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Major disease complications for patients with essential thrombocythemia (ET) include thrombosis and fibrotic or leukemic transformation. Calreticulin (CALR) mutation type 1 frequencies in ET are estimated between 7% and 11% and ET patients carrying CALR type 1 mutation are associated with lower risk of thrombosis but higher risk of myelofibrosis transformation compared to ET patients with JAK2 mutation. Leukemic transformation rates at 20 years are estimated at less than 5% for ET and risk factors for leukemic transformation are advanced age, thrombosis history, leukocytosis, and anemia. Amongst the subtypes of blast phase myeloproliferative neoplasms, acute promyelocytic leukemia is extremely rare. Herein, we present a case of a promyelocytic blast crisis of post-ET myelofibrosis with associated life-threatening splanchnic vein thrombosis. This case suggests that inflammation plays a key role in thrombotic events and fibrotic/leukemic transformation in ET patients, regardless the molecular landscape.

Background: Comprehensive genomic profiling (CGP) has gained an important role in patients with advanced prostate cancer following the introduction of PARP inhibitors in daily clinical practice. Here, we report an overview of CGP results, specifically of BRCA1 and BRCA2 HRD-repair system genes, from patients with prostate cancer analyzed in our institution, and we compare our results with those available from more recent scientific literature. Methods: The study cohort consisted of 70 patients. Somatic DNA was extracted from Formalin-Fixed Paraffin-Embedded (FFPE) tissue using a MagCore Genomic DNA FFPE One-Step Kit for MagCore System. The DNA was quantified by EasyPGX ® Real-Time qPCR and EasyPGX ® Analysis Software (version 4.0.13). Tissue somatic DNA libraries were prepared with Myriapod ® NGS BRCA1-2 panel-NG035 and sequenced in a Mi-Seq ® System. The sequence alignment in hg19 and the variant calling were performed using Myriapod ® NGS Data Analysis Software version 5.0.8 NG900-SW 5.0.8 with a software detection limit (LoD) of 95%. Variants with a coverage of 500 and VAF% ≥ 5 were evaluated. Results: Tumor tissue NGS was unsuccessful in 46/70 patients (66%). Mutations of the BRCA2 gene were detected in 4 of the samples: (1) BRCA2 ex10 c.1244A>G p.His415Arg VAF = 51.03%; (2) BRCA2 ex11 c.5946delT p.Ser1982fs VAF = 72.1%; (3) BRCA2 ex11 c.3302A>G p.His1101Arg VAF = 52.9%; and (4) BRCA2 ex11 c.3195_3198delTAAT p.Asn1066fs VAF = 51.1%. Conclusions: The results from our internal overview seem to support the data and to confirm the performance of the technical issues reported in the literature. Considering the advanced age of our patients, with 84% of men over the age of 65, the application of alternative and less invasive procedures such as liquid biopsy, could be a more suitable solution for some cases.

