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Asbestos fiber exposure triggers chronic inflammation and cancer. Asbestos fibers can adsorb different types of proteins. The mechanism of this adsorption, not yet completely understood, has been studied in detail mainly with serum albumin and was shown to induce structural changes in the bound protein. The findings of these works regarded mainly the changes of the protein structure, independently of any relation with asbestos-related diseases. For the first time, we have focused our attention to the consequences of the interaction between asbestos fibers and ferritin, a protein involved in iron metabolism, which is strongly modified in asbestos-related diseases. Even if it is known that ferritin can be adsorbed by asbestos fibers, the results of this interaction for the ferritin secondary structure has not previously been studied. One consequence of asbestos-ferritin interaction, is the formation of the so-called ferruginous/asbestos bodies (ABs). In the AB-coating material, the secondary structure of ferritin is modified, and at present, it is unclear whether or not this modification is a direct consequence of the asbestos interaction. In the present study, chrysotile asbestos, more than other asbestos fiber types tested, was found to rapidly bind holo-ferritin, and the presence of iron seemed to play a key role in this process, since iron-free apo-ferritin was adsorbed at a lower level, and iron-saturated chrysotile lost its ferritin-adsorbing capacity. To directly study the details of ferritin adsorption on asbestos fibers, High Resolution Transmission Electron Microscopy (HR-TEM) was employed together with FTIR microspectroscopy and Infrared nanoscopy, which to the best of our knowledge, have not previously been used for this purpose. Chrysotile-bound apo-ferritin underwent a significant change in secondary structure, showing a shift from a prevalent α-helix to a β-sheet conformation. Conversely, the adsorbed holo-ferritin structure appeared to be only weakly modified. These findings add a new potential mechanism to the toxic activities of asbestos: the fibers can modify the structure, and very likely, the function of adsorbed proteins. This, in relation to ferritin, could be a key mechanism in cell iron homeostasis alteration, typically reported in asbestos-related diseases., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Many years ago, asbestos fibers were banned and replaced by synthetic vitreous fibers because of their carcinogenicity. However, the toxicity of the latter fibers is still under debate, especially when it concerns the early fiber interactions with biological cell membranes. Here, we aimed to investigate the effects of a synthetic vitreous fiber named FAV173 on the Xenopus laevis oocyte membrane, the cell model we have already used to characterize the effect of crocidolite asbestos fiber exposure. Using an electrophysiological approach, we found that, similarly to crocidolite asbestos, FAV173 was able to stimulate a chloride outward current evoked by step membrane depolarizations, that was blocked by the potent and specific TMEM16A channel antagonist Ani9. Exposure to FAV173 fibers also altered the oocyte cell membrane microvilli morphology similarly to crocidolite fibers, most likely as a consequence of the TMEM16A protein interaction with actin. However, FAV173 only partially mimicked the crocidolite fibers effects, even at higher fiber suspension concentrations. As expected, the crocidolite fibers' effect was more similar to that induced by the co-treatment with (Fe 3+ + H 2 O 2 ), since the iron content of asbestos fibers is known to trigger reactive oxygen species (ROS) production. Taken together, our findings suggest that FAV173 may be less harmful that crocidolite but not ineffective in altering cell membrane properties.

One of the main problems related to ferruginous-asbestos bodies (ABs) exposure is their potential pathogenetic role in asbestos-related diseases. The aim of this study was to examine whether purified ABs, might stimulate inflammatory cells. ABs were isolated by exploiting their magnetic properties, therefore avoiding the strong chemical treatment usually employed for this purpose. This latter treatment, which is based upon the digestion of organic matter with concentrated hypochlorite, may markedly modify the AB structure and consequently also their "in vivo" manifestations. ABs were found to induce secretion of human neutrophil granular component myeloperoxidase, as well as stimulate rat mast cell degranulation. Data demonstrated that by triggering secretory processes in inflammatory cells, purified ABs may play a role in the pathogenesis of asbestos-related diseases by continuing and enhancing the pro-inflammatory activity of the asbestos fibers.

Background: The interaction of asbestos fibers with target cell membranes is still poorly investigated. Here, we detected and characterized an enhancement of chloride conductance in Xenopus oocyte cell membranes induced by exposure to crocidolite (Croc) asbestos fibers., Methods: A two-microelectrode voltage clamp technique was used to test the effect of Croc fiber suspensions on outward chloride currents evoked by step membrane depolarization. Calcium imaging experiments were also performed to investigate the variation of 'resting' oocyte [Ca 2+ ] i following asbestos exposure., Results: The increase in chloride current after asbestos treatment, was sensitive to [Ca 2+ ] e , and to specific blockers of TMEM16A Ca 2+ -activated chloride channels, MONNA and Ani9. Furthermore, asbestos treatment elevated the 'resting' [Ca 2+ ] i likelihood by increasing the cell membrane permeability to Ca 2 in favor of a tonic activation of TMEME16A channels . Western blot analysis confirmed that TMEME16A protein was endogenously present in the oocyte cell membrane and absorbed by Croc., Conclusion: the TMEM16A channels endogenously expressed by Xenopus oocytes are targets for asbestos fibers and represent a powerful tool for asbestos-membrane interaction studies. Interestingly, TMEM16A channels are highly expressed in many types of tumors, including some asbestos-related cancers, suggesting them, for the first time, as a possible early target of crocidolite-mediated tumorigenic effects on target cell membranes.

