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Background Assessing the safety of engineered nanomaterials (ENMs) is an interdisciplinary and complex process producing huge amounts of information and data. To make such data and metadata reusable for researchers, manufacturers, and regulatory authorities, there is an urgent need to record and provide this information in a structured, harmonized, and digitized way. Results This study aimed to identify appropriate description standards and quality criteria for the special use in nanosafety. There are many existing standards and guidelines designed for collecting data and metadata, ranging from regulatory guidelines to specific databases. Most of them are incomplete or not specifically designed for ENM research. However, by merging the content of several existing standards and guidelines, a basic catalogue of descriptive information and quality criteria was generated. In an iterative process, our interdisciplinary team identified deficits and added missing information into a comprehensive schema. Subsequently, this overview was externally evaluated by a panel of experts during a workshop. This whole process resulted in a minimum information table (MIT), specifying necessary minimum information to be provided along with experimental results on effects of ENMs in the biological context in a flexible and modular manner. The MIT is divided into six modules: general information, material information, biological model information, exposure information, endpoint read out information and analysis and statistics. These modules are further partitioned into module subdivisions serving to include more detailed information. A comparison with existing ontologies, which also aim to electronically collect data and metadata on nanosafety studies, showed that the newly developed MIT exhibits a higher level of detail compared to those existing schemas, making it more usable to prevent gaps in the communication of information. Conclusion Implementing the requirements of the MIT into e.g., electronic lab notebooks (ELNs) would make the collection of all necessary data and metadata a daily routine and thereby would improve the reproducibility and reusability of experiments. Furthermore, this approach is particularly beneficial regarding the rapidly expanding developments and applications of novel non-animal alternative testing methods.

Background Metachromatic leukodystrophy (MLD) is a lysosomal enzyme deficiency disorder leading to progressive demyelination and, consecutively, to cognitive and motor decline. Brain magnetic resonance imaging (MRI) can detect affected white matter as T2 hyperintense areas but cannot quantify the gradual microstructural process of demyelination more accurately. Our study aimed to investigate the value of routine MR diffusion tensor imaging in assessing disease progression. Methods MR diffusion parameters (apparent diffusion coefficient [ADC] and fractional anisotropy [FA]) were in the frontal white matter, central region (CR), and posterior limb of the internal capsule in 111 MR datasets from a natural history study of 83 patients (age: 0.5–39.9 years; 35 late-infantile, 45 juvenile, 3 adult, with clinical diffusion sequences of different scanner manufacturers) as well as 120 controls. Results were correlated with clinical parameters reflecting motor and cognitive function. Results ADC values increase and FA values decrease depending on disease stage/severity. They show region-specific correlations with clinical parameters of motor and cognitive symptoms, respectively. Higher ADC levels in CR at diagnosis predicted a disease course with more rapid motor deterioration in juvenile MLD patients. In highly organized tissues such as the corticospinal tract, in particular, diffusion MR parameters were highly sensitive to MLD-associated changes and did not correlate with the visual quantification of T2 hyperintensities. Conclusion Our results show that diffusion MRI can deliver valuable, robust, clinically meaningful, and easily obtainable/accessible/available parameters in the assessment of prognosis and progression of MLD. Therefore, it provides additional quantifiable information to established methods such as T2 hyperintensity.

Aim The study aims to describe cerebral MRI in different onset forms of Niemann-Pick type C (NPC). Systematic MRI analyses in this rare lysosomal storage disease are lacking in the infantile and juvenile onset forms. Methods Thirty-two cerebral MRI scans from 19 patients with NPC were assessed using a newly established and validated scoring system which addresses white matter changes and supratentorial versus infratentorial atrophy. Results Seven scans were from six NPC patients with early infantile onset (15 years). While supratentorial atrophy was the leading sign in the infantile groups, the juvenile and adult forms were characterized by both, infra- and supratentorial atrophy. White matter changes were found in nearly every patient; they increased with the disease duration in the earlier forms and were prominent in the later forms already early in the disease course. Conclusion This is the first systematic and comparative MRI analysis in the different onset groups of NPC using a scoring system. Early during disease course, MRI showed different patterns in infantile compared with juvenile and adult onset NPC patients, for example, only supratentorial atrophy in juvenile versus global atrophy in adult onset patients. MRI changes provide an additional, early biomarker for NPC.

Introduction Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials. Materials and methods In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy. Results Spheroids were exposed to 100 µg mL-1 SiO2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO2 NPs during the process of spheroid formation had no impact on final spheroid size. Conclusion Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation.