Research Question: What is the expression pattern of Raf kinase inhibitory protein (RKIP) in different subtypes of leiomyoma (usual type, cellular, apoplectic or haemorrhagic leiomyoma, leiomyoma with bizarre nuclei and lipoleiomyoma) and leiomyosarcoma specimens, and what is its biological role in leiomyosarcoma cells?, Design: Leiomyoma and leiomyosarcoma specimens underwent immunohistochemistry staining. Leiomyosarcoma SK-LMS-1 cell line was RKIP knocked down and RKIP overexpressed, and cell viability, wound healing migration and clonogenicity assays were carried out., Results: A higher immunohistochemical expression of RKIP was observed in bizarre leiomyomas, than in usual-type leiomyomas. Decreased expression was also found in cellular leiomyoma, with generally absent staining in leiomyosarcomas. Upon RKIP expression manipulation in SK-LMS-1 cell line, no major differences were observed in cell viability and migration capacity over time. RKIP knockout, however, resulted in a significant increase in the cell's ability to form colonies (P = 0.011)., Conclusion: RKIP distinct expression pattern among leiomyoma histotype and leiomyosarcoma, and its effect on leiomyosarcoma cells on colony formation, encourages further studies of RKIP in uterine smooth muscle disorders., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Research Question: Is the hypusinated form of the eukaryotic translation initiation factor 5A (EIF5A) present in human myometrium, leiomyoma and leiomyosarcoma, and does it regulate cell proliferation and fibrosis?, Design: The hypusination status of eIF5A in myometrial and leiomyoma patient-matched tissues was evaluated by immunohistochemistry and Western blotting as well as in leiomyosarcoma tissues by immunohistochemistry. Myometrial, leiomyoma and leiomyosarcoma cell lines were treated with N1-guanyl-1,7-diaminoheptane (GC-7), responsible for the inhibition of the first step of eIF5A hypunization, and the proliferation rate was determined by MTT assay; fibronectin expression was analysed by Western blotting. Finally, expression of fibronectin in leiomyosarcoma tissues was detected by immunohistochemistry., Results: The hypusinated form of eIF5A was present in all tissues examined, with an increasing trend of hypusinated eIF5A levels from normal myometrium to neoplastic benign leiomyoma up to neoplastic malignant leiomyosarcoma. The higher levels in leiomyoma compared with myometrium were confirmed by Western blotting (P = 0.0046). The inhibition of eIF5A hypusination, with GC-7 treatment at 100 nM, reduced the cell proliferation in myometrium (P = 0.0429), leiomyoma (P = 0.0030) and leiomyosarcoma (P = 0.0044) cell lines and reduced the expression of fibronectin in leiomyoma (P = 0.0077) and leiomyosarcoma (P = 0.0280) cells. The immunohistochemical staining of leiomyosarcoma tissue revealed that fibronectin was highly expressed in the malignant aggressive (central) part of the leiomyosarcoma lesion, where hypusinated eIF5A was also highly represented., Conclusions: These data support the hypothesis that eIF5A may be involved in the pathogenesis of myometrial benign and malignant pathologies., (Copyright © 2023 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Patients with early stage cutaneous T cell lymphoma (CTCL) usually have a benign and chronic disease course, characterized by temporally response to conventional skin directed therapies and intrinsic possibility to evolve. Using the combination of psoralen plus ultraviolet A irradiation (PUVA) and low-dose interferon-α (INF), the principal treatment goal is to keep confined the disease to the skin, preventing disease progression. Among 87 patients with early stage IA to IIA MF treated with low-dose IFN-α2b and PUVA in our center, complete remission (CR) were reported in 70 patients (80.5%) and the overall response rate (ORR) was 97.8% (n = 85), with a median time to best response to therapy of 5 months (range, 1-30). Among the responders, only the 8% of patients had a relapse with major event. The median follow-up was 207 months (range, 6-295). Survival data showed a median overall survival (OS) not reached (95% CI; 235-NR months), a disease free survival (DFS) of 210 months (95% CI; 200-226 months) and a median time to next treatment (TTNT) of 38.5 months (95% CI, 33-46 months). The long follow up of this study verifies our preliminary results already published in 2006 and confirms the efficacy of INF-PUVA combination therapy in a real world setting, according conventional (OS and DFS) and emerging (TTNT) clinical endpoint of treatment efficacy., (© 2022 Wiley Periodicals LLC.)

Uterine fibroids represent the most common benign tumors of the uterus. They are considered a typical fibrotic disorder. In fact, the extracellular matrix (ECM) proteins-above all, collagen 1A1, fibronectin and versican-are upregulated in this pathology. The uterine fibroids etiology has not yet been clarified, and this represents an important matter about their resolution. A model has been proposed according to which the formation of an altered ECM could be the result of an excessive wound healing, in turn driven by a dysregulated inflammation process. A lot of molecules act in the complex inflammatory response. Macrophages have a great flexibility since they can assume different phenotypes leading to the tissue repair process. The dysregulation of macrophage proliferation, accumulation and infiltration could lead to an uncontrolled tissue repair and to the consequent pathological fibrosis. In addition, molecules such as monocyte chemoattractant protein-1 (MCP-1), granulocyte macrophage-colony-stimulating factor (GM-CSF), transforming growth factor-beta (TGF-β), activin A and tumor necrosis factor-alfa (TNF-α) were demonstrated to play an important role in the macrophage action within the uncontrolled tissue repair that contributes to the pathological fibrosis that represents a typical feature of the uterine fibroids.

