries by continuing its commitment to these intersectoral partnerships at the international level.

Organohalogen exposure in a Eurasian Eagle owl (Bubo bubo) population from Southeastern Spain: temporal-spatial trends and risk assessment.

Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and organochlorine insecticides (OCs) were analysed in 58 Eurasian Eagle owl (Bubo bubo) unhatched eggs collected between 2004 and 2009 in Southeastern Spain. Levels of p,p'-DDE were found to be higher than in eggs laid by other European owls in the same decade, probably due to the greater agricultural activity in our study area. Compared to other European raptors, exposure to PCBs can be considered intermediate, but low to PBDEs. Land use differences and prey availability were the rationale to divide the study area in two subareas in further assessments. Temporal trends of HCB, p,p'-DDE, ß-HCH, PCBs and PBDEs were significantly different in each subarea, generally increasing over time in the Southern but decreasing or remaining stable in the Northern. On the contrary, levels of cyclodienes tended to decrease in both subareas. Dietary shifts with a greater amount of birds are suggested as a cause for increasing organochlorine loads in raptors. This may explain the increasing trend in the Southern territories. However, due to the proximity of most of these nests to Cartagena, an important industrial city, increasing environmental pollution cannot be ruled out. Although average levels of the compounds analysed are below threshold levels, 17% of the samples exceeded 400 pg g(-1)ww (wet weight), the LOAEC for Total TEQs. Moreover, a negative correlation between TEQ concentrations and the metabolizable fraction of PCBs (F(prob)=0.0018) was found when TEQs values were above 10 pg g(-1)ww. This could be indicative of hepatic enzymes induction in the birds exposed at higher concentrations, which are mainly breeding in the Southern subarea. These females could be suffering from Ah-receptor-related toxic effects, some of which have been related to altered bird reproduction. Finally, a significant negative correlation between p,p'-DDE levels and eggshell thickness (r=-0.469, p<0.001) was observed, with about 17% of eggshell thinning for eggs with p,p'-DDE levels above 100 µg g(-1)lw. The persistence of this degree of thinning over a period of time has been related to population declines in other raptor species.

Effects of C60 nanoparticle exposure on earthworms (Lumbricus rubellus) and implications for population dynamics.

Effects of C60 nanoparticles (nominal concentrations 0, 15.4 and 154 mg/kg soil) on mortality, growth and reproduction of Lumbricus rubellus earthworms were assessed. C60 exposure had a significant effect on cocoon production, juvenile growth rate and mortality. These endpoints were used to model effects on the population level. This demonstrated reduced population growth rate with increasing C60 concentrations. Furthermore, a shift in stage structure was shown for C60 exposed populations, i.e. a larger proportion of juveniles. This result implies that the lower juvenile growth rate due to exposure to C60 resulted in a larger proportion of juveniles, despite increased mortality among juveniles. Overall, this study indicates that C60 exposure may seriously affect earthworm populations. Furthermore, it was demonstrated that juveniles were more sensitive to C60 exposure than adults.

Herpes simplex virus infection of the central nervous system in human immunodeficiency virus-type 1-infected patients.

We report clinical, radiological and virological data from nine consecutive HIV-infected patients with herpes simplex virus (HSV) infection of the central nervous system (CNS). Three patients presented with confusion, two with fever and headache, two with anxiety and depression, one with slow mentation and memory loss and one with expressive dysphasia. Five patients had previous AIDS-defining diagnoses: four of these five patients had previous cutaneous HSV infection. HSV DNA was detected by the polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) in seven patients. HSV infection was diagnosed by brain biopsy (after negative PCR on CSF) in one patient and at autopsy in one patient (after negative CSF PCR and brain biopsy). Seven patients received specific anti-viral therapy; two died of unrelated causes and the other five recovered. Two patients were not treated, in one the diagnosis was made at autopsy and the other recovered spontaneously. HIV-infected patients with CNS HSV infection have a varied presentation. Diagnosis by PCR on CSF identified the majority of cases. With specific treatment the outcome was good.

Mumps epididymo-orchitis with prolonged detection of virus in semen and the development of anti-sperm antibodies.

Epididymo-orchitis is the most common complication of mumps in post-pubertal men. A case of MMR vaccine failure, in whom mumps and mumps-associated unilateral epididymo-orchitis developed, is presented in this article. Mumps virus was isolated from the semen 14 days after onset and mumps RNA was detected in semen for up to 40 days using RT-PCR. Epididymo-orchitis was associated with transient but significant reduction in sperm count and severe abnormalities in sperm morphology. It also led to the appearance of anti-sperm antibodies, which may have potential long-term adverse effects on the patient's fertility. Sequencing of the SH gene of the virus showed this to be a new mumps genotype distinct from the virus circulating currently in the UK.

The public health response to a case of Lassa fever in London in 2000.

