Pleural mesothelioma and lung cancer: the role of asbestos exposure and genetic variants in selected iron metabolism and inflammation genes.

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.

A genetic variant of NLRP1 gene is associated with asbestos body burden in patients with malignant pleural mesothelioma.

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.

Physical interactions between mast cells and eosinophils: a novel mechanism enhancing eosinophil survival in vitro.

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.

Novel approaches for scanning near-field optical microscopy imaging of oligodendrocytes in culture.

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.

Neuropeptides in saliva of subjects with burning mouth syndrome: a pilot study.

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.

A simple and rapid method for isolation of eosinophilic granulocytes from human blood.

A simple and rapid method for the purification of morphologically and functionally intact eosinophils from human blood of both normal and eosinophilic subjects is described. The method is based on a single centrifugation of total blood leukocytes suspended in Percoll with specific gravity 1.0853 g/ml, after erythrocyte removal by dextran sedimentation. The peculiarity of this isolation technique is the maintenance of strictly physiological values of pH and osmolality throughout the entire procedure. Moreover, the cells are not subjected to measures aimed at changing the physical properties of either neutrophils or eosinophils. Because of such characteristics, this isolation method could be usefully exploited for comparative studies of normal and eosinophilic normodense eosinophils and of neutrophils and eosinophils from the same noneosinophilic subject.

A new, one-step assay on whole cell suspensions for peroxidase secretion by human neutrophils and eosinophils.

The degranulation of neutrophils and eosinophils is frequently monitored by assaying myeloperoxidase (MPO) and eosinophil peroxidase (EPO) activity in the cell-free supernatant of degranulating cells, after removal of the cells by centrifugation. This procedure leads to underestimation of the extent of degranulation, since both peroxidases tend to stick to cell surfaces, to test tube walls, and to particulate stimuli used to elicit degranulation, because of their highly cationic nature. In this paper we describe a method for assaying MPO and EPO secretion in whole cell suspensions that avoids separation of the cells from the incubation medium. The least toxic and thus safest among the sensitive peroxidase substrates, 3,3',5,5'-tetramethylbenzidine (TMB), was employed for peroxidase assay. The method we describe here is applied to the detection of peroxidase release by neutrophil and eosinophil cell suspensions incubated in either polypropylene test tubes or flat-bottomed microtiter plate wells. Because of the omission of the centrifugation step, the TMB method offers two major advantages over the currently used techniques: (1) higher estimates of degranulation, which permits the use of a smaller number of cells (in the microassay version, 150,000 neutrophils and 50,000 eosinophils) and smaller amounts of the secretagogues, and (2) rapidity, since the degranulation assay can be performed immediately on completion of the cell incubation with the secretagogue.

Actin-dependent tumour cell adhesion after short-term exposure to the antimetastasis ruthenium complex NAMI-A.

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.

A simple procedure for the purification of eosinophil peroxidase from normal human blood.

A simple procedure to purify human eosinophil peroxidase (EPO) is described. The method uses pure anucleated granule-rich eosinophil fragments (cytosomes) as a suitable starting material from which EPO can be quickly isolated. The enzyme obtained by this procedure has both the biochemical and the spectral properties of EPO and shows a reasonable degree of purity, as judged by its rz value. This procedure, besides its simplicity and reproducibility, offers at least two other advantages over the methods currently used for EPO purification, the possibility of isolating EPO from small amounts of normal human blood and a very high recovery of the enzyme activity.

A simple method to obtain pure granule-rich eosinophil fragments (cytosomes) from normal human blood.

This paper describes a simple, rapid and reproducible method to obtain pure granule-rich eosinophil fragments (cytosomes) with a high yield from normal human blood. The method is based on the treatment of whole blood with saponin and subsequent purification of the cytosomes on Percoll gradient. The enzymatic analysis of the cytosomes shows that the content of 3 granular enzymes is of the same order of magnitude already reported by others for intact eosinophils. This finding suggests that the cytosomes can be employed as starting material for studying the content of the granules or for the isolation of the granular components. The advantages offered by this method over those currently used to obtain eosinophil granules are discussed.

A new method for assessment of serum-induced damage to E. coli.

A simple and rapid spectrophotometric assay for the kinetic evaluation of serum-induced damage to E. coli is described, based on changes in the optical density (OD) of a bacterial suspension. Exposure of antibody-coated E. coli to human absorbed serum results in a diphasic response, namely an increase in OD, which reaches a maximum at about 17 min and is followed by a progressive decrease in OD until a minimum value is reached after 45 min. The increase and the decrease in OD are related to bacterial death and bacterial lysis, respectively.

Oxidation of homovanillic acid as a selective assay for eosinophil peroxidase in eosinophil peroxidase-myeloperoxidase mixtures and its use in the detection of human eosinophil peroxidase deficiency.

