Prenatal exposure to per- and polyfluoroalkyl substances and pregnancy outcome in Austria.

Per- and polyfluoroalkyl substances (PFAS) are a large group of persistent industrial chemicals that can harm reproductive health. PFAS levels were analysed to determine the current sources of exposure and possible associations between prenatal PFAS exposure and adverse pregnancy outcome. Samples from 136 mother-newborn pairs recruited between 2017 and 2019 were analysed for the presence of 31 target PFAS in maternal serum, umbilical cord serum, and placental tissue by high-performance liquid chromatography coupled to a tandem mass spectrometer. Questionnaires and medical records were used to survey sources of exposure and pregnancy outcome, including small for gestational age (SGA), fetal growth restriction (FGR), preeclampsia (PE), preterm birth, large for gestational age (LGA) and gestational diabetes mellitus (GDM). Data were analysed for individual PFAS and sum4PFAS (sum of perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS) serum levels) in logistic regression analyses and categorical regression analyses. Compared to data from a previous Viennese study in 2010-12, sum4PFAS levels were generally lower. Sum4PFAS serum levels of three women (2.2%) exceeded 6.9 µg/L, a level that corresponds to the recently established tolerable weekly intake (TWI) of EFSA for nursing mothers aged 35 years; in the 2010/2012 study it was 13.6%. The large contribution of unidentified extractable organofluorine (EOF) fractions to total PFAS exposure is a concern. Study site, mean maternal corpuscular hemoglobin (MCH), use of facial lotion, and owning upholstered furniture were significantly influencing maternal exposure. While no effect of sum4PFAS on pregnancy outcome could be detected, we found highest placental PFDA levels in SGA births. PFHxS levels in umbilical cord and placenta were highest in preterm births. Further studies are needed to elucidate the relationship of prenatal PFAS exposure and pregnancy outcome, in particular to confirm whether and how placental PFDA levels may contribute to an increased risk for SGA.

Intestinal Shedding of SARS-CoV-2 in Children: No Evidence for Infectious Potential.

The clinical courses of COVID-19 in children are often mild and may remain undiagnosed, but prolonged intestinal virus shedding has been documented, thus potentially enabling fecal-oral transmission. However, the infectious potential of SARS-CoV-2 viruses excreted with feces has remained unclear. Here, we investigated 247 stool specimens from 213 pediatric patients to assess the prevalence of intestinal SARS-CoV-2 shedding in hospitalized children without or with COVID-19 and determined the infectious capacity of stool-borne viruses. Upon RT-qPCR screening, the infectivity of virus-positive samples was tested in cell culture using the Vero-E6 permissive cell line. SARS-CoV-2 RNA was detected by RT-qPCR in 32 (13%) stool specimens, but the analysis of virus-positive samples in cell culture revealed no cytopathic effects attributable to SARS-CoV-2-related cell damage. Our findings do not support the notion of potential fecal-oral SARS-CoV-2 spreading, thus questioning the role of hygienic measures designed to prevent this mode of viral transmission.

Extractable Organofluorine Analysis in Pooled Human Serum and Placental Tissue Samples from an Austrian Subpopulation-A Mass Balance Analysis Approach.

Embryos and fetuses are of major concern due to their high vulnerability. Previous studies demonstrated that human exposure to per- and polyfluoroalkyl substances (PFAS) may be underestimated because only a limited number of known PFAS can be measured. This investigation studied the total PFAS exposure by measuring the extractable organofluorine (EOF) in pooled maternal serum, placental tissue, and cord serum samples (total number of pooled samples: n = 45). The EOF was analyzed using combustion ion chromatography, and the concentrations of known PFAS were determined using ultraperformance liquid chromatography coupled with a tandem mass spectrometer. Using a mass balance analysis approach, the amount of unknown PFAS was estimated between the levels of known PFAS and EOF. The EOF levels ranged from 2.85 to 7.17 ng F/mL (21 PFAS were quantified) in the maternal serum, from 1.02 to 1.85 ng F/g (23 PFAS were quantified) in the placental tissue, and from 1.2 to 2.10 ng F/mL (18 PFAS were quantified) in the cord serum. An average of 24, 51, and 9% of EOF is unidentified in the maternal serum, placental tissue, and cord serum, respectively. The results show that the levels of unidentified EOF are higher in the placental tissue, suggesting accumulation or potential transformation of precursors in the placenta.

Amino Acid Transporter LAT1 (SLC7A5) Mediates MeHg-Induced Oxidative Stress Defense in the Human Placental Cell Line HTR-8/SVneo.

