Novel chemical-physical autopsy investigation in sudden infant death and sudden intrauterine unexplained death syndromes.

Aim: Verify the presence of inorganic nanoparticle entities in brain tissue samples from sudden infant death syndrome (SIDS)/sudden intrauterine unexplained death syndrome (SIUDS) cases. The presence of inorganic debris could be a cofactor that compromises proper brain tissue functionality. Materials & methods: A novel autopsy approach that consists of neuropathological analysis procedures combined with energy dispersive spectroscopy/field emission gun environmental scanning electron microscopy investigations was implemented on 10 SIDS/SIUDS cases, whereas control samples were obtained from 10 cases of fetal/infant death from known cause. Results: Developmental abnormalities of the brain were associated with the presence of foreign bodies. Although nanoparticles were present as well in control samples, they were not associated with histological brain anomalies, as was the case in SIDS/SIUDS. Conclusion: Inorganic particles present in brain tissues demonstrate their ability to cross the hemato-encephalic barrier and to interact with tissues and cells in an unknown yet pathological fashion. This gives a rationale to consider them as cofactors of lethality.

Silver nanoparticles in the fetal brain: new perspectives in understanding the pathogenesis of unexplained stillbirths.

We report, for the first time, the surprising presence of toxic nanoparticles, especially silver, in the brain of a fetus, who died unexpectedly at the end of a regular pregnancy. After an accurate autopsy, including the examination of the fetal annexes, an in-depth anatomopathological study of the nervous system and a search by scanning electron microscopy of nanoparticles in the brain, we highlighted the sequence of events that may have led to this fetal death, triggered primarily by the transition of nanosized xenobiotics from the mother to the fetal bloodstream. From this report emerges the importance of considering the search of nanosubstances in the brain during routine investigations following unexpected and unexplained fetal and infant deaths.

A Novel Forensic Investigation Applied to Bone Remains Exhumed near to Quirra Interforce Firing Range.

The need to implement novel techniques, able to support a causal link between exposure and pathology, has been emerged over the recent years. The application of scanning electron microscope coupled with probe X-ray microanalysis (by means of an energy-dispersive spectroscopy) has been developed by our research group for the bone remains investigation. It was aimed to testify the exposure to microsized and nanosized pollutions, due to military activities in the Quirra interforce firing range, of a Sardinian shepherd, died of acute leukemia. Metallic debris with a combustive morphology and with an oncogenic potential has been surely detected inside his bone marrow canal. This novel technique has proved to be able to bring to light a source of past exposure preserved over time within the bone marrow canal. It can be useful for postmortem analyses, delivering a new avant-garde approach to modern forensic science.

Review: Morphofunctional and biochemical markers of stress in sea urchin life stages exposed to engineered nanoparticles.

We describe the use of different life stages of the Mediterranean sea urchin Paracentrotus lividus for the assessment of the possible risk posed by nanoparticles (NPs) in the coastal water. A first screening for the presence of NPs in sea water may be obtained by checking their presence inside tissues of organisms taken from the wild. The ability of NPs to pass from gut to the coelomic fluid is demonstrated by accumulation in sea urchin coelomocytes; the toxicity on sperms can be measured by embryotoxicity markers after sperm exposure, whereas the transfer through the food chain can be observed by developmental anomalies in larvae fed with microalgae exposed to NPs. The most used spermiotoxicity and embryotoxicity tests are described, as well as the biochemical and histochemical analyses of cholinesterase (ChE) activities, which are used to verify toxicity parameters such as inflammation, neurotoxicity, and interference in cell-to-cell communication. Morphological markers of toxicity, in particular skeletal anomalies, are described and classified. In addition, NPs may impair viability of the immune cells of adult specimens. Molecular similarity between echinoderm and human immune cells is shown and discussed. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1552-1562, 2016.

Toxic effects of colloidal nanosilver in zebrafish embryos.