OBJECTIVES: Evaluation of the Department of Health 1996 guidance, the Memorandum on the Management and Control of Viral Haemorrhagic Fevers. METHODS: Description of the public health management in 2000 of the fifth UK patient confirmed to have Lassa fever. RESULTS: Delayed risk categorisation of the patient occurred for a variety of reasons. DH Guidance was followed once infection control advice was sought. Active surveillance of 125 contacts was extremely resource intense, involving over 3000 communications. Self-monitoring by healthcare workers should be considered in future. Advice on use of ribavirin prophylaxis is not included in the Memorandum, nor advice or templates for information sheets for contacts. Information sheets are now available from the Health Protection Agency in the event of future cases. International aspects not adequately addressed include the need for reliable risk assessment to be carried out before patients are medically evacuated from the country of origin, and the steps required to repatriate UK nationals. Effective and efficient communication is required between national and international organisations involved in such incidents. CONCLUSIONS: If guidelines are unclear or impracticable they will not be followed. It is important that lessons are learned and documented and that national guidance be regularly reviewed.

Ecotoxicological suitability of floodplain habitats in The Netherlands for the little owl (Athene noctua vidalli).

This study describes the actual risks of exposure to contaminants, which little owls (Athene noctua vidalli) face in Dutch river floodplains. The results indicate that PCBs pose a risk: not only are levels in little owls from floodplains higher than levels found in little owls from a reference site but the PCB patterns in owls from the floodplains also indicate induction of hepatic cytochrome P450 enzymes by dioxin like compounds, possibly PCBs. Of the heavy metals, only cadmium is thought to pose a risk in certain conditions, for example, when little owls are feeding only on earthworms over a prolonged period of time. The results do not indicate any effects on the occurrence of prey items of the little owl like for instance earthworm, beetles and shrews. Hence, it is not expected that little owls will be affected by diminishing prey availability due to contamination.

Development and evaluation of an internally controlled semiautomated PCR assay for quantification of cell-free cytomegalovirus.

Quantification of circulating human cytomegalovirus (HCMV) is useful in clinical contexts such as virological surveillance of bone marrow transplant recipients and monitoring of antiviral therapy. This report describes an internally controlled, quantitative, semiautomated, HCMV genome assay that was developed primarily to measure HCMV DNA in the plasma of severely leucopaenic patients. It exhibits greater sensitivity, wider dynamic range and higher sample throughput than a number of previously described commercial and "in-house" assays. Viral DNA extraction from EDTA plasma samples was automated using a BioRobot 9604 (Qiagen). HCMV strain AD169 was used to prepare a calibration curve and murine cytomegalovirus (MCMV) strain Smith was added as internal control to all calibration standards and test samples. Amplification was performed using a set of primers based on the HCMV UL50 region, capable of amplifying both human and murine CMV. The yield of biotinylated polymerase chain reaction (PCR) products was estimated using HCMV-specific and MCMV-specific enzyme-labelled probes and automated chemiluminescence detection. Log-transformed HCMV-to-MCMV signal ratios were calculated and used for quantification of test samples against simultaneously extracted MCMV-spiked calibration standards. Evaluation of the assay sensitivity by Probit analysis demonstrated a 95% probability of detection at 100 HCMV genomes per ml of plasma; the dynamic range was shown to be > or = 4 log(10). A total of 315 samples from 61 bone marrow transplant patients were analysed by both the quantitative PCR (qPCR) and by a previously validated nested nonquantitative PCR (NQPCR). A high level of concordance (90%) was observed between the two assays, although the qPCR assay exhibited slightly greater sensitivity.

PCB concentrations and metabolism patterns in common terns (Sterna hirundo) from different breeding colonies in The Netherlands.

PCB levels in blood of common terns (Sterna hirundo) from Terneuzen, a breeding colony in The Netherlands with relatively low breeding success, were significantly higher than in birds from two Dutch reference colonies, one nearby (Oesterdam) and one further away (the Isle of Griend). However, a detailed analysis of the patterns of PCB congeners in birds from the different colonies indicated that birds from Terneuzen and to a lesser extent birds from the Oesterdam showed Ah-receptor mediated activity of P450 iso-enzymes. It is concluded that the induction of this enzyme activity in Terneuzen may be related to PCBs as well as other non-identified compounds. In contrast to this, it is likely that at the Oesterdam non-identified compounds are mainly responsible for the enzyme activity, resulting in a shift of PCB congener patterns.

Varicella zoster virus-associated neurological disease in HIV-infected patients.