The intestinal microvasculature (iMV) plays multiple pathogenic roles during chronic inflammatory bowel disease (IBD). The iMV acts as a second line of defense and is, among other factors, crucial for the innate immunity in the gut. It is also the therapeutic location in IBD targeting aggravated leukocyte adhesion processes involving ICAM-1 and E-selectin. Specific targeting is stressed via nanoparticulate drug vehicles. Evaluating the iMV in enterocyte barrier models in vitro could shed light on inflammation and barrier-integrity processes during IBD. Therefore, we generated a barrier model by combining the enterocyte cell line Caco-2 with the microvascular endothelial cell line ISO-HAS-1 on opposite sides of a transwell filter-membrane under culture conditions which mimicked the physiological and inflamed conditions of IBD. The IBD model achieved a significant barrier-disruption, demonstrated via transepithelial-electrical resistance (TER), permeability-coefficient (Papp) and increase of sICAM sE-selectin and IL-8. In addition, the impact of a prospective model drug-vehicle (silica nanoparticles, aSNP) on ongoing inflammation was examined. A decrease of sICAM/sE-selectin was observed after aSNP-exposure to the inflamed endothelium. These findings correlated with a decreased secretion of ICAM/E-selectin bearing exosomes/microvesicles, as evaluated via ELISA. Our findings indicate that aSNP treatment of the inflamed endothelium during IBD may hamper exosomal/microvesicular systemic communication.

Jennifer Y Kasper,1 Maria Iris Hermanns,1 Christian Cavelius,2 Annette Kraegeloh,2 Thomas Jung,3 Rolf Danzebrink,3 Ronald E Unger,1 Charles James Kirkpatrick1 1Institute of Pathology, University Medical Center, Mainz, 2Leibniz Institute for New Materials, 3NanoGate AG, Goettelborn, Saarbrücken, Germany Abstract: The microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1). Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin) after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-β) and a nanoparticle (Ba/Gd: coprecipitated BaSO4 and Gd(OH)3), generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (Papp) of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the Papp decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-β, and Ba/Gd). Both the endothelial and epithelial monocultures showed a high inflammatory response in both the upper and lower transwell-compartments. However, in the coculture, inflammatory mediators were only detected on the epithelial side and not on the endothelial side. Thus in the coculture, based on the Papp, the epithelial barrier appears to prevent a potential inflammatory overreaction in the underlying endothelial cells. In summary, this coculture model exhibits in vivo-like features, which cannot be observed in conventional monocultures, making the former more suitable to study interactions with external stimuli. Keywords: intestinal microvasculature, inflammatory bowel disease, intestinal barrier in vitro, Caco-2, ISO-HAS-1, soluble E-selectin, sICAM-1, nanosized gadolinium contrast agent

This paper reports a rapid HILIC-ESI-MS assay to quantify dipalmitoylphosphatidylcholine (DPPC) as component of lung surfactant for nanosafety studies. The technique was used to investigate the concentration-dependent sorption of DPPC to two-sizes of amorphous SiO2 nanoparticles (SiO2-NPs) in a MeOH:H2O (50/50v/v) mixture and in cell culture medium. In MeOH:H2O (50/50v/v), the sorption of DPPC was positively correlated with the nanoparticles concentration. A substantial affinity of small amorphous SiO2-NPs (25nm) to DPPC standard solution compared to bigger SiO2-NPs (75nm) was not confirmed for biological specimens. After dispersion of SiO2-NPs in DPPC containing cell culture medium, the capacity of the SiO2-NPs to bind DPPC was reduced in comparison to a mixture of MeOH:H2O (50/50v/v) regardless from the nanoparticles size. Furthermore, HILIC-ESI-MS revealed that A549 cells internalized DPPC during growth in serum containing medium complemented with DPPC. This finding was in a good agreement with the potential of alveolar type II cells to recycle surfactant components. Binding of lipids present in the cell culture medium to amorphous SiO2-NPs was supported by means of HILIC-ESI-MS, TEM and ICP-MS independently.