Breslow thickness and Clark level are still important factors for cutaneous melanoma, but do not provide a precise prognosis in all cases. It is necessary to find new factors capable of a more accurate prediction of the tumor course. Angiogenesis is essential for tumor development and progression and is regulated by vascular endothelial growth factor A (VEGF-A) and semaphorins (SEMA), in particular, SEMA3A inhibits angiogenesis by affecting VEGF signaling. However, the prognostic role of angiogenetic factors remains unclear. To date, no information is available on SEMA3A in human melanoma. Microvessel density, immunohistochemical and mRNA VEGF and SEMA3A expression level in 60 thin (Breslow thickness ≤ 1.0 mm), 60 intermediate (1.1-4.0 mm) and 50 thick (>4.0 mm) primary human cutaneous melanomas were investigated and related to clinical/pathological parameters and disease-specific survival. No positive association between Breslow thickness, Clark level, metastasis presence and survival was identified; Clark level was poorly related to survival. VEGF and microvessel density were significantly higher in intermediate and thick melanomas and related to Breslow thickness and Clark level but not to metastasis status and survival. On the contrary, SEMA3A was significantly reduced in intermediate and thick melanomas and associated to metastasis and poor survival. VEGF/SEMA3A ratio was higher in the worst prognosis, resulting the most closely related factor with metastasis and survival. SEMA3A expression and VEGF/SEMA3A ratio turned out to be valuable prognostic biomarkers in patients affected by cutaneous melanoma, in particular with Breslow thickness >1 mm. SEMA3A might serve as a candidate tumor suppressor in cutaneous melanoma therapy.

Introduction: Bexarotene is a synthetic retinoid effective in early and advanced stages of mycosis fungoides (MF)/Sezary Syndrome (SS) both in monotherapy and combination schemes. We aimed to assess disease response to low-dose bexarotene and PUVA in maintenance in refractory and/or resistant patients with early and advanced stage MF/SS., Methods: We followed prospectively 21 patients (stages IB-IV): 15 with early stage MF and 6 with advanced disease. "Mini" and standard protocols were respectively applied to patients who failed PUVA or several systemic regimens. The dose of bexarotene and the administration of PUVA were titrated individually and tailored during induction and maintenance according to previous therapy, disease stage and toxicity. We evaluated overall response (OR) at the end of maintenance, safety and event-free survival (EFS)., Results: After induction phase, OR was 85.6%, higher in early MF (93.4%) than in advanced disease (66.6%). At the end of maintenance, OR was 76.2%, including 33.3% of CR. Median EFS for the whole group was 31 months. Bexarotene was well tolerated regarding the side effects, with prophylaxis and progressive drug increase in the induction phase of the protocol. Side effects were mainly of low and moderate grades., Conclusions: We observed a favorable rate of therapeutic effects and few, generally mild, side effects with low doses of bexarotene combined with PUVA.

Background: Vascular events represent the most frequent complications of thrombocytemias. We aimed to evaluate their risk in the WHO histologic categories of Essential Thrombocytemia (ET) and early Primary Myelofibrosis (PMF)., Methods: From our clinical database of 283 thrombocytemic patients, we selected those with available bone marrow histology performed before any treatment, at or within 1 year from diagnosis, and reclassified the 131 cases as true ET or early PMF, with or without fibrosis, according to the WHO histological criteria. Vaso-occlusive events at diagnosis and in the follow-up were compared in the WHO-groups., Results: Histologic review reclassified 61 cases as ET and 72 cases as early PMF (26 prefibrotic and 42 with grade 1 or 2 fibrosis). Compared to ET, early PMF showed a significant higher rate of thrombosis both in the past history (22% vs 8%) and at diagnosis (15.2% vs 1.6%), and an increased leukocyte count (8389 vs 7500/mmc). Venous thromboses (mainly atypical) were relatively more common in PMF than in ET. Patients with prefibrotic PMF, although younger, showed a significant higher 15-year risk of developing thrombosis (48% vs 16% in fibrotic PMF and 17% in ET). At multivariate analysis, age and WHO histology were both independent risk-factors for thrombosis during follow-up; patients >60 yr-old or with prefibrotic PMF showed a significantly higher risk at 20 years than patients <60 yr-old with ET or fibrotic PMF (47% vs 4%, p = 0.005)., Conclusions: Our study support the importance of WHO histologic categories in the thrombotic risk stratification of patients with thrombocytemias., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2020211863144412 .

