Focused X-Ray Histological Analyses to Reveal Asbestos Fibers and Bodies in Lungs and Pleura of Asbestos-Exposed Subjects.

Asbestos bodies are the histological hallmarks of asbestos exposure. Both conventional and advanced techniques are used to evaluate abundance and composition in histological samples. We previously reported the possibility of using synchrotron X-ray fluorescence microscopy (XFM) for analyzing the chemical composition of asbestos bodies directly in lung tissue samples. Here we applied a high-performance synchrotron X-ray fluorescence (XRF) set-up that could allow new protocols for fast monitoring of the occurrence of asbestos bodies in large histological sections, improving investigation of the related chemical changes. A combination of synchrotron X-ray transmission and fluorescence microscopy techniques at different energies at three distinct synchrotrons was used to characterize asbestos in paraffinated lung tissues. The fast chemical imaging of the XFM beamline (Australian Synchrotron) demonstrates that asbestos bodies can be rapidly and efficiently identified as co-localization of high calcium and iron, the most abundant elements of these formations inside tissues (Fe up to 10% w/w; Ca up to 1%). By following iron presence, we were also able to hint at small asbestos fibers in pleural spaces. XRF at lower energy and at higher spatial resolution was afterwards performed to better define small fibers. These analyses may predispose for future protocols to be set with laboratory instruments.

The crocidolite fibres interaction with human mesothelial cells as investigated by combining electron microscopy, atomic force and scanning near-field optical microscopy.

In this study, we have performed a morphological analysis of crocidolite fibres interaction with mesothelial cells (MET5A) by combining conventional electron microscopy with atomic force (AFM) and scanning near-field optical microscopy (SNOM). After 6-h exposure at a crocidolite dose of 5 µg cm(-2), 90% of MET5A cells interact with fibres that under these conditions have a low cytotoxic effect. SEM images point out that fibres can be either engulfed by the cells that lose their typical morphology or they can accumulate over or partially inside the cells, which preserve their typical spread morphology. By using AFM we are able to directly visualize the entry-site of nanometric-sized fibres at the plasma membrane of the spread mesothelial cells. More importantly, the crocidolite fibres that are observed to penetrate the plasma membrane in SNOM topography can be simultaneously followed beneath the cell surface in the SNOM optical images. The analysis of SNOM data demonstrates the entrance of crocidolite fibres in proximity of nuclear compartment, as observed also in the TEM images. Our findings indicate that the combination of conventional electron microscopy with novel nanoscopic techniques can be considered a promising approach to achieve a comprehensive morphological description of the interaction between asbestos fibres and mesothelial cells that represents the early event in fibre pathogenesis.

Peroxidase-like activity of ferruginous bodies isolated by exploiting their magnetic property.

Ferruginous bodies (FB) are polymorphic structures whose formation is macrophage dependent, and are composed of a core, which may consist of an asbestos fiber coated with proteins, among which ferritin is the main component. Within ferritin, the ferric and ferrous ions are coordinated as ferrihydrite, which is the main iron (Fe) storage compound. However, when ferritin accumulates in some tissues following Fe overload it also contains magnetite along with ferrihydrite, which endows it with magnetic properties. Recently studies showed that magnetite exerts peroxidase-like activity, and since ferruginous bodies display magnetic properties, it was postulated that these particular structures may also contain magnetite within the ferritin coating, and thus may also exert peroxidase-like activity. Histochemical analysis for peroxidase of isolated FB smears demonstrated positive staining. Samples isolated from 4 different autopsy lung fragments were also able to oxidize 3,3',5,5'-tetramethyl-benzidine to a blue colored compound that absorbs at 655 nm. This activity was (1) azide and heat insensitive with optimal pH from 5 to 6, and (2) highly variable, changing more than 25-fold from one sample to another. These findings, together with evidence that the peroxidase-like activity of ferruginous bodies has a hydrogen peroxide and substrate requirement different from that of human myeloperoxidase, can exclude that this enzyme gives a significant contribution to the formation of FB. Standard Fe-rich asbestos fibers also express a peroxidase-like activity, but this appears negligible compared to that of ferruginous bodies. Strong acidification of standard Fe-containing asbestos fibers or magnetically isolated ferruginous bodies liberates a high amount of peroxidase-like activity, which is probably accounted for by the release of Fe ions. Further, FB also damage mesothelial cells in vitro. Data suggest that FB exert peroxidase-like activity and cytotoxic activity against mesothelial cells, and hence may be an important factor in pathogenesis of asbestos-related diseases.