Inflammatory activation of alveolar macrophages by ambient particles can be facilitated via Toll-like receptors (TLR). The action of TLR agonists and antagonists has been reported to depend on the formation of nanoparticulate structures. Aim of the present study was to identify the signaling pathways induced by nanoparticulate structures in human macrophages, which might be critical for inflammatory cell activation. Methods Studies were performed in primary human alveolar macrophages or in differentiated THP-1 macrophages. Silica nanoparticles were prepared by Stober synthesis and characterized by dynamic light scattering and scanning electron microscopy. Mycobacterial DNA was isolated from Mycobacterium bovis BCG, and nanoparticle formation was assessed by atomic force microscopy and dynamic light scattering. Actin polymerization was measured by phalloidin–TRITC staining, and cell activation was determined by reverse transcription quantitative PCR analysis, L929 cytotoxicity assay (cytokine induction), and pull-down assays (Rho GTPases). Results In contrast to immune stimulatory sequence ISS 1018, BCG DNA spontaneously formed nanoparticulate structures and induced actin polymerization as did synthetic silica nanoparticles. Co-incubation with silica nanoparticles amplified the responsiveness of macrophages toward the TLR9 ligand ISS 1018. The activation of Rac1 was induced by silica nanoparticles as well as BCG DNA and is suggested as the critical signaling event inducing both cytoskeleton changes as well as inflammatory cell activation. Conclusion Nanoparticles can induce signaling pathways, which amplify an inflammatory response in macrophages.

Superparamagnetic iron oxide nanoparticles (SPION) are widely used both clinically and experimentally for diverse in vivo applications, such as contrast enhancement in magnetic resonance imaging, hyperthermia and drug delivery. Biomedical applications require particles to have defined physical and chemical properties, and to be stable in biological media. Despite a suggested low cytotoxic action, adverse reactions of SPION in concentrations relevant for biomedical use have not yet been studied in sufficient detail. In the present work we employed Endorem®, dextran-stabilized SPION approved as an intravenous contrast agent, and compared its action to a set of other nanoparticles with potential for magnetic resonance imaging applications. SPION in concentrations relevant for in vivo applications were rapidly taken up by endothelial cells and exhibited no direct cytotoxicity. Electric cell impedance sensing measurements demonstrated that SPION, but not BaSO4/Gd nanoparticles, impaired endothelial integrity, as was confirmed by increased intercellular gap formation in endothelial monolayers. These structural changes induced the subcellular translocation and inhibition of the cytoprotective and anti-atherosclerotic enzyme endothelial NO-synthase and reduced NO production. Lipopolysaccharide-induced inflammatory NO production of macrophages was not affected by SPION. In conclusion, our data suggest that SPION might substantially alter endothelial integrity and function at therapeutically relevant doses, which are not cytotoxic.

Attention deficit with or without hyperactivity (AD[H]D) is a common comorbidity of neurofibromatosis type 1 (NF 1). We tested the hypothesis that permanent medication with methylphenidate can improve cognitive functioning in children with NF 1 and comorbid AD(H)D. We retrospectively analyzed data of a clinical sample of patients with NF 1 with or without AD(H)D, who underwent standardized neuropsychological diagnostics twice (age range: T1, 6-14 years; T2, 7-16 years; mean interval, 49.09 months). A total of 16 children without AD(H)D (nine females) were compared with 14 unmedicated children with AD(H)D (eight females) and to 13 medicated children with AD(H)D (two females). Effects of medication and attention on cognitive outcome (IQ) were tested by repeated measures analysis of covariance (rmANCOVA). Medicated children with NF 1 improved significantly in full-scale IQ from T1 to T2 (IQ[T1] = 80.38, IQ[T2] = 98.38, confidence interval [diff]: -25.59 to -10.40, p 0.0001), this effect was not evident for the other groups. With attention measures as covariates, the effect remained marginally significant. Children and adolescents with NF 1 and comorbid AD(H)D may profit from MPH medication regarding general cognition. This effect could be specific for the group of patients with NF 1, and cannot be explained solely by improvements in attention. Controlled, prospective studies are warranted to corroborate our findings.

Ventriculitis may complicate neurosurgical procedures, for example, due to shunt or external ventricular drainage infection. Clearance of the infection with subsequent intravenous antibiotic therapy and shunt reinsertion, if necessary, are the standard treatment procedures with a high rate of success. Here, we report on a protracted complication, the development of destructive subependymal cysts, illustrate its treatment and discuss the pathomechanisms. The 2-year-and-9-month-old girl was admitted 5 weeks after a shunt revision with symptoms of shunt infection. Ventriculitis caused by Streptococcus salivarius (S. salivarius) was diagnosed and intravenous antibiotic treatment was performed. A new shunt system was implanted after clearance of infection and the girl did not show clinical signs of infection thereafter. A routine follow-up magnetic resonance imaging (MRI) revealed progressive and space-occupying multifocal subependymal cysts with partial destruction of the corpus callosum including compression of the ventricular system. Endoscopic broad-based laser fenestration of all cysts resulted in sustained regression of cavity formation. The cognitive development of the girl assessed 2 years afterward was completely normal. We conclude that routine follow-up MRI investigations are recommended 6 months after successful treatment of ventriculitis to detect protracted postinflammatory destructive subependymal cyst formations. Endoscopic broad-based laser-assisted cyst fenestration can stop progression and lead to regression of postinfectious subependymal cysts.