A variety of consumer products containing silver nanoparticles (Ag NPs) are currently marketed. However, their safety for humans and for the environment has not yet been established and no standard method to assess their toxicity is currently available. The objective of this work was to develop an effective method to test Ag NP toxicity and to evaluate the effects of ion release and Ag NP size on a vertebrate model. To this aim, the zebrafish animal model was exposed to a solution of commercial nanosilver. While the exposure of embryos still surrounded by the chorion did not allow a definite estimation of the toxic effects exerted by the compound, the exposure for 48 h of 3-day-old zebrafish hatched embryos afforded a reliable evaluation of the effects of Ag NPs. The effects of the exposure were detected especially at molecular level; in fact, some selected genes expressed differentially after the exposure. The Ag NP toxic performance was due to the combined effect of Ag(+) ion release and Ag NP size. However, the effect of NP size was particularly detectable at the lowest concentration of nanosilver tested (0.01 mg l(-1)) and depended on the solubilization media. The results obtained indicate that in vivo toxicity studies of nanosilver should be performed with ad hoc methods (in this case using hatched embryos) that might be different depending on the type of nanosilver. Moreover, the addition of this compound to commercial products should take into consideration the Ag NP solubilization media.

Effects of nanosilver exposure on cholinesterase activities, CD41, and CDF/LIF-like expression in zebrafish (Danio rerio) larvae.

Metal nanosolicoparticles are suspected to cause diseases in a number of organisms, including man. In this paper, we report the effects of nanosilver (Ag, 1-20 nm particles) on the early development of the zebrafish, a well-established vertebrate model. Embryos at the midgastrula stage were exposed to concentrations ranging from 100 to 0.001 mg/L to verify the effects on different endpoints: lethality, morphology, expression of cholinergic molecules, and development of the immune system. (1) Relative risk of mortality was exponential in the range between 0.001 and 10 mg/L. Exposure to 100 mg/L caused 100% death of embryos before reaching the tail-bud stage. (2) Developmental anomalies were present in the 72 h larvae obtained from embryos exposed to nanosilver: whole body length, decreased eye dimension, and slow response to solicitation by gentle touch with a needle tip, with a significant threshold at 0.1 mg/L. (3) Dose-dependent inhibition of acetylcholinesterase activity was significant among the exposures, except between 1 mg/L and 10 mg/L. (4) The distribution of CD41+ cells and of CDF/LIF-like immunoreactivity was altered according to the Ag concentration. The possible effect of nanosilver in impairing immune system differentiation through the inhibition of molecules related to the cholinergic system is discussed.

Developmental abnormalities and changes in cholinesterase activity in sea urchin embryos and larvae from sperm exposed to engineered nanoparticles.

The objective of this study is to examine the toxicity of engineered nanoparticles (NPs) that are dispersed in sea water by using an in vivo model. Because many products of nanotechnology contain NPs and are commonly used and well-established in the market, the accidental release of NPs into the air and water is quite possible. Indeed, at the end of their life cycle, some NPs are inevitably released into waste water and can reach marine ecosystem and affect the organisms there. Although there are few data on the presence of NPs in the marine environment, our awareness of their potential impact on environmental and organismal health is growing. Shallow-water benthonic organisms such as sea urchins provide planktonic larvae as a trophic base for finfish juveniles and are exposed to water from estuaries and precipitation. Such organisms can therefore be directly affected by NPs that are dispersed into those media. We evaluated the effects of exposure to different concentrations of nanosilver, titanium oxide and cobalt NPs on the sperm of the sea urchin Paracentrotus lividus by analyzing the functionality and the morphology and biochemistry of the first developmental stages of the sea urchin. Sperm were exposed to sea water containing suspensions of NPs ranging from 0.0001 mg/L to 1 mg/L. Fertilization ability was not affected, but developmental anomalies were identified in embryos from the gastrula to pluteus stages, including morphological alterations of the skeletal rods. In addition, the enzymatic activity (cholinesterase, ChE) of the larvae was measured. Acetylcholinesterase (AChE) and propionylcholinesterase activity (PrChE) was affected in all of the exposed samples. The results did not vary consistently with the concentration of NP, but controls were significantly different from exposed samples. Exposure of sea urchin to these NPs may cause neurotoxic damage, and the altered ChE activity may be involved in skeletogenic aberrations. In conclusion, the sea urchin represents a suitable and sensitive model for testing the toxicity and effects of engineered NPs that are dispersed in sea water.