Asbestos fibers interact with many different proteins and may affect either their structure or functions. The aim of this study was to determine whether ferritin absorbed onto fibers might modify its ferroxidase activity. By measuring apo-ferritin ferroxidase activity, data demonstrated that ferritin in the presence of fibers did not significantly modify this enzymatic activity. However, fibers in the absence of ferritin promoted ferrous iron oxidation. Evidence suggests that asbestos fibers may promote iron oxidation and subsequently affect cellular iron homeostatic mechanisms.

The variant enrichment analysis (VEA), a recently developed bioinformatic workflow, has been shown to be a valuable tool for whole-exome sequencing data analysis, allowing finding differences between the number of genetic variants in a given pathway compared to a reference dataset. In a previous study, using VEA, we identified different pathway signatures associated with the development of pulmonary toxicities in mesothelioma patients treated with radical hemithoracic radiation therapy. Here, we used VEA to discover novel pathways altered in individuals exposed to asbestos who developed or not asbestos-related diseases (lung cancer or mesothelioma). A population-based autopsy study was designed in which asbestos exposure was evaluated and quantitated by investigating objective signs of exposure. We selected patients with similar exposure to asbestos. Formalin-fixed paraffin-embedded (FFPE) tissues were used as a source of DNA and whole-exome sequencing analysis was performed, running VEA to identify potentially disrupted pathways in individuals who developed thoracic cancers induced by asbestos exposure. By using VEA analysis, we confirmed the involvement of pathways considered as the main culprits for asbestos-induced carcinogenesis: oxidative stress and chromosome instability. Furthermore, we identified protective genetic assets preserving genome stability and susceptibility assets predisposing to a worst outcome.

Radical hemithoracic radiotherapy (RHR), after lung-sparing surgery, has recently become a concrete therapeutic option for malignant pleural mesothelioma (MPM), an asbestos-related, highly aggressive tumor with increasing incidence and poor prognosis. Although the toxicity associated to this treatment has been reduced, it is still not negligible and must be considered when treating patients. Genetic factors appear to play a role determining radiotherapy toxicity. The aim of this study is the identification of biological pathways, retrieved through whole exome sequencing (WES), possibly associated to the development of lung adverse effects in MPM patients treated with RHR. The study included individuals with MPM, treated with lung-sparing surgery and chemotherapy, followed by RHR with curative intent, and followed up prospectively for development of pulmonary toxicity. Due to the strong impact of grade 3 pulmonary toxicities on the quality of life, compared with less serious adverse events, for genetic analyses, patients were divided into a none or tolerable pulmonary toxicity (NoSTox) group (grade ≤2) and a severe pulmonary toxicity (STox) group (grade = 3). Variant enrichment analysis allowed us to identify different pathway signatures characterizing NoSTox and Stox patients, allowing to formulate hypotheses on the protection from side effects derived from radiotherapy as well as factors predisposing to a worst response to the treatment. Our findings, being aware of the small number of patients analyzed, could be considered a starting point for the definition of a panel of pathways, possibly helpful in the management of MPM patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Crovella, Revelant, Muraro, Moura, Brandão, Trovò, Steffan, Zacchi, Zabucchi, Minatel and Borelli.)

Endometriosis is a local pelvic inflammatory process, frequently associated with infertility, with altered function of immune-related cells in the peritoneal environment. Mast cells are known to be key players of the immune system and have been recently involved in endometriosis and in infertility, with their mediators directly suppressing sperm motility. In this study, we evaluated the mast cell population and their mediators in the peritoneal fluid of infertile patients with endometriosis and their impact on human sperm motility. Peritoneal fluids, collected by laparoscopy from 11 infertile patients with endometriosis and 9 fertile controls were evaluated for the presence of mast cells, tryptase levels and their effect on sperm motility. Furthermore, an in vitro model of mast cells-sperm interaction in peritoneal fluid was set up, using LAD2 cell line as a mast cell model, and analyzed from a functional as well as a morphological point of view. Mast cell peritoneal fluid population and its main mediator, tryptase, is more represented in endometriosis confirming an involvement of these cells in this disease. Anyway it appears unlikely that tryptase enriched peritoneal fluid, which fails to inhibit sperm motility, could contribute to endometriosis associated infertility. Despite of this, sperm interaction with the mast cell surface (LAD2) induced a significantly mast cell-degranulation response in the peritoneal fluid from endometriosis which could directly modulate sperm function other than motility. This evidence lead us to suppose that there is, between these elements, an interrelationship which deserves further studies., (Copyright © 2020 Borelli, Martinelli, Luppi, Vita, Romano, Fanfani, Trevisan, Celsi, Zabucchi, Zanconati, Bottin and Ricci.)

