The first investigative science-based evidence of Morgellons psychogenesis.

Morgellons disease is an infrequent syndromic condition, that typically affects middle-aged white women, characterized by crawling sensations on and under the skin, associated with itchy rashes, stinging sores, fiber-like filaments emerging from the sores, severe fatigue, concentrating difficulty, and memory loss. The scientific community is prone to believe that Morgellons is the manifestation of various psychiatric syndromes (Munchausen, Munchausen by proxy, Ekbom, Wittmaack-Ekbom). Up until now, no investigative science-based evidence about its psychogenesis has ever been provided. In order to close this gap, we have analyzed the filaments extracted from the skin lesions of a 49-year-old Caucasian female patient, by using a Field Emission Gun-Environmental Electron Scanning Microscope equipped with an X-ray microprobe, for the chemico-elemental characterization of the filaments, comparing them with those collected during a detailed indoor investigation, with careful air monitoring, in her apartment. Our results prove the self-introduction under the epidermis of environmental filaments. For the first time in the literature, we have scientifically demonstrated the self-induced nature of Morgellons disease, thereby wiping out fanciful theories about its etiopathogenesis.

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.

ESEM Detection of Foreign Metallic Particles inside Ameloblastomatous Cells.

Ameloblastoma is a borderline tumor of odontogenic origin, with a high recurrence rate and possible local aggressiveness. The etiopathogenetic factors involved in its occurrence are not still defined and our study has been precisely aimed to search for novel factors associated with its development. Sections cut from paraffin blocks, containing the representative specimens of 18 different ameloblastomas, collected in a 15-year period (1999-2014), have been observed by an environmental scanning electron microscope, in order to search micro- and nano-sized particles and to identify their composition. In all the neoplastic cases, micro- and nano-sized metallic debris, differing in size and composition, have been detected inside the ameloblastomatous cells. On the contrary, the total absence of metallic particles in the healthy control cases has been emerged. Our results reveal a relationship between ameloblastoma and metallic particulate. The cigarette smoke and the routine dental practice appear the most probable source for the presence of these biopersistant inorganic particles inside the neoplastic cells.

Heavy Metal Bioaccumulation in an Atypical Primitive Neuroectodermal Tumor of the Abdominal Wall.

Heavy metals are able to interfere with the function of vital cellular components. Besides in trace heavy metals, which are essential at low concentration for humans, there are heavy metals with a well-known toxic and oncogenic potential. In this study, for the first time in literature, we report the unique adulthood case of an atypical primitive neuroectodermal tumor of the abdominal wall, diagnosed by histology and immunohistochemistry, with the molecular hybridization support. The neoplasia occurred in a patient chronically exposed to a transdermal delivery of heavy metal salts (aluminum and bismuth), whose intracellular bioaccumulation has been revealed by elemental microanalysis.

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.

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.

Innovative scanning electron microscopic investigation in blood samples of patients affected by leukaemia: A physical-chemical-biological approach.

The study proposes an innovative, interdisciplinary observation on blood samples of patients coming from the region Marche (Italy) suffering from acute myeloid leukaemia (AML) by means of a scanning electron microscope coupled with an X-ray microprobe of an Energy Dispersive Spectroscope. A novel protocol of blood preparation was studied and prepared to identify exogenous, potentially toxic foreign bodies coming from an environmental contamination. The results on the four different blood fractions of 38 blood samples (erythrocytes, leukocytes, platelets, and plasma) indicate that the circulating blood of leukaemic patients shows the systematic presence of physical contaminants, with a frequency and concentration which are statistically meaningful as compared to the healthy controls. The chemical compositions of the particles were identified as well as the frequency of the elements they were composed of. The analysis of these chemical compositions demonstrated that these patients had undergone a remarkable environmental, occupational, industrial exposure at some time. A possible pathological mechanism based on a nano-bio-interaction between the internalized environmental particulate matter and the blood components is hypothesized and discussed.

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.

Nanopathology and its applications within the forensic discipline.

The impact of nanopathology in medicine necessarily involves also the anatomo-pathological diagnostics, because of the current large spread of nanoparticles in the environment and the wide spectrum of correlated human diseases. The main entrance gates of nanoparticles into the body are respiratory inhalation, gastro-intestinal absorption and injection of polluted drugs. In all these cases, their penetration in the lymphatic or blood streams are possible, with subsequent systemic translocation. Different diseases can be generated by nanoparticles exposure, from a direct contact irritation to the onset of granulomatous diseases. Interestingly, they can also act as endocrine disruptors on the autocrine and paracrine systems. At cellular level, nanoparticles can damage the DNA content leading to a subsequent tumorigenesis. In the forensic setting, they can be searched in case of known exposure to inorganic particulate matter or in case of diseases of unknown origin, from granulomatous reactions to foreign inclusions in neoplastic tissues. The combined physical-histopathological studies allow to relate possible environmental/industrial pollution with the pathology and offer a novel tool for forensic investigations, but, overall, they represent new technical evidences for lawyers to present in a court.

In Vivo Uptake of Rare Earth Metals by Triple-Negative Breast Cancer Cells.