Synchrotron soft X-ray imaging and fluorescence microscopy reveal novel features of asbestos body morphology and composition in human lung tissues.

BACKGROUND: Occupational or environmental exposure to asbestos fibres is associated with pleural and parenchymal lung diseases. A histopathologic hallmark of exposure to asbestos is the presence in lung parenchyma of the so-called asbestos bodies. They are the final product of biomineralization processes resulting in deposition of endogenous iron and organic matter (mainly proteins) around the inhaled asbestos fibres. For shedding light on the formation mechanisms of asbestos bodies it is of fundamental importance to characterize at the same length scales not only their structural morphology and chemical composition but also to correlate them to the possible alterations in the local composition of the surrounding tissues. Here we report the first correlative morphological and chemical characterization of untreated paraffinated histological lung tissue samples with asbestos bodies by means of soft X-ray imaging and X-Ray Fluorescence (XRF) microscopy, which reveals new features in the elemental lateral distribution. RESULTS: The X-ray absorption and phase contrast images and the simultaneously monitored XRF maps of tissue samples have revealed the location, distribution and elemental composition of asbestos bodies and associated nanometric structures. The observed specific morphology and differences in the local Si, Fe, O and Mg content provide distinct fingerprints characteristic for the core asbestos fibre and the ferruginous body. The highest Si content is found in the asbestos fibre, while the shell and ferruginous bodies are characterized by strongly increased content of Mg, Fe and O compared to the adjacent tissue. The XRF and SEM-EDX analyses of the extracted asbestos bodies confirmed an enhanced Mg deposition in the organic asbestos coating. CONCLUSIONS: The present report demonstrates the potential of the advanced synchrotron-based X-ray imaging and microspectroscopy techniques for studying the response of the lung tissue to the presence of asbestos fibres. The new results obtained by simultaneous structural and chemical analysis of tissue specimen have provided clear evidence that Mg, in addition to Fe, is also involved in the formation mechanisms of asbestos bodies. This is the first important step to further thorough investigations that will shed light on the physiopathological role of Mg in tissue response to the asbestos toxicity.

Carcinoma of the uterine cervix with squamous and sebaceous differentiation.

Sebaceous neoplasms, including carcinoma, can exceptionally arise in extracutaneous sites. We present the third known case of carcinoma with sebaceous differentiation in the uterine cervix. Histologic and immunohistochemical features suggested a metaplastic process within an otherwise usual squamous cell carcinoma. We speculate that, by analogy with the skin where the epidermis and the 3 types of adnexa have a common embryologic origin from basal cell layer of the superficial ectoderm, it is possible that endocervical reserve cells, in addition to the well-known capacity of squamous differentiation, retain the potential to give rise to appendages including sebaceous glands.

Apoptosis-related factors (TRAIL, DR4, DR5, DcR1, DcR2, apoptotic cells) and proliferative activity in ameloblastomas.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a recently identified death factor that acts as a potent apoptosis inducer in ameloblastomas. MATERIALS AND METHODS: The expression of TRAIL and its receptors (TRAIL-R), and the location of apoptotic cells were evaluated in 15 cases of ameloblastoma using immunohistochemistry and an in situ DNA nick-end labeling method. The proliferative activity of ameloblastomas was analyzed by determining the Ki-67 labeling index. RESULTS: TRAIL and TRAIL-R were diffusely expressed in ameloblastomas, without clear correlation with the location of apoptotic cells. Apoptosis and proliferation were opposite in the peripheral and central components of the ameloblastomas. In some ameloblastoma variants, apoptosis and proliferation seemed to modify in the same direction. CONCLUSION: TRAIL and its receptors might be involved in neoplastic transformation of odontogenic epithelium and might suggest some intrinsic regulation of neoplastic cell proliferation and death in ameloblastomas, thus explaining their slow growth and inability to metastasize.