Photodynamic therapy (PDT) has become an emerging novel therapeutic approach for treating localized microbial infections, particularly those sustained by multidrug-resistant strains. Given the irreplaceable role played by professional phagocytes in limiting infections, such as polymorphonuclear neutrophils, any newly designed antimicrobial therapeutic approach must not interfere with their function. The present investigation presents a detailed analysis of the effect of PDT on the viability and several functional responses of human polymorphonuclear neutrophils loaded with methylene blue (MB), one of the more commonly used photosensitizers in antimicrobial PDT. Taking advantage of the use of a specifically-designed optical LED array for illuminating MB-loaded human polymorphonuclear neutrophils, a number of cell functions have been assayed under miniaturized, strictly controlled and reproducible experimental conditions. The major findings of this study are the following: (1) MB-PDT increases human neutrophils adhesion and does not modify myeloperoxidase release; (2) MB-PDT markedly enhances reactive oxygen species generation that is independent of superoxide-forming phagocytic oxidase and very likely ascribable to LED-dependent excitation of accumulated methylene blue; (3) MB-PDT almost abolishes human neutrophils candidacidal activity by hindering the engulfing machinery. This in vitro study may represent a valuable reference point for future research on PDT applications for treating localized microbial infections., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The so-called amphibole asbestos fibers are enriched with mineral iron ions, able to stimulate ROS production. We recently reported that crocidolite asbestos was able to interact with the cell membranes of Xenopus laevis oocytes, to alter their electrical membrane properties. Here, we found that applied iron ions (Fe 3+ ) or H 2 O 2 (for ROS generation) mimicked these effects, suggesting that at least one effect of iron-containing asbestos fiber exposure was mediated by ROS production. Furthermore, combined Fe 3+ and H 2 O 2 acted synergistically, producing a membrane effect stronger than that induced by these factors alone. Similar to crocidolite, these changes peaked within 30 minutes of incubation and vanished almost completely after 120 min. However, in the presence of cytochalasin D, which inhibits membrane actin repair mechanisms, crocidolite or applied Fe 3+ /H 2 O 2 invariably produced oocyte cell death. While the electrophysiological modifications induced by crocidolite suggested a modification of an intrinsic chloride ion channel, the morphological appearance of the treated oocytes also indicated the formation of membrane "pores"; the effects of asbestos exposure may therefore consist of multiple (not necessarily exclusive) underlying mechanisms. In conclusion, using Xenopus oocytes allowed us for the first time, to focus on a specific membrane effect of crocidolite asbestos exposure, which deserves to be tested also on human lung cell lines. Much available evidence suggests that asbestos fibers damage cells through the production of ROS. Our present data confirm that crocidolite fibers can indeed trigger ROS-mediated damaging effects in the oocyte cell membrane, provided iron ions and H 2 O 2 are available for ROS production.

Two of the major cancerous diseases associated with asbestos exposure are malignant pleural mesothelioma (MPM) and lung cancer (LC). In addition to asbestos exposure, genetic factors have been suggested to be associated with asbestos-related carcinogenesis and lung genotoxicity. While genetic factors involved in the susceptibility to MPM were reported, to date the influence of individual genetic variations on asbestos-related lung cancer risk is still poorly understood. Since inflammation and disruption of iron (Fe) homeostasis are hallmarks of asbestos exposure affecting the pulmonary tissue, this study aimed at investigating the association between Fe-metabolism and inflammasome gene variants and susceptibility to develop LC or MPM, by comparing an asbestos-exposed population affected by LC with an "asbestos-resistant exposed population". A retrospective approach similar to our previous autopsy-based pilot study was employed in a novel cohort of autoptic samples, thus giving us the possibility to corroborate previous findings obtained on MPM by repeating the analysis in a novel cohort of autoptic samples. The protective role of HEPH coding SNP was further confirmed. In addition, the two non-coding SNPs, either in FTH1 or in TF , emerged to exert a similar protective role in a new cohort of LC exposed individuals from the same geographic area of MPM subjects. No association was found between NLRP1 and NLRP3 polymorphisms with susceptibility to develop MPM and LC. Further research into a specific MPM and LC "genetic signature" may be needed to broaden our knowledge of the genetic landscape attributed to result in MPM and LC.

Background: Exposure to mineral fibers is of substantial relevance to human health. A key event in exposure is the interaction with inflammatory cells and the subsequent generation of pro-inflammatory factors. Mast cells (MCs) have been shown to interact with titanium oxide (TiO₂) and asbestos fibers. In this study, we compared the response of rat peritoneal MCs challenged with the asbestos crocidolite and nanowires of TiO₂ to that induced by wollastonite employed as a control fiber., Methods: Rat peritoneal MCs (RPMCs), isolated from peritoneal lavage, were incubated in the presence of mineral fibers. The quantities of secreted enzymes were evaluated together with the activity of fiber-associated enzymes. The ultrastructural morphology of fiber-interacting RPMCs was analyzed with electron microscopy., Results: Asbestos and TiO₂ stimulate MC secretion. Secreted enzymes bind to fibers and exhibit higher activity. TiO₂ and wollastonite bind and improve enzyme activity, but to a lesser degree than crocidolite., Conclusions: (1) Mineral fibers are able to stimulate the mast cell secretory process by both active (during membrane interaction) and/or passive (during membrane penetration) interaction; (2) fibers can be found to be associated with secreted enzymes-this process appears to create long-lasting pro-inflammatory environments and may represent the active contribution of MCs in maintaining the inflammatory process; (3) MCs and their enzymes should be considered as a therapeutic target in the pathogenesis of asbestos-induced lung inflammation; and (4) MCs can contribute to the inflammatory effect associated with selected engineered nanomaterials, such as TiO₂ nanoparticles., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

The presence of asbestos bodies (ABs) in lung parenchyma is considered a histopathologic hallmark of past exposure to asbestos fibers, of which there was a population of longer fibers. The mechanisms underlying AB formation are complex, involving inflammatory responses and iron (Fe) metabolism. Thus, the responsiveness to AB formation is variable, with some individuals appearing to be poor AB formers. The aim of this study was to disclose the possible role of genetic variants of genes encoding inflammasome and iron metabolism proteins in the ability to form ABs in a population of 81 individuals from North East Italy, who died after having developed malignant pleural mesothelioma (MPM). This study included 86 genetic variants distributed in 10 genes involved in Fe metabolism and 7 genetic variants in two genes encoding for inflammasome molecules. Genotypes/haplotypes were compared according to the number of lung ABs. Data showed that the NLRP1 rs12150220 missense variant (H155L) was significantly correlated with numbers of ABs in MPM patients. Specifically, a low number of ABs was detected in individuals carrying the NLRP1 rs12150220 A/T genotype. Our findings suggest that the NLRP1 inflammasome might contribute in the development of lung ABs. It is postulated that the NLRP1 missense variant may be considered as one of the possible host genetic factors contributing to individual variability in coating efficiency, which needs to be taken when assessing occupational exposure to asbestos.