The placental barrier can protect the fetus from contact with harmful substances. The potent neurotoxin methylmercury (MeHg), however, is very efficiently transported across the placenta. Our previous data suggested that L-type amino acid transporter (LAT)1 is involved in placental MeHg uptake, accepting MeHg-L-cysteine conjugates as substrate due to structural similarity to methionine. The aim of the present study was to investigate the antioxidant defense of placental cells to MeHg exposure and the role of LAT1 in this response. When trophoblast-derived HTR-8/SVneo cells were LAT1 depleted by siRNA-mediated knockdown, they accumulated less MeHg. However, they were more susceptible to MeHg-induced toxicity. This was evidenced in decreased cell viability at a usually noncytotoxic concentration of 0.03 µM MeHg (~6 µg/L). Treatment with ≥0.3 µM MeHg increased cytotoxicity, apoptosis rate, and oxidative stress of HTR-8/SVneo cells. These effects were enhanced under LAT1 knockdown. Reduced cell number was seen when MeHg-exposed cells were cultured in medium low in cysteine, a constituent of the tripeptide glutathione (GSH). Because LAT1-deficient HTR-8/SVneo cells have lower GSH levels than control cells (independent of MeHg treatment), we conclude that LAT1 is essential for de novo synthesis of GSH, required to counteract oxidative stress. Genetic predisposition to decreased LAT1 function combined with MeHg exposure could increase the risk of placental damage.

Human placental cell line HTR-8/SVneo accumulates cadmium by divalent metal transporters DMT1 and ZIP14.

Cadmium (Cd) is a global pollutant that accumulates in the placenta and can cause placental dysfunction. Although iron transporters have been suggested to participate in placental Cd uptake, it is still unknown which transporters are actually involved in this process. We specifically aimed to study the role of three iron transporters in the uptake of Cd into the placental cell line HTR-8/SVneo. For this purpose, Divalent Metal Transporter (DMT)1 and ZRT/IRT like protein (ZIP)8 and ZIP14 were downregulated and changes in cellular Cd levels analysed in relation to controls. As clearly shown by the reduction of the Cd content by ∼60% in DMT1- and ZIP14-downregulated cells, the two proteins are essential for Cd accumulation in HTR-8/SVneo cells. Using a validated antibody, we show DMT1 to be localised in situ in trophoblast and stromal cells. We further wanted to investigate how placental cells cope with Cd loading and which metallothionein (MT) isoforms they express. Cd-exposed cells accumulate Cd in a dose-dependent manner and upregulate MT2A accordingly (up to 15-fold induction upon 5 µM CdCl2 treatment for 72 h). 5 µM Cd exposure for 72 h decreased cell number to 60%, an effect that was aggravated by MT2A depletion (cell number reduced to 30%) indicating additive effects. In conclusion, our data suggest that DMT1 and ZIP14 are required for Cd uptake into human placental cells that upregulate MT2A to store and detoxify the metal. Cd storage in the placenta reduces Cd transport to the fetus, which, however, could impair placental functions and fetal development.

In vitro function and in situ localization of Multidrug Resistance-associated Protein (MRP)1 (ABCC1) suggest a protective role against methyl mercury-induced oxidative stress in the human placenta.

Methyl mercury (MeHg) is an organic highly toxic compound that is transported efficiently via the human placenta. Our previous data suggest that MeHg is taken up into placental cells by amino acid transporters while mercury export from placental cells mainly involves ATP binding cassette (ABC) transporters. We hypothesized that the ABC transporter multidrug resistance-associated protein (MRP)1 (ABCC1) plays an essential role in mercury export from the human placenta. Transwell transport studies with MRP1-overexpressing Madin-Darby Canine Kidney (MDCK)II cells confirmed the function of MRP1 in polarized mercury efflux. Consistent with this, siRNA-mediated MRP1 gene knockdown in the human placental cell line HTR-8/SVneo resulted in intracellular mercury accumulation, which was associated with reduced cell viability, accompanied by increased cytotoxicity, apoptosis, and oxidative stress as determined via the glutathione (GSH) status. In addition, the many sources claiming different localization of MRP1 in the placenta required a re-evaluation of its localization in placental tissue sections by immunofluorescence microscopy using an MRP1-specific antibody that was validated in-house. Taken together, our results show that (1) MRP1 preferentially mediates apical-to-basolateral mercury transport in epithelial cells, (2) MRP1 regulates the GSH status of placental cells, (3) MRP1 function has a decisive influence on the viability of placental cells exposed to low MeHg concentrations, and (4) the in situ localization of MRP1 corresponds to mercury transport from maternal circulation to the placenta and fetus. We conclude that MRP1 protects placental cells from MeHg-induced oxidative stress by exporting the toxic metal and by maintaining the placental cells' GSH status in equilibrium.

Don't trust an(t)ybody - Pitfalls during investigation of candidate proteins for methylmercury transport at the placental interface.