Heavy metals nanoparticles in fetal kidney and liver tissues.

The proliferation of the nanotechnologies with the production of engineered nanoparticles presents a dilemma to regulators regarding hazard identification mostly for human health. We investigated the presence of inorganic micro and nanosized contamination in fetal liver and kidney tissues by Field Emission Gun-Environmental Scanning Electron Microscope (FEGESEM) innovative observations. An observational study in 16 fetuses, complicated (n=8) or not (n=8) by neural tube defects, whose mothers obtained the authorization for abortion between 21-23 weeks of gestation was carried out. Heavy metals concentrations in maternal blood were undetectable. FEGESEM assessment showed particles of iron, silicon, aluminum and magnesium in different tissues analyzed. The mean size and the number of the foreign bodies detected in kidney and liver tissues were higher in NTD fetuses as well as the number of total particles (P < 0.05, for all). The present study shows first the presence of xenobiotic, nanoscaled contamination, not detectable in maternal blood in fetuses. Data are suggestive and open-up a new clue for further investigations to elucidate the relationship between pollution at nanoscale stage and multiorgan damage.

Sarcoidosis in a dental surgeon: a case report.

INTRODUCTION: Although the causes of sarcoidosis are still unknown, past and current studies have provided evidence that this disease may be associated with occupational exposure to specific environmental agents. We describe a case of sarcoidosis in a dental surgeon with long exposure to inorganic dusts. To the best of our knowledge, this is the first report of this kind in the literature. CASE PRESENTATION: At the beginning of 2000, a 52-year-old Caucasian man, who worked as a dental surgeon, presented with shortness of breath during exercise, cough and retrosternal pain. After diagnosis of sarcoidosis, a scanning electronic microscopy with X-ray microanalysis of biopsy specimens was used in order to determine whether the disease could be traced to an occupational environmental agent. Results showed the presence of inorganic dust particles within sarcoidotic granulomas, and demonstrated that the material detected was identical to that found in a powder used by our patient for several years. CONCLUSIONS: Although these results cannot be considered as definitive proof, they do however provide strong evidence that this disease may be associated with material used by dental surgeons.

Toxicity of antimony trioxide nanoparticles on human hematopoietic progenitor cells and comparison to cell lines.

Nanoparticles (NPs) are materials with one dimension in the range of 1-100 nm. The toxicity of NPs remains widely unknown and still poses concerns, due to the peculiar characteristics of materials in the nano-size range. We analyze the toxicity of seven NPs ((Fe2O3, Fe3O4, Sb2O3, Au, TiO2, Co, and Ag) on primary cultures of human hematopoietic progenitor cells from the bone marrow of healthy donors with CFU assays, and show that antimony oxide (Sb2O3) NPs and cobalt (Co) NPs have a toxic effect, while the other NPs have no effect at the tested concentrations (5, 25 and 100 microg/ml). While Co NPs suspension is toxic to both erythroid and granulocytic-monocytic precursors, Sb2O3 NPs at 5 microg/ml are specifically toxic to erythroid colony development, suggesting a highly selective type of toxicity. With liquid culture assays we show that Sb2O3 NPs impair the proliferation of erythroid progenitors, while no toxic effect is observed when Sb2O3 NPs are added during erythroid differentiation. CFU assays and liquid culture assays on seven human cell lines of hematopoietic origin (K562, HL-60, CEM, CEM-R, Thp-1, Jurkat, and Molt-4) show that, contrary to what observed on primary cultures of bone marrow progenitors, Sb2O3 NPs have no toxic effect on proliferation of any of the cell lines, raising concerns about the use of immortalized cell lines for nanotoxicology tests.