Background: Mast cells are key effector cells in allergic reactions. When activated to degranulate, they release a plethora of bioactive compounds from their secretory granules, including mast cell-restricted proteases such as tryptase. In a previous study, we showed that tryptase, in addition to its intragranular location, can be found within the nuclei of mast cells where it truncates core histones at their N-terminal ends., Objective: Considering that the N-terminal portions of the core histones constitute sites for posttranslational modifications of major epigenetic impact, we evaluated whether histone truncation by tryptase could have an impact on epigenetic events in mast cells., Methods: Mast cells were cultured from wild-type and tryptase null mice, followed by an assessment of their profile of epigenetic histone modifications and their phenotypic characteristics., Results: We show that tryptase truncates nucleosomal histone 3 and histone 2B (H2B) and that its absence results in accumulation of the epigenetic mark, lysine 5-acetylated H2B. Intriguingly, the accumulation of lysine 5-acetylated H2B was cell age-dependent and was associated with a profound upregulation of markers of non-mast cell lineages, loss of proliferative control, chromatin remodeling as well as extensive morphological alterations., Conclusions: These findings introduce tryptase-catalyzed histone clipping as a novel epigenetic regulatory mechanism, which in the mast cell context may be crucial for maintaining cellular identity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

This data article contains data related to the research article entitled, "Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice" [1]. Asbestos fibers disrupt iron homeostasis in the human and mouse lung, leading to the deposition of iron (Fe) onto longer asbestos fibers which forms asbestos bodies (AB) [2]. Similar to Fe, calcium (Ca) is also deposited in the coats of the AB. This article presents data on iron and calcium in the mouse lung after asbestos exposure detected by histochemical evaluation.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with poor prognosis. The development of MPM is frequently linked to inhalation of asbestos fibers. A genetic component of susceptibility to this disease is suggested by the observation that some individuals develop MPM following lower doses of asbestos exposure, whereas others exposed to higher quantities do not seem to be affected. This hypothesis is supported also by frequent reports of MPM familial clustering. Despite the widely recognized role of iron (Fe) in cellular asbestos-induced pulmonary toxicity, the role of the related gene polymorphisms in the etiology of MPM has apparently not been evaluated. Eighty-six single-nucleotide polymorphisms (SNPs) of 10 Fe-metabolism genes were examined by exploiting formalin-fixed paraffin-embedded postmortem samples from 77 patients who died due to MPM (designated AEM) and compared with 48 who were exposed to asbestos but from died in old age of cause other than asbestos (designated AENM). All subjects showed objective signs of asbestos exposure. Three SNPs, localized in the ferritin heavy polypeptide, transferrin, and hephaestin genes, whose frequencies were distributed differently in AEM and AENM populations, were identified. For ferritin and transferrin the C/C and the G/G genotypes, respectively, representing intronic polymorphisms, were significantly associated with protection against MPM and need to be considered as possible genetic markers of protection. Similarly, the C/C hephaestin SNP, a missense variation of this multicopper ferroxidase encoding gene, may be related, also functionally, with protection against MPM. In conclusion, it is proposed that three Fe metabolism-associated genes, significantly associated with protection against development of MPM, may serve as protective markers for this aggressive tumor.

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 β-sheet structures. The occurrence of conformational changes in the proteic component of the asbestos coating provides new insights into long-term asbestos effects.

The mode of interaction of asbestos fibres with cell membranes is still debatable. One reason is the lack of a suitable and convenient cellular model to investigate the causes of asbestos toxicity. We studied the interaction of asbestos fibres with Xenopus laevis oocytes, using electrophysiological and morphological methods. Oocytes are large single cells, with a limited ability to endocytose molecular ligands; we therefore considered these cells to be a good model for investigating the nature of asbestos/membrane interactions. Electrophysiological recordings were performed to compare the passive electrical membrane properties, and those induced by applying positive or negative voltage steps, in untreated oocytes and those exposed to asbestos fibre suspensions. Ultrastructural analysis visualized in detail, any morphological changes of the surface membrane caused by the fibre treatment. Our results demonstrate that Amosite and Crocidolite-type asbestos fibres significantly modify the properties of the membrane, starting soon after exposure. Cells were routinely depolarized, their input resistance decreased, and the slow outward currents evoked by step depolarizations were dramatically enhanced. Reducing the availability of surface iron contained in the structure of the fibres with cation chelators, abolished these effects. Ultrastructural analysis of the fibre-exposed oocytes showed no evidence of phagocytic events. Our results demonstrate that asbestos fibres modify the oocyte membrane, and we propose that these cells represent a viable model for studying the asbestos/cell membrane interaction. Our findings also open the possibly for finding specific competitors capable of hindering the asbestos-cell membrane interaction as a means of tackling the long-standing asbestos toxicity problem., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Mast cells (MC) play a key role in triggering the inflammatory process and share some functions with professional phagocytes. It is not clear whether or not the phagocytic process in MC follows the same route and has the same meaning of that of professional phagocytes. Herein we analyze in detail the structure of the phagosome in rat peritoneal mast cells (RPMC). The ultrastructural analysis of the phagosome, containing either model particles or bacteria, reveals that these vacuoles are very tight, and in several areas, their membrane seems to have dissolved. RPMC express NOD1 and NOD2 proteins whose role is to recognize intracellular foreign components and induce the production of pro-inflammatory mediators. Following Escherichia coli ingestion, both these molecules are found on the phagosome membrane and on ingested pathogens, together with phagosome maturation markers. These findings suggest that in RPMC the ingested cargo can, through interruptions of the phagosome membrane, interact directly with NODs, which act as switches in the process of cytokine production.