Biochemical assays for peroxidase activity do not usually distinguish between different peroxidases. The guaiacol assay, for example, which is one of the most commonly used assays for peroxidase activity, is sensitive to both eosinophil peroxidase (EPO) and the peroxidase of neutrophils, i.e., myeloperoxidase (MPO), thus preventing distinction of the two peroxidases in mixed neutrophil-eosinophil populations. In this paper we describe a simple and sensitive method for selective assays of EPO in EPO-MPO mixtures or mixed populations of neutrophils and eosinophils. The method is based on the peroxidase-mediated oxidation of homovanillic acid (HVA) under appropriate assay conditions in which EPO is still very active in catalyzing the reaction whilst MPO-mediated HVA oxidation is almost undetectable. Optimal assay conditions were as follows: pH 10.5, 10 microM hydrogen peroxide, 0.8 mM HVA and an incubation time of 120 min at 37 degrees C. Under these conditions the assay permits EPO activities as low as 0.025 guaiacol U/ml to be measured even in the presence of 0.175 guaiacol U/ml of MPO. In mixed neutrophil-eosinophil cell suspensions the test permits the detection of as few as 5 X 10(3) eosinophils even in the presence of about 700 X 10(3) neutrophils (eosinophils: neutrophils ratio 1:140) with no appreciable interference by the latter cells. The method described here has been applied to studies of human EPO deficiency and proved to be successful in the identification of individuals with partial EPO deficiency, which is not feasible with non quantitative methods (for example, cytochemistry) or unselective biochemical assay of peroxidase activity.

A simple reliable assay for myeloperoxidase activity in mixed neutrophil-eosinophil cell suspensions: application to detection of myeloperoxidase deficiency.

Quantitation of myeloperoxidase (MPO) activity by guaiacol peroxidation (GP) assay is profoundly affected by the peroxidase present in eosinophils (EPO) that contaminate the granulocyte suspensions. Inclusion of 3-amino-1,2,4-triazole (AMT) in the GP assay permits quantitation of MPO activity in mixed neutrophil-eosinophil suspension because of the differential inhibition of EPO and MPO by AMT. Results show that: (1) the peroxidase activity of eosinophil-free granulocyte suspensions is not appreciably affected by AMT; (2) in the presence of AMT the peroxidase activities of granulocyte preparations containing different numbers of eosinophils are similar on a neutrophil basis, regardless of the number of eosinophils and correspond with the activity of eosinophil-free granulocyte suspensions; (3) AMT almost completely inhibits the activity of partially purified EPO, only slightly affecting the catalytic activity of partially purified MPO; (4) AMT completely inhibits the residual peroxidase activity of granulocyte suspensions from MPO-deficient subjects contributed by contaminating eosinophils. The GP assay in the presence of AMT was used to study the pattern of hereditary transmission of MPO deficiency. The genealogy derived on the basis of this assay was compatible with an autosomal recessive inheritance, in agreement with previously reported results, while no definite pattern of inheritance could be established by use of the GP assay without AMT. We suggest that the GP assay supplemented with AMT is the method of choice for detection of MPO deficiency, particularly partial deficiency.

Uptake of human eosinophil peroxidase and myeloperoxidase by cells involved in the inflammatory process.

We have recently shown that human neutrophils bind and internalize human eosinophil peroxidase (EPO) but not myeloperoxidase (MPO). In the present work, we studied the interactions of human EPO and MPO with other cells that may be involved in the inflammatory process, i.e., lymphocytes, monocytes, platelets, fibroblasts, and endothelial cells. The results indicate that EPO is bound by all the cell types considered, but is efficiently internalized only by lymphocytes, monocytes, and endothelial cells. Conversely, MPO binds appreciably only to fibroblasts and endothelial cells, although with a lower affinity than EPO, but its internalization by any of the cell types studied is hardly detectable. Furthermore, both peroxidases bind strongly to collagen fibers, whereas only EPO binds to elastin. The results suggest that EPO, owing to its high cytophilia, exerts its biological activity close to the site at which it is released from the eosinophil.

Observations on the antimetastatic action of lysozyme in mice bearing Lewis lung carcinoma.

The effects of the oral administration of 100 mg/kg/day of lysozyme chloride on lung metastasis development were studied in mice bearing Lewis lung carcinoma. Lysozyme was administered to mice by supplying the daily amount of lysozyme with the powdered food. Lysozyme treatment reduces lung metastasis development, by significantly reducing the number of metastases of large dimension (diameters greater than 2mm) and by causing a significant increase of the percentage of animals free of large metastases, as compared with untreated controls. Correspondingly, the same animals show a pronounced increase of the number of multinuclear giant cells in the spleen; this parameter appears to be inversely correlated with the antimetastatic effect. These effects support the hypothesis that the antimetastatic effect of orally administered lysozyme depends upon spleen activation and perhaps upon induction of multinuclear giant cells of macrophage origin. This effect is consistent with previous findings indicating the occurrence of host-mediated effects in the antitumor action of lysozyme administered through the oral route.