Investigation of the presence of inorganic micro- and nanosized contaminants in bread and biscuits by environmental scanning electron microscopy.

A European project called "Nanopathology" allowed to develop a new diagnostic tool through which the presence of inorganic particulate matter in pathological human tissues of the digestive tract could be shown. This unexpected evidence induced the authors to put forward the hypothesis that that sort of contamination was present in ingested food. In order to demonstrate this hypothesis, 86 samples of wheat bread and 49 of wheat biscuits from 14 different countries were analyzed by means of an Environmental Scanning Electron Microscopy to detect inorganic, micro-, and nano-scaled contaminants. The X-ray microprobe of an Energy Dispersion Spectroscope was employed to identify their chemical composition. The results indicate that 40% of the samples analyzed contained foreign bodies as ceramic and metallic debris of probable environmental or industrial origin. Because of the great variety of chemical composition of the particulate matter, those contaminants were listed according to the most expressed element. The majority of these debris are not biodegradable, some are chemically toxic, and none of them have any nutritive value. The work discusses the possible origin of such a pollution and the role that it can play on human life.

ESEM evaluations of muscle/nanoparticles interface in a rat model.

In order to examine the influence that shape and chemistry of different materials have on the incitement of a tissue reaction, we implanted five materials (the two metals Ni and Co, the two ceramics TiO2 and SiO2, and the polymer poly vinyl-chloride) as nanoparticles or bulk, in the dorsal muscles of 50 rats. After 6 or 12 months, rats were euthanized and the implanted materials were excised together with the surrounding tissue. After a first histological evaluation, the specimens were prepared for environmental scanning electron microscopy (ESEM) and for energy dispersive spectroscopy (EDS), in order to analyse the chemical composition of the implanted material after the biological interaction had occurred, and to evaluate the possible corrosion and diffusion of the materials at tissue interface. The results indicate that the metals at nanoscale size have a carcinogenic effect, while the bulk materials only induce a foreign-body reaction. The ESEM observations show a chemical transformation of the materials. Corrosion of the metals and subsequent recombination of the released ions in a sort of organic-inorganic crystals is showed and verified by the EDS analyses. Finally, our hypotheses of the involved pathological mechanism are suggested.

Biological tolerance of different materials in bulk and nanoparticulate form in a rat model: sarcoma development by nanoparticles.

In order to study the pathobiological impact of the nanometre-scale of materials, we evaluated the effects of five different materials as nanoparticulate biomaterials in comparison with bulk samples in contact with living tissues. Five groups out of 10 rats were implanted bilaterally for up to 12 months with materials of the same type, namely TiO2, SiO2, Ni, Co and polyvinyl chloride (PVC), subcutaneously with bulk material on one side of the vertebral column and intramuscularly with nanoparticulate material on the contralateral side. At the end of each implantation time, the site was macroscopically examined, followed by histological processing according to standard techniques. Malignant mesenchymal tumours (pleomorphic sarcomas) were obtained in five out of six cases of implanted Co nanoparticle sites, while a preneoplastic lesion was observed in an animal implanted with Co in bulk form. In the Ni group, all animals rapidly developed visible nodules at the implanted sites between 4 and 6 months, which were diagnosed as rhabdomyosarcomas. Since the ratio of surface area to volume did not show significant differences between the Ni/Co group and the TiO2/SiO2/PVC group, we suggested that the induction of neoplasia was not mediated by physical effects, but was mediated by the well-known carcinogenic impact of Ni and Co. The data from the Co group show that the physical properties (particulate versus bulk form) could have a significant influence on the acceleration of the neoplastic process.

Bone augmentation with bioactive glass in three cases of dental implant placement.