Globozoospermia is a rare disorder characterized by the presence of sperm with round head, lacking acrosome. Coiling tail around the nucleus has been reported since early human studies, but no specific significance has conferred it. By contrast, studies on animal models suggest that coiling tail around the nucleus could represent a crucial step of defective spermatogenesis, resulting in round-headed sperm. No observations, so far, support the transfer of this hypothesis to human globozoospermia. The purpose of this work was to compare ultrastructural morphology of human and mouse model globozoospermic sperm. Sperm have been investigated by using scanning and transmission electron microscopy. The images that we obtained show significant similarities to those described in GOPC knockout mice, an animal model of globozoospermia. By using this model as reference, we were able to identify the probable steps of the tail coiling process in human globozoospermia. Although we have no evidence that there is the same pathophysiology in man and knocked-out mouse, the similarities between these ultrastructural observations in human and those in the experimental model are very suggestive. This is the first demonstration of the existence of relevant morphological homologies between the tail coiling in animal model and human globozoospermia.

Mast cells (MCs) reside in tissues that are common targets of Candida spp. infections, and can exert bactericidal activity, but little is known about their fungicidal activity. MCs purified from rat peritoneum (RPMC) and a clinical isolate of C. albicans, were employed. Ingestion was evaluated by flow cytometry (FACS) and optical microscopy. The killing activity was assayed by FACS analysis and by colony forming unit method. RPMC degranulation was evaluated by β-hexosaminidase assay and phosphatidylserine externalization by FACS. Phagocytosing RPMC were also analyzed by transmission electron microscopy. Herein, we show that the killing of C. albicans by RPMC takes place in the extracellular environment, very likely through secreted granular components. Ultrastructural analysis of the ingestion process revealed an unusual RPMC-C. albicans interaction that could allow fungal survival. Our findings indicate that MCs have a positive role in the defense mechanism against Candida infections and should be included among the cell types involved in host-defense against this pathogen.

A hallmark feature of mast cells is their high content of cytoplasmic secretory granules filled with various preformed compounds, including proteases of tryptase-, chymase-, and carboxypeptidase A3 type that are electrostatically bound to serglycin proteoglycan. Apart from participating in extracellular processes, serglycin proteoglycan and one of its associated proteases, tryptase, are known to regulate cell death by promoting apoptosis over necrosis. Here we sought to outline the underlying mechanism and identify core histones as primary proteolytic targets for the serglycin-tryptase axis. During the cell death process, tryptase was found to relocalize from granules into the cytosol and nucleus, and it was found that the absence of tryptase was associated with a pronounced accumulation of core histones both in the cytosol and in the nucleus. Intriguingly, tryptase deficiency resulted in defective proteolytic modification of core histones even at baseline conditions, i.e. in the absence of cytotoxic agent, suggesting that tryptase has a homeostatic impact on nuclear events. Indeed, tryptase was found in the nucleus of viable cells and was shown to cleave core histones in their N-terminal tail. Moreover, it was shown that the absence of the serglycin-tryptase axis resulted in altered chromatin composition. Together, these findings implicate histone proteolysis through a secretory granule-derived serglycin-tryptase axis as a novel principle for histone modification, during both cell homeostasis and cell death.

Mast cell degranulation requires N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and mammalian uncoordinated18 (Munc18) fusion accessory proteins for membrane fusion. However, it is still unknown how their interaction supports fusion. In this study, we found that small interfering RNA-mediated silencing of the isoform Munc18-2 in mast cells inhibits cytoplasmic secretory granule (SG) release but not CCL2 chemokine secretion. Silencing of its SNARE-binding partner syntaxin 3 (STX3) also markedly inhibited degranulation, whereas combined knockdown produced an additive inhibitory effect. Strikingly, while Munc18-2 silencing impaired SG translocation, silencing of STX3 inhibited fusion, demonstrating unique roles of each protein. Immunogold studies showed that both Munc18-2 and STX3 are located on the granule surface, but also within the granule matrix and in small nocodazole-sensitive clusters of the cytoskeletal meshwork surrounding SG. After stimulation, clusters containing both effectors were detected at fusion sites. In resting cells, Munc18-2, but not STX3, interacted with tubulin. This interaction was sensitive to nocodazole treatment and decreased after stimulation. Our results indicate that Munc18-2 dynamically couples the membrane fusion machinery to the microtubule cytoskeleton and demonstrate that Munc18-2 and STX3 perform distinct, but complementary, functions to support, respectively, SG translocation and membrane fusion in mast cells.

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., (© 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.)

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.