Isolation of a murine metastatic cell line and preliminary test of sensitivity to the anti-metastasis agent NAMI-A.

We have isolated a new cell line (metGM) obtained from the spontaneous lung metastases of the mouse MCa mammary carcinoma. MetGM is a stable cell line which, after one year from its isolation, grows in vitro in suspension, forming cell aggregates, with cells that show irregular blabbing borders, active protein synthesis and convoluted nuclei and which have the capacity of invading matrigel membranes on which they give rise to a network of branching colonies. The preliminary study of the effects of the anti-metastasis ruthenium complex NAMI-A on metGM showed no direct cytotoxicity, with a mild reduction of cell proliferation, independent of the concentration of the ruthenium complex and not evident before 24 hours from treatment. A 10% DNA fragmentation was also measured on metGM cells 24 hours after challenge for 1 hour with 10(-5)M NAMI-A, suggesting that this compound is probably capable of apoptosis in a metastasis-derived cell line. Besides these effects on a limited percent of the cell population, NAMI-A changed the shape of the metGM cells and these alterations might account for the non-cytotoxic anti-metastatic properties of this innovative ruthenium complex. Thus MetGM appears to be a novel cell line suitable for the in vitro study of compounds endowed with anti-metastatic properties and for the development of new drugs with this activity.

In vitro growth of TS/A adenocarcinoma and of the gene transfected TS/A-IL4 line on biological substrates.

The growth capacity and adaptation of TS/A and TS/A-IL4 lines on laminin, fibronectin, collagens I and IV and matrigel compared to plastics were studied by flow cytometry. On plastic plates, TS/A-IL4 grows in vitro more slowly than the TS/A line and shows a more differentiated phenotype. TS/A-IL4 cells loose the capacity to bind lymphocytes and peroxidase positive cells obtained from mice implanted with the same tumour. The ratio between fibroblast- and epithelial-like cells of TS/A adenocarcinoma is subjected to marked modifications depending on the substrate on which the two cell lines are grown. IL4 release per cell unit is increased by collagen I as is the number of CD54 positive cells, suggesting that, at least in part, the in vivo rejection of TS/A-IL4 tumor might be ascribed to the stimulatory effect of the tissue on the IL4 release by tumor cells. The overall result is that gene modified TS/A-IL4 line shows marked changes of behaviour, most of them depending on the substrate on which tumor cells are growing.

Antiinflammatory action of hydrosoluble dimethyltriazenes on the carrageenin-induced edema in guinea pigs.

The antiinflammatory activity of three hydrosoluble aryldimethyltriazenes has been examined on the carrageenin induced edema in guinea pig. The administration of equitoxic dosages of p-(3,3-dimethyl-1-triazeno)benzoic acid potassium salt (DM-COOK) and p-(3,3-dimethyl-1-triazeno)sulfonic acid sodium salt (DM-SO3Na) 1 h after carrageenin application, causes 4 h later a similar and statistically significant reduction of paw swelling by about 40% whereas, p-alanylphenyl-3,3-dimethyl-1-triazeno (DM-ALA(OH)) is inactive. Of the two active compounds, DM-COOK displays interesting properties, being rapidly active and causing a peak of inhibition higher than that caused by DM-SO3Na. The antiinflammatory activity of DM-COOK is comparable with that caused by 5 mg/kg indomethacin and 200 mg/kg phenylbutazone. However, DM-COOK, unlike indomethacin, causes an inhibition of leukocyte migration into the peritoneal cavity induced by casein treatment, thus indicating a different mechanism of action. This effect needs clarification and seems not to be correlated to cytotoxicity of the drug for migrating white blood cells, as evidenced by "in vitro' examination.

Paracrine effects of IL-4 transfection on TS/A adenocarcinoma cells mediate reduced in vivo growth.

The in vitro/in vivo growth capacity and phenotype of TS/A and the IL4-transfected TS/A-IL4 cell lines were studied by cell cycle analysis, expression of ICAM-1/CD54, transferrin receptor/CD71 and E-cadherin and by histology of the primary tumors. TS/A-IL4, unlike the TS/A line, shows in vitro a marked increase in the fibroblastoid cell type and a decreased E-cadherin expression. Administration of conditioned medium containing IL4 obtained from the TS/A-IL4 cell line, stimulates CD54 expression in the TS/A cell line. TS/A-IL4 tumors grow more slowly in vivo and are ultimately rejected. These processes are accompanied by a marked increase in collagen and extracellular matrix proteins and increased recruitment and degranulation of mast cells. The paracrine effect of IL4, released by the transfected tumor cells, might be responsible for the reduced in vivo growth of the TS/A cell line in the presence of TS/A-IL4 cells.