Michael Busse,1 David Stevens,3 Annette Kraegeloh,2 Christian Cavelius,2 Mathias Vukelic,1 Eduard Arzt,2 Daniel J Strauss1,2 1Systems Neuroscience and Neurotechnology Unit, Saarland University, Faculty of Medicine, Neurocenter, and Saarland University of Applied Sciences, Homburg/Saarbruecken, Germany; 2Leibniz Institute for New Materials, Saarbruecken, Germany; 3Department of Physiology, Saarland University, Faculty of Medicine, Homburg/Saarbruecken, Germany Background: Beside the promising application potential of nanotechnologies in engineering, the use of nanomaterials in medicine is growing. New therapies employing innovative nanocarrier systems to increase specificity and efficacy of drug delivery schemes are already in clinical trials. However the influence of the nanoparticles themselves is still unknown in medical applications, especially for complex interactions in neural systems. The aim of this study was to investigate in vitro effects of coated silver nanoparticles (cAgNP) on the excitability of single neuronal cells and to integrate those findings into an in silico model to predict possible effects on neuronal circuits. Methods: We first performed patch clamp measurements to investigate the effects of nanosized silver particles, surrounded by an organic coating, on excitability of single cells. We then determined which parameters were altered by exposure to those nanoparticles using the Hodgkin–Huxley model of the sodium current. As a third step, we integrated those findings into a well-defined neuronal circuit of thalamocortical interactions to predict possible changes in network signaling due to the applied cAgNP, in silico. Results: We observed rapid suppression of sodium currents after exposure to cAgNP in our in vitro recordings. In numerical simulations of sodium currents we identified the parameters likely affected by cAgNP. We then examined the effects of such changes on the activity of networks. In silico network modeling indicated effects of local cAgNP application on firing patterns in all neurons in the circuit. Conclusion: Our sodium current simulation shows that suppression of sodium currents by cAgNP results primarily by a reduction in the amplitude of the current. The network simulation shows that locally cAgNP-induced changes result in changes in network activity in the entire network, indicating that local application of cAgNP may influence the activity throughout the network. Keywords: coated silver nanoparticles, modeling, patch clamp recordings, neuronal circuit model, neuromodulatory effect, nanocarriers, nonviral vectors, Llinás model

Long-term follow-up data on preterm infants with breast milk-acquired postnatal cytomegalovirus (CMV) infection are sparse.To systematically evaluate the long-term cognitive outcome and prevalence of cerebral palsy (CP) in patients after postnatal CMV infection.All surviving infants1500 g born in our centre between 1 June 1995 and 1 June 2000, and with postnatal CMV infection acquired at up to 3 months of corrected age, were eligible for our study; this included neurological and neurocognitive assessment, using the Kaufman Assessment Battery for Children (K-ABC) at the age of4 years. A blinded and controlled matched-pairs design was used with gestational age, gender and date of birth as matching criteria.Of 50 eligible children, 42 (84%) could be tested. There was no difference in the prevalence of cerebral palsy. Following CMV infection during their hospital stay, infants had significantly lower results in the simultaneous processing scale of the K-ABC (p=0.029) after correction for additional risk factors like socioeconomic status (SES). Results for the sequential and achievement scales were only slightly reduced (p0.05).It seems possible that breast milk-acquired CMV infection has a detrimental influence on cognitive development of preterm infants.

Fucosidosis is a rare autosomal recessive lysosomal storage disease, resulting from a deficiency of alpha- L-fucosidase. We report on the clinical and MRI findings of a girl with this disorder. Developmental delay became obvious at an age between 6 and 12 months. Cranial MRI at 16 months revealed severe global hypomyelination of both supra- and infratentorial white matter but no involvement of basal ganglia or thalamus. No clinical signs typical for fucosidosis were present at this time, and psychomotor development still progressed slowly. Since the age of 2 years, progressive neurological deterioration occurred. The diagnosis was established by severely decreased activity of alpha- L-fucosidase in plasma and leukocytes and confirmed by the detection of compound heterozygosity for two missense mutations of the FUCA1 gene. A follow-up imaging at the age of 4 years showed progression of neuroradiological abnormalities, particularly progressive involvement of basal ganglia and thalami. The course of this patient and her MRI findings enlarge the clinical and neuroradiological spectrum of fucosidosis.