Epithelial-to-mesenchymal transition (EMT) is the fundamental process by which an epithelial cell loses its epithelial characteristics including cell polarity and acquires mesenchymal and stemness-related features. Therefore, we investigated whether malignant pleural mesothelioma (MPMs) histologies were associated with specific patterns of expression of a selected set of genes related to EMT, cell polarity and stemness features. The association between MPM histologies and genes expression were explored using active and passive Principal Components Analysis-based biplots and PAM analysis that provided evidence that with respect to normal tissues, MPMs histologies were better characterized by specific patterns of expression of genes involved in EMT activation, cell polarity and stemness., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Mast cells (MCs) and eosinophils (Eos) are the key effector cells of the allergic reaction. Although classically associated with different stages of the response, the cells co-exist in the inflamed tissue in the late and chronic phases in high numbers and are likely to cross-talk. While some mediators of MCs are known to affect Eos biology and vice versa, paracrine and physical interplay between the two cells has not been described yet. We aimed to investigate whether intercellular MC-Eos communication could take place in the allergic response and exert functional bidirectional changes on the cells., Methods: Tissue sections from various allergic disorders were specifically stained for both cells. Human cord blood-derived MCs and peripheral blood Eos, co-cultured under different conditions, were studied by advanced microscopy and flow cytometry., Results: Several co-localized MC-Eos pairs were detected in human nasal polyps and asthmatic bronchi, as well in mouse atopic dermatitis. In vitro, MCs and Eos formed stable conjugates at high rates, with clear membrane contact. In the presence of MCs, Eos were significantly more viable under several co-culture conditions and at both IgE-activated and steroid-inhibited settings. MC regulation of Eos survival required communication through soluble mediators but was even more dependent on physical cell-cell contact., Conclusions: Our findings provide the first evidence for a complex network of paracrine and membrane interactions between MCs and Eos. The prosurvival phenotype induced by this MC-Eos interplay may be critical for sustaining chronic allergic inflammation., (© 2010 John Wiley & Sons A/S.)

Background: FAS has been shown to be involved in the regulation of many immune processes by induction of cellular apoptosis. However, accumulated evidence shows that FAS signaling also exhibits nonapoptotic functions, such as induction of cell proliferation and differentiation. FAS is the only death receptor known to be expressed on murine mast cells (MCs)., Objective: To evaluate the role of FAS on murine MC maturation., Methods: Mouse bone marrow-derived MCs (BMMCs) or peritoneal MCs were derived from FAS-deficient, FASlpr/lpr, and congenic wild-type strains. The MC degranulation and cytokine release after IgE activation was assessed by measuring β-hexosaminidase, interleukin 13, and tumor necrosis factor α release. Transmission electron microscopy analysis was performed to evaluate the level of BMMC maturation. The surface markers and intracellular preformed mediators were measured as well., Results: Our data reveal that FAS deficiency has an impact on IgE-dependent activation of BMMCs, resulting in a significant decrease in β-hexosaminidase, interleukin 13, and tumor necrosis factor α release. The total content of preformed mediators (eg, tryptase and β-hexosaminidase) was reduced in BMMCs derived from FAS-deficient mice. We also found that the level of FcεRI in peritoneal mast cells from FAS-deficient mice was significantly diminished. FAS deficiency also influenced the kinetics of BMMC maturation as was revealed by transmission electron microscopy analysis., Conclusion: Our data show that FAS has an impact on the regulation of mouse MC maturation in vitro., (Copyright © 2011 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Introduction: Bacillus Calmette-Guerin (BCG) is used to treat high risk superficial bladder cancer, but its anti-tumor effect remains incompletely defined. Recently a role for polymophonuclear (PMN) neutrophils has been suggested. To investigate the role of granulocytes, we monitored the activation state of these cells in the urine of BCG-treated patients., Materials and Methods: Ten patients with stage T1, grade 3 (T1G3) transitional cell carcinoma received an 8 week course of BCG after undergoing transurethral resection of the bladder. Cytological and enzymatic analyses of urine samples collected before and 2 hours after the physiological expulsion of BCG were performed. The activation state of urine granulocytes and the presence of activating factors within the urine samples were monitored., Results: BCG immunotherapy stimulated, through soluble factors, the activation of PMN neutrophils, which transmigrated into the bladder, and the degree of activation of the PMN neutrophils was related directly to the number of epithelial cells detached from the urothelial layer., Conclusions: This study suggests that PMN neutrophils can participate in reducing the recurrence of bladder cancer by promoting urothelial cell turnover proportionally to their degree of activation. Our results provide further evidence to support the role of PMN neutrophils in BCG immunotherapy.

We have hypothesized that mast cells (MC) and eosinophils (Eos), the main effectors of allergy, can form an effector unit. These cells co-exist in the inflamed tissues during the late and chronic stages of allergy and have been shown to be capable of influencing each other's survival and activity via soluble mediators. We have recently described couples of receptor-ligands that are expressed on either/both of these cells and that imply a physical interaction. In this study, we have investigated the existence of short-term (60 min) in vitro interactions between human peripheral blood Eos and cord-blood-derived MC by transmission electron microscopy. We have found that MC and Eos adhere to each other; the lipid body content and the granule morphology of co-cultured MC and Eos, respectively, are altered, and the level of Eos peroxidase (EPO) released by co-cultured Eos is elevated. Moreover, the transfer of EPO from Eos to MC and tryptase from MC to Eos has been observed. Our results thus indicate that, when co-cultured, MC and Eos show signs of physical contact and of reciprocal activation. This is the first in vitro evidence of functional physical interactions between human MC and Eos, interactions that might also occur in vivo during allergic diseases.