Background: Aberrant DNA methylation at CpG islands within promoters is increasingly recognised as a common event in human cancers and has been associated with the silencing of important tumour suppressor genes. Epigenetic therapy using hypomethylating agents has demonstrated clinical effectiveness; the drugs azacitidine and decitabine have been approved for the treatment of MDS., Method: We investigated the association between global DNA methylation and clinical outcome in MDS. We evaluated 134 MDS bone marrow trephine biopsies (BMTB) by immunohistochemistry and compared the results with those from an age-matched group of normal BMTB. Immunohistochemistry was performed on paraffin-embedded sections using the anti-5-methylcytosine (5mc) antibody., Results: Our results showed that the 5mc immunostaining score (M-score) of patients with MDS was higher than those of normal controls and that overall survival significantly correlated with global DNA methylation, age and IPSS score. Therefore, we found that patients with high levels of methylation had a shorter median overall survival (OS) compared with patients with lower levels. These immunohistochemistry results were confirmed by analysing global DNA methylation on LINE-1 sequences using the COBRA method and pyrosequencing., Conclusion: This study reports that global DNA methylation detected by immunohistochemistry predicts OS in MDS., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mineral fibers are potential carcinogens to humans. In order to help clarify the etiology of the pathological effects of asbestos, cellular reactions to natural and synthetic asbestos fibers were compared using a lung alveolar cancer cell line (A549 epithelial cells), considered the first target of inhaled micro-environmental contaminants. Natural asbestos tremolite (NAT) fibers were collected from rocks in NW Italy. Synthetic asbestos tremolite (SAT) was iron-free and therefore considered as standard tremolite. Both fibers, subjected to mineralogical characterization by X-ray powder diffractometry, electron microscopy and energy dispersive spectrometry, fell within the definition of respirable and potentially carcinogenic fibers. Several signs of functional and structural cell damage were found after treatment with both fibers, documented by viability, motility, and morphological perturbations. Phalloidin labeling showed irregular distribution of cytoskeletal F-actin, whereas immunohistochemical investigations showed abnormal expression of VEGF, Cdc42, β-catenin, assessed as risks indicators for cancer development. Both fibers caused significant loss of viability, even compared to UICC crocidolite, but, while SAT fibers exerted a more direct cytotoxic effect, survival of damaged cells expressing high VEGF levels was detected after NAT contact. This in vitro pilot study outlines potential health risks of NAT fibers in vivo related to their iron content, which could trigger signaling networks connected with cell proliferation and neoplastic transformation., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Background: Neoplastic T-cell recruitment into the skin is a critical step in the pathogenesis of mycosis fungoides (MF), and the cutaneous T-cell attracting chemokine, CTACK/CCL27, might be involved., Objectives: To investigate the clinical and prognostic significance of CTACK/CCL27 levels in patients with early-stage MF., Methods: Serum samples and skin biopsy specimens were collected from 15 patients at the time of diagnosis and after the end of treatment with psoralen plus ultraviolet A/interferon alfa-2b combination therapy. Serum samples were also collected from 20 healthy donors as controls. CTACK/CCL27 serum levels were analysed by enzyme-linked immunosorbent assays. CTACK/CCL27 tissue expression was determined by immunohistochemistry on skin biopsy specimens taken at diagnosis and after therapy. Event-free survival was taken as the primary clinical outcome., Results: In patients with MF at diagnosis, CTACK/CCL27 serum levels were not significantly different from healthy controls, whereas CTACK/CCL27 expression in the skin was increased in 87% of cases compared with normal controls. After therapy, all patients obtained a clinical complete remission, serum levels did not change significantly and tissue expression remained abnormal in 80% of patients, even if complete histological remission was recorded. Serum levels were not significantly different in cases with different intensity of cutaneous immunostaining. Eight patients experienced a relapse: the combination of high CTACK/CCL27 levels both in sera and skin increased the probability of experiencing an event at 51 months from 36% to 83%., Conclusions: Our data seem to indicate that CTACK/CCL27 levels in skin and sera after therapy might be correlated with risk of recurrence., (© 2012 The Authors. BJD © 2012 British Association of Dermatologists.)