Varicella zoster virus (VZV) is an uncommon but well recognized cause of neurological disease in HIV-infected patients. Analysis of cerebrospinal fluid (CSF) using the polymerase chain reaction (PCR) in HIV-infected patients presenting with neurological disease has increasingly allowed diagnosis of VZV-associated pathology. We report clinical, radiological and virological data from 15 consecutive patients with VZV-associated neurological disease. Clinical presentation was varied, including meningo-encephalitis in 9 and isolated cranial nerve palsies in 6. VZV deoxyribonucleic acid (DNA) was detected by PCR in CSF of 11/15; pleocytosis was present in only 6/15, raised protein in 11/15. Magnetic resonance imaging (MRI) appearances were focal signal abnormalities in 8, meningeal enhancement in 2 and normal in 2. With specific anti-VZV therapy 10 patients recovered fully. The predictive value of PCR on CSF for diagnosis of VZV-associated neurological disease should take into account the patient's clinical presentation, concurrent infections and response to anti-VZV therapy.

Prophylaxis with a nevirapine-containing triple regimen after exposure to HIV-1.

Evidence suggests that nevirapine, a non-nucleoside reverse-transcriptase inhibitor, might be very effective in the prevention of HIV-1 integration and the reduction of risk of HIV-1 acquisition after exposure. We used a triple combination regimen, including nevirapine, for prophylaxis after occupational or sexual exposure to HIV-1 infection. Of 57 individuals who started therapy, only 41 returned for follow-up. Five had a grade three or four drug-induced hepatitis, two of whom also had a rash. This high rate of major adverse events raises concerns over the safety of such a regimen for its use in this population.

Evaluation of diagnostic methods for the detection of cytomegalovirus in recipients of allogeneic stem cell transplants.

BACKGROUND: Although several diagnostic methods are available for the surveillance of patients at risk of human cytomegalovirus (CMV) infection and disease, little data is available on their comparative performances in the diagnostic setting. OBJECTIVES: To compare different assays for CMV detection, especially assays based on (quantitative) DNA and mRNA detection. STUDY DESIGN: Eight allogeneic bone marrow and stem cell transplant recipients at high risk for developing CMV disease (donor CMV-negative, recipient positive) were regularly tested for 7-20 weeks post-transplant by spin-amplification rapid culture from urine (viruria), antigenemia (pp65 assay), pp67 mRNA in whole blood (NASBA), and CMV DNA both qualitatively (in-house PCR, whole blood) and quantitatively (in-house PCR, plasma; Cobas Amplicor CMV Monitor Test, plasma and whole blood; Hybrid Capture, whole blood). RESULTS: Four patients (50%) suffered CMV reactivation during follow-up. Out of 104 sample dates, 41 (39.4%) yielded a positive CMV result in at least one assay. Out of the 28 samples tested by all assays, the highest percentage of positive results was obtained with the in-house quantitative PCR (60.7%), followed by the Hybrid Capture system (39.3%), the Cobas Amplicor CMV Monitor Test, plasma version (35.7%), the Cobas Amplicor CMV Monitor Test, whole blood version (32.1%), in-house qualitative PCR (28.6%), and the mRNA assay (21.4%). Viruria was positive in one sample and pp65 antigenemia was found in two samples. CONCLUSIONS: Despite a considerable incidence of CMV reactivations, pre-emptive anti-CMV chemotherapy prevented the development of CMV disease with the exception of one case. The molecular assays had superior sensitivity to conventional ones. The antigenemia assay proved unsuitable for the surveillance of hematological transplant patients. However, none of the tests recognized all timepoints with CMV reactivation. Further comparative studies are needed to determine their respective diagnostic values.

Extended routine polymerase chain reaction surveillance and pre-emptive antiviral therapy for cytomegalovirus after allogeneic transplantation.

Pre-emptive treatment strategies based on sensitive screening for cytomegalovirus (CMV) infection up to day +100 after allogeneic transplantation have been shown to reduce the incidence of CMV disease during the period of surveillance. However, the use of ganciclovir has been associated with delays in immune reconstitution and an increased incidence of late CMV disease after day +100. In the present study, 81 patients undergoing allogeneic transplantation received polymerase chain reaction (PCR)-guided pre-emptive therapy based on detection of CMV DNA by PCR on 2 consecutive weeks up to day +180. Thirty-three of the 52 high-risk patients (CMV-seropositive donor or recipient) received a total of 45 treatment episodes up to day +100. Three of these patients (5.7%) developed CMV disease, with one fatality. Twelve of the surviving 44 high-risk patients (27%) required pre-emptive treatment between days +101 and +192, but none of these patients developed late CMV disease with a median follow-up of 402 d (range 117-952 d). Antiviral therapy was stopped after a single negative PCR result with no subsequent episodes of CMV disease while patients remained off antiviral treatment. As all initial episodes of CMV DNA detection occurred within 60 d of transplantation, it may be possible to discontinue monitoring beyond day +100 in patients who have remained CMV PCR negative before this. Thus, we have confirmed that PCR-guided pre-emptive therapy results in a low incidence of CMV disease before day +100 and that discontinuing treatment on the basis of viral clearance as determined by CMV PCR appears to be safe practice. In addition, we have observed no episodes of late CMV disease with an extension of surveillance to 26 weeks.