While investigating placental mercury transport, we validated specificity of commercial antibodies against four candidate transporters (Large neutral amino acids transporter (LAT)1, LAT2, 4F2 cell-surface antigen heavy chain (4F2hc), and multidrug resistance-associated protein (MRP)2) by immunoblotting and small interfering RNA (siRNA)-mediated protein knockdown. An anti-4F2hc- and one anti-LAT1-antibody were specific. Another anti-LAT1-antibody reacted with LAT2. Two anti-LAT2-antibodies detected mainly albumin in placental lysates. A specific anti-MRP2-antibody hardly detected MRP2 in human placentas, contradicting published data. We recommend testing any unknown antibody by western blotting for 1/specificity for the protein of interest using e.g. siRNA knockdown and 2/cross-reactivity with albumin.

Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters.

BACKGROUND: The capacity of the human placenta to handle exogenous stressors is poorly understood. The heavy metal mercury is well-known to pass the placenta and to affect brain development. An active transport across the placenta has been assumed. The underlying mechanisms however are virtually unknown. OBJECTIVES: Uptake and efflux transporters (17 candidate proteins) assumed to play a key role in placental mercury transfer were examined for expression, localization and function in human primary trophoblast cells and the trophoblast-derived choriocarcinoma cell line BeWo. METHODS: To prove involvement of the transporters, we used small interfering RNA (siRNA) and exposed cells to methylmercury (MeHg). Total mercury contents of cells were analyzed by Cold vapor-atomic fluorescence spectrometry (CV-AFS). Localization of the proteins in human term placenta sections was determined via immunofluorescence microscopy. RESULTS: We found the amino acid transporter subunits L-type amino acid transporter (LAT)1 and rBAT (related to b(0,+) type amino acid transporter) as well as the efflux transporter multidrug resistance associated protein (MRP)1 to be involved in mercury kinetics of trophoblast cells (t-test P<0.05). CONCLUSION: The amino acid transporters located at the apical side of the syncytiotrophoblast (STB) manage uptake of MeHg. Mercury conjugated to glutathione (GSH) is effluxed via MRP1 localized to the basal side of the STB. The findings can well explain why mercury is transported primarily towards the fetal side.

Foreign body aspiration - Sometimes a tough nut to crack.

Foreign body aspiration (FBA) is a dangerous and potentially life-threatening event. We report the case of a 24-month old boy, who was initially presented with an episode of obstructive bronchitis to the family pediatrician. Then, while being treated with empiric antibiotics, he aspirated a peanut. Although resulting in a coughing episode, the mother did initially not ascribe any relevancy to it. Since the diagnosis of obstructive bronchitis had already been established, only an in-depth history taking session with the mother could help figure out, why the boy's symptoms got worse instead of better. This article underlines the importance of accurate history taking and clinical examination.

Reversible cytotoxic oedema in the splenium of the corpus callosum related to tetracycline therapy.

We report a symptomatic girl with reversible circumscribed cytotoxic oedema in the splenium of the corpus callosum (CC) that occurred, to our knowledge, for the first time in relation to tetracycline treatment. After stopping tetracycline therapy the girl recovered completely and the CC lesion, clearly visible on diffusion-weighted MR imaging (DWI), disappeared. Reversible circumscribed cytotoxic oedema (CCO) of the splenium of the CC is a well-defined entity that is found to be associated with administration of antiepileptic drugs, alterations in therapy using arginin-vasopressin and metronidazole or infections with influenza and rotavirus. CCO of splenium of the CC is clearly visible on DWI, shows no enhancement after administration of contrast medium and is completely reversible in most cases.

Smoking, cereal consumption, and supplementation affect cadmium content in breast milk.

Breast milk might be a source of potentially toxic metals such as cadmium (Cd). The purpose of the present study is to provide data pertaining to the influence of maternal lifestyles on Cd concentrations in breast milk in the Austrian setting. Breast milk was obtained from 124 Austrian women. Each participant provided 10 ml of milk. A second group of eight mothers were recruited to investigate changes in their milk cadmium levels at 1, 3, 5, 7 and 9 weeks post partum. The study participants filled a questionnaire concerning nutrition, supplementation, and smoking habits. The samples were analyzed using GF-Atomic Absorption Spectrophotometer (AAS). The mean Cd content in breast milk was among the lowest in Europe (0.086 +/- 0.085 microg/l, 95% CI: 0.07-0.10; n = 124). Increased Cd levels in breast milk were found to be significantly associated with frequent cereal consumption and smoking. Smokers had a two-fold higher concentration than did non-smokers (0.15 versus 0.07 microg/l; P = 0.000). In contrast, low Cd levels in breast milk were associated with the intake of supplements containing trace elements or vitamins and trace elements (P < 0.05). This protective effect of supplements on Cd levels was only observed in non-smokers. The Cd levels registered in the present investigation were far below critical levels. We conclude that the current maternal Cd levels in Austria signify no risk for the breastfed infant of a healthy mother. Further research will have to focus on the specific effects of supplementation and smoking on Cd concentrations.