This study reports the clinical use of a bioactive bone graft material, PerioGlas, in the treatment of dental extraction sites before dental implant placement, to effect bone regeneration and to give early fixation to the implant.PerioGlas, granules, ranging from 90 to 710 mm, are implanted after tooth extraction in three patients; after 6 months bone biopsies were performed in the site of the glass implantation and observed under Electron Scanning Microscopy. All the granules showed a biodegradation involving precipitation of calcium phosphate that worked as a scaffold for osteoblasts colonization. All cases examined showed the bioactivity of PerioGlas granules resulting in new bone formation and biodegradation of the glass. After a two-year clinical follow-up all the implants were successfully loaded and appeared stable.

In-vivo short- and long-term evaluation of the interaction material-blood.

Through an innovative electron microscopy technique, thrombi and fibrotic tissue taken from 14 explanted vena cava filters were observed. Twelve cases showed the presence of micro-and nano-sized inorganic, non biodegradable nor biocompatible particles which did not belong to the metal the device was made of and which could be the sole cause or, more likely, a pre-existing cause for thrombosis. In two cases, those debris activated immunological reactions typical of a foreign body. The presence of inorganic particles in the blood was never detected before and their effects on human health are hardly known. Their thrombogenicity should be added to the Virchow's Triad as a fourth factor and could be the explanation to many of the cases of pulmonary embolism where no thrombotic focus could be demonstrated.

Effects of nano-scaled particles on endothelial cell function in vitro: studies on viability, proliferation and inflammation.

Recent studies give support for a connection between the presence of inorganic particles (of microm and nm size) in different organs and tissues and the development of inflammatory foci, called granulomas. As the potential source of particles (e.g. porcelain dental bridges) and the location of particle detection were topographically far apart, a distribution via the blood stream appears highly probable. Thus, endothelial cells, which line the inner surface of blood vessels, would come into direct contact with these particles, making particle-endothelial interactions potentially pathogenically relevant. The objective of this study was to evaluate the effects that five different nano-scaled particles (PVC, TiO2, SiO2, Co, Ni) have on endothelial cell function and viability. Therefore, human endothelial cells were exposed to different amounts of the above-mentioned particles. Although most particle types are shown to be internalised (except Ni-particles), only Co-particles possessed cytotoxic effects. Furthermore, an impairment of the proliferative activity and a pro-inflammatory stimulation of endothelial cells were induced by exposure to Co- and, to a lesser extent, by SiO2-particles. If a pro-inflammatory stimulation of endothelial cells occurs in vivo, a chronic inflammation could be a possible consequence.

Biocompatibility of micro- and nano-particles in the colon. Part II.

Pathological colonic tissues were investigated with an Environmental Scanning Electron Microscope technique to verify the presence of inorganic, non-biodegradable pollutants, i.e. micro- and nano-debris of exogenous origin, after debris in liver and kidney had been discovered. In all, 18 samples of colon tissues affected by cancer and Crohn's disease were evaluated and found in all the cases to contain micro- and nano-particles. Their chemistry, detected with an X-ray microprobe, indicated a heterogeneous nature, whereas the size of the particles was homogeneous. Three control samples of healthy, young, cadavers were analysed and showed the absence of debris within the normal, healthy colon mucosa. The study reveals the presence of particulate debris, generally considered as biocompatible, in pathological specimens of human colon. The findings suggest a possible link between the presence of such particles and the underlying pathology in the cases analysed.

Innate defence functions of macrophages can be biased by nano-sized ceramic and metallic particles.