Rare earth metals (REM) are a group of 17 chemical elements in the periodic table, namely scandium (Sc), yttrium (Y) and the lanthanides. In relation to atomic volume and geological behavior, the lanthanides are further subdivided into light, medium and heavy REM. They find many applications in the technological field; however, their impact on the human health is still conflicting and, for many aspects, unknown. During a research program carried on 113 cases of female breast cancer, immunohistochemically categorized in Her2-positive (29 cases), Her2-negative (57 cases) and triple negative (27 cases), aimed to evaluate the role of environmental particulate in carcinogenesis by elemental microanalysis, for the first time in literature we have detected a REM uptake, in detail europium (Eu), dysprosium (Dy) and praseodymium (Pr), inside the neoplastic cells belonging to a single triple negative breast cancer. Curiously, the woman affected by this form of malignancy had worked in the ceramic industry, a well-known source of REM, during her life, and she was the one and only patient of our series to be dedicated to this activity. The medical repercussions of our findings are here discussed: in fact, a REM detection in only 1 of 113 examined cases seems to exclude active roles in breast carcinogenesis and discloses new possibilities for therapeutic developments in triple negative breast cancer.

Functionalization of a nanostructured hydroxyapatite with Cu(II) compounds as a pesticide: in situ transmission electron microscopy and environmental scanning electron microscopy observations of treated Vitis vinifera L. leaves.

BACKGROUND: The present study evaluated a biocompatible material for plant protection with the aim of reducing the amount of active substance applied. We used a synthetic hydroxyapatite (HA) that has been studied extensively as a consequence of its bioactivity and biocompatibility. An aggregation between HA nanoparticles and four Cu(II) compounds applied to Vitis vinifera L. leaves as a pesticide was studied. Formulations were characterized by X-ray diffraction (XRD), dynamic light scattering (DLS) and electron microscopy and applied in planta to verify particle aggregation and efficiency in controlling the pathogen Plasmopara viticola. RESULTS: The XRD patterns showed different crystalline phases dependig on the Cu(II) compound formulated with HA particles, DLS showed that nanostructured particles are stable as aggregates out of the nanometer range and, in all formulations, transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM) microscopy showed large aggregates which were partially nanostructured and were recognized as stable in their micrometric dimensions. Such particles did not show phytotoxic effects after their application in planta. CONCLUSION: A formulation based on HA and a soluble Cu(II) compound showed promising results in the control of the fungal pathogen, confirming the potential role of HA as an innovative delivery system of Cu(II) ions. The present work indicates the possibility of improving the biological activity of a bioactive substance by modifying its structure through an achievable formulation with a biocompatible material. © 2018 Society of Chemical Industry.

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.

The Uncontrolled Sialylation is Related to Chemoresistant Metastatic Breast Cancer.

Among the scientific communities, there is a convergence of results supporting a direct relationship between dysregulated sialylation and poor prognosis in many human cancers. For this reason, we have retrospectively investigated 169 cases of invasive ductal carcinoma of the breast, coming from female patients aged between 31 and 76 years old. The whole series was subdivided into two prognostic groups: the first group consisted of 138 patients, who showed a post-treatment survival time more than 5 years, while the second group was made up by 31 patients, died within 5 years despite of chemotherapy. All the surgical specimens were fixed in 10 % neutral buffered formalin, paraffin embedded and, then, submitted to routinely haematoxylin/eosin staining and to a further histochemical (Alcian Blue, DDD-Fast Blue B, Mercury Orange), immunohistochemical (ST3GAL5 sialyltransferase, Ki67, c-erbB2, ER, PR) and chemico-elemental characterization. In the 31 cases of breast cancer belonging to the second group, an overexpression of sialomucins and sialyltransferases has been detected. Our results lead us to support that in aggressive chemoresistant breast cancers, the altered expression of sialic acid, due to an uncontrolled sialylation, creates an excessive negative charge on cell membranes, which stimulates repulsion between neoplastic cells and their subsequent access into the blood stream. This event implies an early metastatization and a rapid disease progression with fatal outcome. The early application of Alcian Blue stain on diagnostic biopsies of breast cancer is able to cheaply reveal the sialomucin accumulations, providing for the disease course.

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.

Uptake and translocation of metals and nutrients in tomato grown in soil polluted with metal oxide (CeO2, Fe3O4, SnO2, TiO2) or metallic (Ag, Co, Ni) engineered nanoparticles.

The influence of exposure to engineered nanoparticles (NPs) was studied in tomato plants, grown in a soil and peat mixture and irrigated with metal oxides (CeO2, Fe3O4, SnO2, TiO2) and metallic (Ag, Co, Ni) NPs. The morphological parameters of the tomato organs, the amount of component metals taken up by the tomato plants from NPs added to the soil and the nutrient content in different tomato organs were also investigated. The fate, transport and possible toxicity of different NPs and nutrients in tomato tissues from soils were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES). The tomato yield depended on the NPs: Fe3O4-NPs promoted the root growth, while SnO2-NP exposure reduced it (i.e. +152.6 and -63.1 % of dry matter, respectively). The NP component metal mainly accumulated in the tomato roots; however, plants treated with Ag-, Co- and Ni-NPs showed higher concentration of these elements in both above-ground and below-ground organs with respect to the untreated plants, in addition Ag-NPs also contaminated the fruits. Moreover, an imbalance of K translocation was detected in some plants exposed to Ag-, Co- and Fe3O4-NPs. The component metal concentration of soil rhizosphere polluted with NPs significantly increased compared to controls, and NPs were detected in the tissues of the tomato roots using electron microscopy (ESEM-EDS).

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.

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.

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).