Objective Niemann Pick Type C (NPC) is a rare autosomal-recessive, lysosomal disorder. The diagnosis is often delayed because of non-specific first clinical symptoms and the lack of easily accessible biomarkers. There are no systematic descriptions of MRI in NPC of infantile and juvenile onset. Methods We collected 30 MRI-brain-scans from 18 patients with NPC. We established a scoring-system for MRI in NPC which addresses white-matter-changes (4 points for periventricular and central involvement in the frontal and par.-occ. lobes) and supra- (3 points for enlargement of internal, external spaces and thin corpus callosum) as well as infratentorial atrophy (3 points for mild, severe vermis atrophy and hemispheric atrophy). MRIs were evaluated in 4 age groups: early infantile ≤2 yrs, infantile 2–6 yrs, juvenile 6–15 yrs, adult >15 yrs. Results White-matter-changes were found in nearly every patient. Localization was occipital and central early in the disease which then extended to more periventricular and frontal changes. Atrophy was the characteristic feature in all patients. In both infantile groups (10 scans) supratentorial atrophy was predominant, also during disease course of up to 6 years. Whereas juvenile patients (8 scans) were more characterized by infratentorial atrophy and the adult group (12 scans) showed a combination of infratentorial and supratentorial atrophy even early during disease course. Conclusion For the first time we show systematic descriptions of MRI in infantile and juvenile patients with NPC in comparison to patients with adult onset. Subtle white-matter-changes were a common and early finding, but atrophy seems to be the more distinct feature. It was seen early during disease course clearly differing between the age groups. Early onset (

contact and the ability to walk; kept slight right upper limb hypertonia and occasional chorea. Lab results: minor pleocytosis (11 cells/mm3) on 1st CSF examination, normal CSF immunoelectrophoresis and normal neurotransmitters profile; negative auto-antibodies and complement fractions study; normal extensive metabolic and infectious investigations. 1st MR (D5) normal; 2nd MR (D13) showed T2 hypersignal on cerebellar cortex; 4th MR (D40) revealed atrophy of cerebellar hemispheres and slight cerebral diffuse atrophy. Recently, anti NMDAR antibodies sampling in frozen CSF from 1st lumbar puncture evidenced strong positivity; this study was repeated in blood and CSF, confirming strong positivity. Investigations to exclude occult neoplasia, were negative. The patient was treated with high dose steroids and intravenous imunne globulin. Conclusion: anti-NMDAR auto-immune encephalitis was described recently. Very few cases were reported in children. Our patient is one of the youngest. This disorder should be considered in the differential diagnosis of encephalitis with prominent psychiatric and extrapyramidal symptoms. This disorder, though severe, is frequently associated with a good therapeutic response.

Background Exposure of the airways to carbonaceous nanoparticles can contribute to the development of immune diseases both via the aggravation of the allergic immune response in sensitized individuals and by adjuvant mechanisms during the sensitization against allergens. The cellular and molecular mechanisms involved in these adverse pathways are not completely understood. We recently described that the reduction of carbon nanoparticle-induced lung inflammation by the application of the compatible solute ectoine reduced the aggravation of the allergic response in an animal system. In the current study we investigated the influence of carbon nanoparticles on the sensitization of animals to ovalbumin via the airways. Ectoine was used as a preventive strategy against nanoparticle-induced neutrophilic lung inflammation. Methods Balb/c mice were repetitively exposed to the antigen ovalbumin after induction of airway inflammation by carbon nanoparticles, either in the presence or in the absence of ectoine. Allergic sensitization was monitored by measurement of immunoglobulin levels and immune responses in lung and lung draining lymph nodes after challenge. Furthermore the role of dendritic cells in the effect of carbon nanoparticles was studied in vivo in the lymph nodes but also in vitro using bone marrow derived dendritic cells. Results Animals exposed to antigen in the presence of carbon nanoparticles showed increased effects with respect to ovalbumin sensitization, to the allergic airway inflammation after challenge, and to the specific TH2 response in the lymph nodes. The presence of ectoine during the sensitization significantly reduced these parameters. The number of antigen-loaded dendritic cells in the draining lymph nodes was identified as a possible cause for the adjuvant effect of the nanoparticles. In vitro assays indicate that the direct interaction of the particles with dendritic cells is not able to trigger CCR7 expression, while this endpoint is achieved by lung lavage fluid from nanoparticle-exposed animals. Conclusions Using the intervention strategy of applying ectoine into the airways of animals we were able to demonstrate the relevance of neutrophilic lung inflammation for the adjuvant effect of carbon nanoparticles on allergic sensitization. Electronic supplementary material The online version of this article (doi:10.1186/s12989-015-0093-5) contains supplementary material, which is available to authorized users.