Mortality and morbidity in extremely preterm infants (22 to 26 weeks of gestation): Austria 1999-2001.

OBJECTIVE: The aim of this retrospective study was to analyze the mortality and morbidity for extremely preterm infants with a gestational age from 22 to 26 weeks. All infants were born in Austria during the years 1999-2001. METHODS: Data were collected from 16 neonatal intensive care units in Austria. Main outcome criteria were mortality, the rates of chronic lung disease (CLD) and severe retinopathy of prematurity (ROP, stage > or =3) to determine the short-term outcome; the rate of cerebral palsy (CP) at the corrected age of twelve months to assess the long-term outcome. RESULTS: Overall, 796 preterm infants with a gestational age less than 27 weeks were born in Austria and 581 (73%) were registered as live-born infants. Of those live born, 508 (87%) were analyzed. The mortality rates were 83%, 76%, 43%, 26% and 13% for 22, 23, 24, 25 and 26 weeks' gestation, respectively. The rates of CLD were 33% (22 weeks), 36% (23 weeks), 42% (24 weeks), 31% (25 weeks) and 22% (26 weeks). The rates of ROP of stage > or =3 were 0% (22 weeks), 29% (23 weeks), 23% (24 weeks), 18% (25 weeks) and 10% (26 weeks). The rates of CP at the corrected age of 12 months were 33%, 50%, 33%, 26% and 25% for 22, 23, 24, 25 and 26 weeks' gestation, respectively. CONCLUSIONS: The results of this national study are in accordance with the international literature: mortality and morbidity increased with decreasing gestational age.

Tick-borne encephalitis in childhood--consensus 2004.

Tick-borne encephalitis (TBE) is a communicable disease caused by a flavi-virus, ticks being the main vectors. The nervous system is affected, four clinical features of different severity are observed: meningitis, meningoencephalitis, meningoencephalomyelitis, meningoradiculoneuritis. TBE is a preventable disease, which is rapidly becoming a growing public health problem in Europe. So far no causal treatment is possible but an efficient, safe vaccination is available. During the 6th meeting of the International Scientific Working Group on TBE with the main conference issue "Tick-borne encephalitis in childhood" an international consensus was achieved. In countries where TBE is endemic--and not prevented by immunization--both children and adults are affected. The disease in children is generally milder, although severe illness may occur and even lead to permanent impairment of the quality of life due to neuropsychological sequelae. Therefore immunization should be offered to all children living in or traveling to endemic areas.

Lead and mercury in breast milk.

OBJECTIVE: Heavy metals are potentially toxic substances, especially for the susceptible infant. Exposure to mercury (Hg) and lead (Pb) may result in neurotoxic and nephrotoxic impairment and in anemia. Previous data on breast milk Pb and Hg contents are sparse or missing for the Austrian population. No evaluations of the influence of mothers' lifestyles on Pb and Hg levels in breast milk are available. METHODS: Five- to 10-mL individual samples of breast milk were provided from healthy mothers in Vienna (urban; n = 59), Linz (industrial; n = 47), and Tulln (rural; n = 59). A questionnaire about area of residence, maternal nutrition, smoking habits, and dental fillings was filled out by the lactating mothers. Milk samples and infant formulas were lyophilized, wet-ashed with nitric acid (65%), and analyzed with atomic absorption spectrophotometry. Spiked skim milk powder was used as reference material. Statistical analysis included the Kruskal-Wallis test and multiple robust regression analysis. RESULTS: Breast milk showed low Hg and Pb concentrations (Hg: 1.59 +/- 1.21 1g/l, n = 116; Pb: 1.63 +/- 1.66 6g/l, n = 138). Eight percent of the breast milk samples marginally exceeded the screening level of 3.5 micro g/L for Hg. Austrian Pb values declined strongly during the last 20 years. Bivariate comparison revealed that the factors significantly related to metal levels in breast milk were area of residence (Hg, Pb), prematurity (Hg), consumption of fish (Pb) and cereals (Hg), vitamin supplementation (Hg), and smoking (Pb). The Hg and Pb contents of cow milk and infant formulas were far below respective guideline values. CONCLUSIONS: Neither Hg nor Pb concentrations exceeded critical levels. There are no reports on infants harmed by the intake of milk from unexposed mothers. We conclude that even theoretical risks from current Hg or Pb levels for the breastfed infant of a healthy mother can be ruled out.