Chemical reactivity of asbestos tremolite from Italy and USA localities and Union Internationale Contre le Cancer (UICC) crocidolite was studied in relation to Fe content, oxidation state, and structural coordination. Direct correlation between amount of Fe(2+) at the exposed M(1) and M(2) sites of the amphibole structure and fiber chemical reactivity was established. The in vitro toxicity of the same samples was investigated on human alveolar A549 cell line. Relationship between crystal-chemical features and cell toxicity is not straightforward. UICC crocidolite has Fe content and chemical reactivity largely higher than that of tremolite samples, but all show comparable in vitro toxic potential. Results obtained evidenced that Fe topochemistry is not a primary factor for induced cell toxicity, though it accounts for asbestos chemical reactivity (and possibly genotoxicity).

Background: Recently, human germinal center-associated lymphoma (HGAL) gene protein has been proposed as an adjunctive follicular marker to CD10 and BCL6., Methods: Our aim was to evaluate immunoreactivity for HGAL in 82 cases of follicular lymphomas (FLs)--67 nodal, 5 cutaneous and 10 transformed--which were all analysed histologically, by immunohistochemistry and PCR., Results: Immunostaining for HGAL was more frequently positive (97.6%) than that for BCL6 (92.7%) and CD10 (90.2%) in FLs; the cases negative for bcl6 and/or for CD10 were all positive for HGAL, whereas the two cases negative for HGAL were positive with BCL6; no difference in HGAL immunostaining was found among different malignant subtypes or grades., Conclusions: Therefore, HGAL can be used in the immunostaining of FLs as the most sensitive germinal center (GC)-marker; when applied alone, it would half the immunostaining costs, reserving the use of the other two markers only to HGAL-negative cases., (© 2011 Goteri et al; licensee BioMed Central Ltd.)

In fast growing organisms, like fish larvae, fatty acids provided through live prey are essential to satisfy high energy demand and are required to promote growth. Therefore, in recent decades, a great amount of research has been directed towards the development of lipid enrichment in order to improve larval fish survival and growth. However, in fish, the biochemical and molecular processes related to highly unsaturated fatty acid (HUFA) administration are still poorly understood. In the current study, the false percula clownfish, a short larval phase marine species, was used as an experimental model and the effects of a standard and a HUFAs-enriched diet were tested through a molecular, biochemical, ultrastructural and morphometric approach. Our results support the hypothesis that HUFA administration may improve larval development through the presence of better structured mitochondria, a higher synthesis of energy compounds and coenzymes with a central position in the metabolism, with respect to controls. This higher energy status was confirmed by better growth performance and a shorter larval phase in larvae fed with an enriched diet with respect to the control. This strategy of rapid growth and early energy storage may be considered positively adaptive and beneficial to the survival of this species., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Asbestos fibres can be transformed into potentially non-hazardous silicates by high-temperature treatment via complete solid-state transformation. A549 cells were exposed to standard concentrations of raw cement asbestos (RCA), chrysotile and cement asbestos subjected to an industrial process at 1200 degrees C (Cry&#95;1200 and KRY.AS, respectively), raw commercial grey cement (GC). Cell growth rate and viability (MTT test) were detected in vitro. RCA and KRY.AS subjected to comprehensive microstructural study by electron microscopy were further in vitro assayed to compare their cytotoxic potential by morphostructural studies, proliferation index (Ki-67 antigen), apoptosis induction (AO/EB staining) assays and detection of intracellular reactive oxygen species (ROS) with the fluorescent DCFA dye. More severe cytotoxic damage was induced by RCA than by KRY.AS after each incubation period. Exposure to KRY.AS and GC resulted in comparable cell growth rates and cytotoxic effects. Cells incubated with RCA showed greater apoptotic induction and ROS production and a lower cell proliferation index than those exposed to KRY.AS. Chrysotile asbestos and RCA subjected to heat treatment underwent complete microstructure transformation. The final product of heat treatment of cement asbestos, KRY.AS, was considerably more inert and had lower cytotoxic potential than the original asbestos material in all in vitro tests., (Copyright 2010 Elsevier Ltd. All rights reserved.)