Nano-sized particles of ceramic and metallic materials are generated by high-tech industrial activities, and can be generated from worn-out replacement and prosthetic implants. The interaction with the human body of such nanoparticles has been investigated, with a particular emphasis on innate defence mechanisms. Human macrophages (PMA-differentiated myelomonocytic U-937 cells) were exposed in vitro to non-toxic concentrations of TiO(2), SiO(2), ZrO(2), or Co nanoparticles, and their inflammatory response (expression of TLR receptors and co-receptors, and cytokine production) was examined. Expression of TLR receptors was generally unaffected by exposure to the different nanoparticles, except for some notable cases. Exposure to nanoparticles of ZrO(2) (and to a lesser extent TiO(2)), upregulated expression of viral TLR receptors TLR3 and TLR7. Expression of TLR10 was also increased by TiO(2) and ZrO(2) nanoparticles. On the other hand, TLR9 expression was decreased by SiO(2) nano-particles, and expression of the co-receptor CD14 was inhibited by Co nanoparticles. Basal and LPS-induced production of cytokines IL-1beta, TNF-alpha, and IL-1Ra was examined in macrophages exposed to nanoparticles. SiO(2) nanoparticles strongly biased naive macrophages towards inflammation (M1 polarisation), by selectively inducing production of inflammatory cytokines IL-1beta and TNF-alpha. SiO(2) nanoparticles also significantly amplified the inflammatory phenotype of LPS-polarised M1 macrophages. Other ceramic nanoparticles had little influence on cytokine production, either in resting macrophages, or in LPS-activated cells. Generally, Co nanoparticles had an overall pro-inflammatory effect on naive macrophages, by reducing anti-inflammatory IL-1Ra and inducing inflammatory TNF-alpha. However, Co nanoparticles reduced production of IL-1beta and IL-1Ra, but not TNF-alpha, in LPS-polarised M1 macrophages. Thus, exposure to different nanoparticles can modulate, in different ways, the defence/inflammatory capacities of macrophages. A thorough analysis of these biasing effects may shed light on the mechanisms of pathogenesis of several diseases based on dysregulation of the immune response (allergies, autoimmunity, tumours).

Carbon-coated stents implanted in porcine iliac and renal arteries: histologic and histomorphometric study.

PURPOSE: To test in a pig model the biocompatibility and effectiveness of carbon-coated renal and iliac artery stent systems during implantation procedures and at different follow-up periods. MATERIALS AND METHODS: Twenty-two miniature pigs received carbon-coated balloon-expandable stainless-steel stents in their renal and iliac arteries. Animals were killed at 7, 30, or 180 days for evaluation of acute, subacute, and chronic biologic response to the implanted devices. Histologic, histomorphometric, and scanning electron microscopy (SEM) analyses were performed to assess inflammatory reaction, endothelialization process, and neointimal growth. RESULTS: Forty-four iliac stents and 42 renal stents were successfully implanted. Mural thrombi were not observed by angiography or histologic examinations. Histologically, no significant inflammatory reaction was detected: the stents appeared covered by a thin monolayer of endothelial cells even at 7-day follow-up. The neointima showed homogeneous growth and moderate thickness at 30-day and 180-day explantations (0.09 mm +/- 0.06 and 0.15 mm +/- 0.13, respectively, for renal arteries; 0.12 mm +/- 0.04 and 0.21 mm +/- 0.12, respectively, for iliac arteries). Internal and external elastic laminae were intact in 82 of 86 cases (95%) of stent-implanted arteries. Histologic validations were obtained with SEM observations for each follow-up group. CONCLUSIONS: This study showed good technical results of deployment of carbon-coated renal and iliac stents and very satisfactory biologic behavior in terms of tissue and hematologic compatibility. The devices do not induce thrombus formation.

Biocompatibility of micro- and nanoparticles. Part I: in liver and kidney.

An accidental discovery led us to correlate a pathology of "unknown origin" with the presence of micro- and nano-particles of inorganic nature in the human tissues. Bioptic samples of livers and kidneys affected by cryptogenic granulomas were observed through a new type of electron scanning microscope called environmental scanning electron microscope and X-ray-microanalyzed with an energy dispersive system. All the randomly selected samples showed the presence of inorganic samples, sometimes varying in chemical composition, but consistent in size (larger in the liver, smaller in the kidney). This article illustrates the results obtained on randomly picked samples and proposes our new investigative method as an aid to achieve a medical diagnosis. Two more items are discussed: the possible, until now unexplained, origin of a pathology as deriving from the presence of exogenous particles, so a new word "nano-pathology" was developed and, as a consequence, the relativity of the concept of biocompatibility as depending on the size of the material considered and its local concentration. The implications of the discovery are briefly discussed.