SV40 multiple tissue infection and asbestos exposure in a hyperendemic area for malignant mesothelioma.

To assess the presence of SV40 in malignant mesothelioma tissue, 19 formalin-fixed paraffin-embedded pleural cancer samples of patients from a hyperendemic area of northeastern Italy were analyzed retrospectively. A total of 48 other tissues from the malignant mesothelioma subjects were investigated. The SV40 load was determined by real-time quantitative PCR. Exposure to asbestos was evaluated through a careful review of the occupational history of patients, supplemented by histology and isolation of asbestos bodies. Three of 19 (15.8%) malignant mesothelioma tissues harbored SV40 genomic signals. Two patients with SV40-positive malignant mesothelioma had viral sequences in another tissue. Overall, 3 of 18 (16.7%) normal liver tissues tested positive for SV40, as did 1 of 8 (12.5%) kidney tissues. SV40 viral loads were higher in malignant mesothelioma than in normal cells (P = 0.045). This survey shows that SV40 sustains infections in multiple tissues in malignant mesothelioma patients from a geographic area affected with asbestos-related mesothelioma.

Bone regeneration with adipose derived stem cells in a rabbit model.

It has been shown that stem cells are able to calcify both in vitro and in vivo once implanted under the skin, if conveniently differentiated. Nowadays, however, a study on their efficiency in osseous regeneration does not exist in scientific literature and this very task is the real aim of the present experimentation. Five different defects of 6 mm in diameter and 2 mm in depth were created in the calvaria of 8 white New Zealand rabbits. Four defects were regenerated using 2 different conveniently modified scaffolds (Bio-Oss® Block and Bio-Oss Collagen®, Geistlich), with and without the aid of stem cells. After the insertion, the part was covered with a collagen membrane fixed by 5 modified titan pins (Altapin®). The defect in the front was left empty on purpose as an internal control to each animal. Two animals were sacrificed respectively after 2, 4, 6, 10 weeks. The samples were evaluated with micro-CT and histological analysis. Micro-CT analysis revealed that the quantity of new bone for samples with Bio-Oss® Block and stem cells was higher than for samples with Bio-Oss® Block alone. Histological analysis showed that regeneration occurred in an optimal way in every sample treated with scaffolds. The findings indicated that the use of adult stem cells combined with scaffolds accelerated some steps in normal osseous regeneration.

Ferruginous bodies resolved by synchrotron XRF in a dog with peritoneal malignant mesothelioma.

Mesothelioma is a malignant tumor mainly correlated to occupational asbestos exposure. Rare reports describe its occurrence also in animals, mainly linked to asbestos in the environment. Asbestos exposure is demonstrated by the appearance of characteristic histological hallmarks: asbestos containing ferruginous bodies that are iron-based structures forming around fibers and also other dust particles. Here we present a clinical case of a suspect of mesothelioma in the peritoneum of a dog with parallel histological observation of ferruginous bodies. To possibly correlate the dog tumor to environmental exposure, we performed X-ray fluorescence (XRF) analyses at two different synchrotrons to resolve the ferruginous bodies' composition. While the histological examination diagnoses a tubulo-papillary mesothelioma, the XRF analyses show that ferruginous bodies contain Si particles, resembling formations of exogenous origin; however, the morphology is unlikely that of asbestos fibers. We speculate that the peritoneal mesothelioma of this dog could be related to environmental exposure to non-asbestos material.