Objective: A neuropathic basis has been suggested for burning mouth syndrome (BMS) and an altered concentration of neuropeptides has been reported in lingual oral mucosa and saliva in this disease. The aims of this study were to compare the levels of nerve growth factor (NGF), substance P (SP) and degranulation products from mast cells and neutrophils in the saliva of BMS subjects with those of control subjects., Material and Methods: Salivary flow rate, protein concentration, NGF peptide and mRNA, SP, mast cells tryptase, neutrophil myeloperoxidase and calprotectin were analyzed in saliva of 20 BMS subjects and of 20 age- and gender-matched healthy subjects., Results and Conclusions: NGF peptide and tryptase activity were shown to be significantly and persistently higher in saliva of BMS subjects, with respect to control values. Conversely the salivary levels of SP were shown to be significantly lower, while neutrophil markers didn't show any change. We conclude that the neuropathic origin of the disease is confirmed at salivary level. Furthermore, the higher tryptase activity indicates a possible involvement of mast cells. The salivary neuropeptide concentration in BMS subjects, together with mast cell derived compounds, could be useful biomarkers for diagnosis and monitoring of this disease.

In this paper we show that rat peritoneal mast cells (RPMC) adhere to rat oligodendrocytes (ODC) in culture and switch on a bi-directional signal affecting both adhering cell and its target. Following heterotypic interaction, RPMC release granule content and ODC show morphological changes and enter the apoptotic programme. Altogether, these findings indicate that the interaction of MC with ODC could play a role in the mechanism of CNS damage induced by the inflammatory reaction., (Copyright 2009 Elsevier B.V. All rights reserved.)

Newborn rat oligodendrocyte cultures were investigated by scanning near-field optical microscope (SNOM), a versatile new tool able to map cell membranes in 3D and simultaneously obtain images of the cytoplasm. Topography, error, transmission and reflection signals were acquired to describe cell morphology with nanometer-scale resolution. Oligodendrocytes were studied as a model because their extensive membrane processes (typical of their physiological role in myelination) made them particularly suitable to test the sensitivity of the new method. Furthermore, we combined a classical histochemical method with SNOM, to identify specific intracellular proteins at high definition. In particular, with this technique, cytoskeleton elements of oligodendrocytes, such as microtubules, were observed with tubulin antibodies. Images obtained with SNOM were also compared with those from conventional scanning electron microscopy (SEM) and optical microscopy. Our results showed that SNOM allowed to observe cell nanostructures otherwise undetectable all together with other microscopies. In conclusion, SNOM, combined with rapid and non-invasive methods of specimen preparation, appears to be a powerful tool that can offer new possibilities in the field of neuroscience imaging at nano-scale level.

Human neutrophil granule exocytosis mobilizes a complex set of secretory granules. This involves different combinations of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins to facilitate membrane fusion. The control mechanisms governing the late fusion steps are still poorly understood. Here, we have analyzed SNARE-interacting Sec1/Munc18 (SM) family members. We found that human neutrophils express Munc18-2 and Munc18-3 isoforms and that Munc18-2 interacts with the target-SNARE syntaxin 3. Munc18-2 was associated preferentially with primary granules but could also be found with secondary and tertiary granules, while Munc18-3 was majorily associated with secondary and tertiary granules. Ultrastructural analysis showed that both Munc18-2 and Munc18-3 were often located in close proximity to their respective SNARE-binding partners syntaxin 3 and syntaxin 4. Both isoforms were also found in plasma membrane fractions and in the cytosol, where they associate with cytoskeletal elements. Upon stimulation, Munc18-2 and Munc18-3 redistributed and became enriched on granules and in the plasma membrane. Munc18-2 primary granule exocytosis can be blocked by introduction of Munc18-2-specific antibodies indicating a crucial role in primary granule fusion. Our results suggest that Munc18-2 acts as a regulator of primary granule exocytosis, while Munc18-3 may preferentially regulate the fusion of secondary granules.

A role for mast cells (MC) in the pathogenesis of multiple sclerosis (MS) has been suggested, based on the analysis of human lesions and on an animal model of the disease (EAE). What role MC play in the development of MS is not well understood. We hypothesized that the link connecting MC with demyelinating diseases may be represented by their interaction with myelin. Here we show that myelin can activate mast cells. This process could be a key event in the mast cell function required for inducing EAE in mice and possibly in MS in man.

Inflammatory responses by mast cells are characterized by massive exocytosis of prestored granular mediators followed by cytokine/chemokine release. The vesicular trafficking mechanisms involved remain poorly understood. Vesicular-associated membrane protein-8 (VAMP-8), a member of the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) family of fusion proteins initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. Here we show that in bone marrow-derived mast cells (BMMCs) VAMP-8 partially colocalized with secretory granules and redistributed upon stimulation. This was associated with increased SNARE complex formation with the target t-SNAREs, SNAP-23 and syntaxin-4. VAMP-8-deficient BMMCs exhibited a markedly reduced degranulation response after IgE+ antigen-, thapsigargin-, or ionomycin-induced stimulation. VAMP-8-deficient mice also showed reduced plasma histamine levels in passive systemic anaphylaxis experiments, while cytokine/chemokine release was not affected. Unprocessed TNF accumulated at the plasma membrane where it colocalized with a VAMP-3-positive vesicular compartment but not with VAMP-8. The findings demonstrate that VAMP-8 segregates secretory lysosomal granule exocytosis in mast cells from cytokine/chemokine molecular trafficking pathways.