Iron-related toxicity of single-walled carbon nanotubes and crocidolite fibres in human mesothelial cells investigated by Synchrotron XRF microscopy.

Carbon nanotubes (CNTs) are promising products in industry and medicine, but there are several human health concerns since their fibrous structure resembles asbestos. The presence of transition metals, mainly iron, in the fibres seems also implicated in the pathogenetic mechanisms. To unravel the role of iron at mesothelial level, we compared the chemical changes induced in MeT-5A cells by the exposure to asbestos (crocidolite) or CNTs at different content of iron impurities (raw-SWCNTs, purified- and highly purified-SWCNTs). We applied synchrotron-based X-Ray Fluorescence (XRF) microscopy and soft X-ray imaging (absorption and phase contrast images) to monitor chemical and morphological changes of the exposed cells. In parallel, we performed a ferritin assay. X-ray microscopy imaging and XRF well localize the crocidolite fibres interacting with cells, as well as the damage-related morphological changes. Differently, CNTs presence could be only partially evinced by low energy XRF through carbon distribution and sometimes iron co-localisation. Compared to controls, the cells treated with raw-SWCNTs and crocidolite fibres showed a severe alteration of iron distribution and content, with concomitant stimulation of ferritin production. Interestingly, highly purified nanotubes did not altered iron metabolism. The data provide new insights for possible CNTs effects at mesothelial/pleural level in humans.

A procedure for the isolation of asbestos bodies from lung tissue by exploiting their magnetic properties: a new approach to asbestos body study.

The role of asbestos bodies (and associated proteinacious coating) in asbestos associated diseases is not well understood. Currently employed methods of isolation of these bodies employ harsh chemicals that lead to destruction of their proteinacious coating. In this work a method was developed that enabled the purification of whole, integral, unmodified asbestos bodies (AB) by exploiting their magnetic properties. Albumin and ferritin were found to be the major proteins associated with AB isolated from lung tissue of mesothelioma patients. Magnetically isolated AB were shown to be cytotoxic and to activate free radical production from inflammatory cells at a higher extent than that induced by bodies obtained by chemical digestion. The finding that hypochlorite-treated AB induce DNA damage, while AB obtained by the method described in this article failed to do so, together with the differential behavior of these bodies toward inflammatory cells, suggests that native asbestos bodies should be used to investigate the pathogenetic role of these structures.

Puzzling Results from BAP1 Germline Mutations Analysis in a Group of Asbestos-Exposed Patients in a High-risk Area of Northeast Italy.

BACKGROUND: Germline mutations of the oncosuppressor gene breast cancer 1-associated protein 1 (BAP1) were recently related to an autosomal-dominant tumor predisposition syndrome (BAP1-TPDS), characterized by uveal melanoma, malignant mesothelioma (MM), cutaneous melanoma, and other malignancies. The demonstration that BAP1 mutations are strongly associated with MM has provided a real breakthrough in the study of genetic predisposition in MM, that may explain why only a fraction of asbestos-exposed individuals go on to develop MM. MATERIALS AND METHODS: To evaluate the possible role of BAP1 mutations in the epidemiology of sporadic MM, and their relationship with asbestos exposure, we determined the prevalence of germline BAP1 mutations by the Sanger method in a group of 29 asbestos-exposed patients, 21 of which were diagnosed with MM. They were residents of Trieste, a ship-building town in Northeast Italy with a very high incidence of mesothelioma. RESULTS: We identified non-obviously pathogenetic germline sequence variants of BAP1 in 3/29 patients and in 2/21 MM cases (10%). CONCLUSION: Non obviously pathogenic germline sequence variants of BAP1 were found. Nevertheless, limitations of predictive web tools allowed us to comment on some interesting peculiarities of our findings.

Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice.