Following i.v. BCG infection, a new population of macrophages are recruited in the rabbit lung. These macrophages, known as activated macrophages, substitute the resident macrophages and can play a key role in the defence against mycobacteria. We report here that BCG-activated alveolar macrophages are equipped with a more active hexose monophosphate pathway, which can maintain an optimal intracellular concentration of NADPH and GSH, and allow to produce mycobactericidal free radicals and to become resistant to mycobacterium-induced programmed cell death. These findings suggest that sustaining the anti-oxidant properties of macrophages could represent a candidate process to be considered as a good therapeutic target in fighting Mycobacterium spp infections.

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.

Objectives: The antitumour effect of bacillus Calmette-Guérin (BCG) still remains relatively undefined. Most investigations on its mechanism of action have focused on mononuclear cells; little consideration has been given to granulocytes. We analysed urine of patients with bladder cancer during 8 wk of intravesical BCG prophylaxis. The number of polymorphonuclear neutrophils (PMNs) and urothelial cells (UCs) was evaluated. We examined the in vitro response of the T24 UC line to human PMNs after BCG treatment., Methods: Seventeen patients were enrolled in the study. Cytologic analyses were performed on urine samples collected before each BCG instillation and after 2 h from the first voided urine after BCG instillation. Elastase activity was determined on these samples to evaluate PMN activation. PMN-induced damage was measured on the T24 cell line treated with BCG., Results: After BCG treatment, a large number of PMNs transmigrated through the urothelium and PMNs adherent to detached UCs were found. One patient, who did not respond with significant PMN transmigration, experienced recurrent disease. The number of eosinophils that transmigrated was low, with the exception of three patients with recurrent disease. In vitro, PMNs adhered to BCG-primed T24 cells and damaged the monolayer., Conclusions: The results agree with recent evidence that PMNs may play an important role in the antitumour action of BCG during the BCG induction period. This role is probably nonspecific because both normal UCs in vivo and tumour cells in vitro appeared to be injured. As suggested by results obtained from a limited number of patients, a high number of eosinophils in the urine may indicate therapy failure.

Imidazolium trans-imidazoledimethylsulphoxidetrachlororuthenate (NAMI-A) was tested in vitro on the pro-adhesive properties, evaluated as resistance to trypsin treatment, which is a bona fide measure of adhesion strength, of KB and HeLa carcinoma cell lines and on human polymorphonuclear neutrophils (HPMN). NAMI-A increased the pro-adhesive activity of KB cells at 0.001 mM concentration, after few minutes incubation and this effect was not influenced by the vehicle used for cell challenge, neither did it depend on NAMI-A concentration or on temperature. The same effect occurred on HeLa cells at 0.01 mM NAMI-A. This effect, detected at concentrations up to 100 times lower than those necessary to block cells at the G(2)-M premitotic phase of cell cycle, or to inhibit matrix metalloproteinase release or cell invasion, was not related to ruthenium uptake by tumour cells. HeLa cells and healthy HPMN, following short exposure to 0.1 mM NAMI-A, assumed a different shape, with the extrusion of filopodia (HeLa) and of large lamellopodia (HPMN), which increased their interactions with the substrate. This effect was attributed to stabilisation, altered turnover and sensitivity to cytochalasin D of actin filaments. Provided that adhesion is associated with cell motility and invasion, these data suggest that NAMI-A may exert antimetastatic properties at concentrations lower than those observed in the lungs at the end of a conventional intraperitoneal treatment in vivo.

Secretion in neutrophils is thought to be regulated in different ways for the different granule types. Specific granules are endowed with proteins which are related to docking and fusion events and are absent on azurophilic granules. Furthermore, even if secretion of content from all neutrophil granules is a Ca(2+)-dependent process, a higher concentration of cytosolic calcium is required for azurophilic than for specific granule secretion. In this paper we show that human neutrophils and promyelocitic cells express neuronal calcium sensor-1 (NCS-1), a calcium binding protein involved in exocytosis in various cell types. Both mRNA and protein were found in mature cells and precursors. NCS-1 is shown to be mainly associated with azurophilic granules and, therefore could play an instrumental role in the calcium-dependent secretion of azurophilic granules.

NAMI-A is a ruthenium complex endowed with a selective effect on lung metastases of solid metastasizing tumors. The aim of this study is to provide evidence that NAMI-A's effect is based on the selective sensitivity of the metastasis cell, as compared with other tumor cells, and to show that lungs represent a privileged site for the antimetastatic effects. The transplantation of Lewis lung carcinoma cells, harvested from the primary tumor of mice treated with 35 mg/kg/day NAMI-A for six consecutive days, a dose active on metastases, shows no change in primary tumor take and growth but a significant reduction in formation of spontaneous lung metastases. Transmission electron microscopy examination of lungs and kidney shows NAMI-A to selectively bind collagen of the lung extracellular matrix and also type IV collagen of the basement membrane of kidney glomeruli. The half lifetime of NAMI-A elimination from the lungs is longer than for liver, kidney, and primary tumor. NAMI-A bound to collagen is active on tumor cells as shown in vitro by an invasion test, using a modified Boyden chamber and Matrigel, and it inhibits the matrix metallo-proteinases MMP-2 and MMP-9 at micromolar concentrations, as shown in vitro by a zimography test. These data show NAMI-A to significantly affect tumor cells with metastatic ability. Binding to collagen allows NAMI-A to exert its selective activity on metastatic cells during dissemination and particularly in the lungs. These data also stress the wide spectrum of daily doses and treatment schedules at which NAMI-A is active against metastases.