Human exposure to asbestos can cause a wide variety of lung diseases that are still a current major health concern, even if asbestos has been banned in many countries. It has been shown in many studies that asbestos fibers, ingested by alveolar macrophages, disrupt lung iron homeostasis by sequestering iron. Calcium can also be deposited on the fibers. The pathways along which iron and above all calcium interact with fibers are still unknown. Our aim was that of investigating if the iron accumulation induced by the inhaled asbestos fibers also involves calcium ions accumulation. Lung sections of asbestos-exposed mice were analyzed using an extremely sensitive procedure available at the synchrotron facilities, that provides morphological and chemical information based on X-ray fluorescence microspectroscopy (µ-XRF). In this study we show that (1) where conventional histochemical procedures revealed only weak deposits of iron and calcium, µ-XRF analysis is able to detect significant deposits of both iron and calcium on the inhaled asbestos fibers; (2) the extent of the deposition of these ions is proportionally directly related and (3) iron and calcium deposition on inhaled asbestos fibers is concomitant with the appearance of inflammatory and hyperplastic reactions.

Intra-abdominal ovarian-type mucinous cystadenoma associated with fallopian tube-like structure and aberrant epididymal tissue in a male patient.

Ovarian-type mucinous tumors may occasionally occur in the retroperitoneum, pancreas, and liver exclusively or almost exclusively in women. In men, only few cases of such neoplasms arising within or around the testis have been reported. We describe a unique case of an ovarian-type mucinous cystadenoma occurring in the peritoneal cavity of a 65-year-old male patient with secondary adrenocortical insufficiency and hypogonadism. There was a typical fallopian tube enclosed in the capsule of the tumor. Contiguous to that, accessory ectopic epididymal tissue was found. We have interpreted this case as the result of a minor disorder of embryonic development involving structures of both müllerian and wolffian origin. The disproportion of sexual hormones might have had a role in inducing intra-abdominal müllerian remnants to give rise to the mucinous cystadenoma.

[Inflammatory pseudotumour of the spleen: a case report and review of the literature]. / Pseudotumore infiammatorio della milza: presentazione di un caso e revisione della letteratura.

The case of a 64-year-old man presenting dysuria and haematuria is described. The cause of these symptoms was related to a splenomegaly compressing the left kidney and renal pelvis. A splenectomy was performed. Macro- and microscopic examination of the mass revealed an inflammatory pseudotumour of the spleen, composed of a variable mixture of polyclonal lymphocytes, eosinophils, neutrophils, plasmacells, foamy hystiocytes, giant cells and fibrous tissue. The aetiology is unknown. An inflammatory pseudotumour of the spleen is clearly a reactive lesion resulting from a variety of causes, particularly vascular thrombosis, infections or autoimmune mechanisms. Splenectomy is both diagnostic and curative.

Differential protein folding and chemical changes in lung tissues exposed to asbestos or particulates.

Environmental and occupational inhalants may induce a large number of pulmonary diseases, with asbestos exposure being the most risky. The mechanisms are clearly related to chemical composition and physical and surface properties of materials. A combination of X-ray fluorescence (µXRF) and Fourier Transform InfraRed (µFTIR) microscopy was used to chemically characterize and compare asbestos bodies versus environmental particulates (anthracosis) in lung tissues from asbestos exposed and control patients. µXRF analyses revealed heterogeneously aggregated particles in the anthracotic structures, containing mainly Si, K, Al and Fe. Both asbestos and particulates alter lung iron homeostasis, with a more marked effect in asbestos exposure. µFTIR analyses revealed abundant proteins on asbestos bodies but not on anthracotic particles. Most importantly, the analyses demonstrated that the asbestos coating proteins contain high levels of ß-sheet structures. The occurrence of conformational changes in the proteic component of the asbestos coating provides new insights into long-term asbestos effects.

Evidence for a proangiogenic activity of TNF-related apoptosis-inducing ligand.

Starting from the observation that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2L protein is expressed in both malignant and inflammatory cells in some highly vascularized soft tissue sarcomas, the angiogenic potential of TRAIL was investigated in a series of in vitro assays. Recombinant soluble TRAIL induced endothelial cell migration and vessel tube formation to a degree comparable to vascular endothelial growth factor (VEGF), one of the best-characterized angiogenic factors. However, the proangiogenic activity of TRAIL was not mediated by endogenous expression of VEGF. Although TRAIL potentiated VEGF-induced extracellular signal-regulated kinase (ERK) phosphorylation and endothelial cell proliferation, the combination of TRAIL + VEGF did not show additive effects with respect to VEGF alone in inducing vessel tube formation. Thus, although TRAIL has gained attention as a potential anticancer therapeutic for its ability to induce apoptosis in a variety of cancer cells, our present data suggest that TRAIL might also play an unexpected role in promoting angiogenesis, which might have therapeutic implications.

Correlation between vascular endothelial growth factor, angiogenesis, and tumor-associated macrophages in invasive ductal breast carcinoma.

Angiogenic and anti-angiogenic factors, secreted by tumor, inflammatory, and stromal cells play an important role in regulation of neovascularization. Among the most important of these is vascular endothelial growth factor (VEGF), a specific mitogen for endothelium, which increases vascular permeability and induces proteolytic enzymes necessary for vascular remodeling. Tumor-associated macrophages (TAMs) can express complex functions related to tumor biology, including growth, proliferative rate, stroma formation and dissolution, and neovascularization. The aim of this study was to define, using immunohistochemical analysis, the microvessel density (MVD), VEGF expression, and TAMs level in 97 human invasive ductal breast carcinomas not otherwise specified (NOS), investigate a possible relationship between them and then correlate their values with tumor grade, mitotic activity index (MAI), tumor size and lymph-node status. Statistical analysis showed a strong positive relationship between MVD and VEGF expression ( P<0.001). Furthermore, both MVD and VEGF expression were significantly correlated with tumor grade and lymph-node status, and TAMs infiltration with MAI. TAM level showed a significant positive connection with VEGF expression and MVD. These in situ observations suggest that VEGF stimulates angiogenesis in human invasive ductal breast carcinoma NOS and attracts macrophages to the tumor locus, which then may be involved in angiogenesis promotion. The expression of this angiogenic molecule, and MVD and TAM level, can provide additional prognostic significance and help in the identification of patients who need postoperative adjuvant therapy.

Do Ki67, S-phase, S + G2M and DNA ploidy, evaluated by flow cytometry, reveal locoregional metastasis in oral cavity and oropharynx carcinomas?

Tumour cell proliferation is an important biological prognostic parameter to be considered alongside clinical and histopathological parameters. It has been evaluated by immunohistochemistry using proliferative markers (PCNA, Ki67, etc.) and by flow cytometry considering DNA content, growth fraction (S + G2M) and S-phase fraction. Our aim was to evaluate by flow cytometry both Ki67 and S-phase fraction, S + G2M, DNA content in patients surgically treated for oral cavity and/or oropharynx carcinomas. The study, performed on archival material, analysed the significance and prognostic reliability of these biological parameters to reveal locoregional metastasis and evaluated their possible correlation with clinical and histopathological parameters. In conclusion, tumour cell proliferation seems not to be useful in revealing the presence of locoregional metastasis in oral cavity and oropharynx carcinomas. Thus, new approaches are required.

Atypical fibroxanthoma and malignant fibrous histiocytoma of the skin.

A case of cutaneous atypical fibroxanthoma (AFX) which metastasized, leading to patient death, is described. This is the third reported case in which a lesion qualified as AFX behaves in such an aggressive manner, comparable with that of malignant fibrous histiocytoma (MFH). Ki-67 proliferation index, p53 and bcl-2 protein expression and DNA ploidy were also consistent with a highly malignant potential. The controversial issue of the biological behavior of AFX and its relationship with MFH is discussed. The authors conclude that MFH and AFX probably represent a single entity with a split biological "personality".