In the last years, physical and chemical methods of plasmid delivery have revolutionized the efficiency of nonviral gene transfer, and the success of gene therapy is largely dependent upon the development of gene-delivery methods. The nonviral techniques that lead to a direct transfer of DNA into tissue fragments, like electroporation (EP) and lipofection delivery systems are still insufficiently investigated. Our aim was to test the efficiency of EP and lipofection protocols in endometrial and testicular tissue fragments, using a naked plasmid DNA encoding green fluorescent protein (GFP). Because the transfection efficiency depends upon several factors, we tried to optimize the transfection conditions by testing different lipofectamine 2000 and plasmid ratios, electrical parameters, and culture after transfection. Our results show that these two nonviral methods of gene delivery are feasible and efficient in gene transfection of endometrial and testicular tissue biopsies. We found that the most performing ratio of plasmid:lipofectamine was 10:50 for transient lipofection, whereas two pulses for 10 s at 960 microF of capacitance, 200 V of voltage were the most favorable electrical parameters for EP efficiency in the presence of 5 microL of phMGFP plasmid. After lipofection and EP, the highest GFP intensity was observed respectively after 48 and 72 h of tissue fragment culturing. In conclusion, nonviral methods are attractive for an improvement of the gene therapy and our protocol could provide useful indications for in vivo gene therapy applications.

The purpose of the study was to investigate the biological risk of asbestiform antigorite, which is a fibrous variety of antigorite, one of the natural mineral fibres of the serpentine group to which asbestos chrysotile belongs. Asbestiform antigorite is very abundant and commonly found associated with asbestos chrysotile in serpentinites, a kind of rock outcropping present in many geographical locations worldwide. In this study we evaluated the morphological, immunohistochemical and functional effects of antigorite fibres in alveolar epithelial cancer cells (A549), a standardized human cell line currently used as a model to study cytotoxicity induced by pharmacological agents. The antigorite fibres were identified and characterized morphologically and chemically by X-ray powder diffractometry, transmission and scanning electron microscopy, both with annexed energy dispersive spectrometry. The effects of 50 microg/ml of antigorite in A549 lung cells treated at 24 and 48 h resulted in increased synthesis of VEGF, Cdc42 and beta-catenin that represent potential risks for cancer development. Phalloidin labelling showed an irregular distribution of filamentous actin resulting from antigorite contact. Our studies indicate potential cellular toxicity of antigorite in vivo, providing the opportunity to elucidate the effect of asbestos on cancer induction and possible modes of therapy., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Asbestos fibers, such as chrysotile and crocidolite, are known to have cytotoxic effects on different cell types. In vivo exposure to asbestos fibers can induce both fibrotic and malignant lung diseases , however, the mechanisms linking exposure to the subsequent development of the diseases are unknown. Numerous investigations suggest the involvement of reactive oxygen species (ROS). ROS are known to damage biological macromolecules including proteins, cell membrane lipids and nucleic acids; alterations of these essential cellular components can alter cell function and can drive the cell to neoplastic transformation or to cell death. Because the mitochondrial respiratory chain is an important source of ROS and RNS (reactive nitogen species) in the cells, we have investigated the effects of aqueous extracts of asbestos (natural and synthetic) fibers on some mitochondrial activities. Our data show that crocidolite fibers release substances in solution that may interfere directly with the mitochondrial cytochrome oxidase complex. Moreover, the calcium ions released from these fibers induce opening of the permeability transition pore of the inner membrane leading to a possible cytotoxic effect due to the release of apoptotic factors normally localized in the mitochondrial intermembrane space. In addition, crocidolite extracts enhance the mitochondrial production of ROS. No significant biochemical effects are exerted by chrysotile, either natural or synthetic, on isolated mitochondria. Nevertheless, all asbestos fibers tested induce morphological alterations visualized by transmission electron microscopy and morphometric analysis.

The in vitro biological response to fluoro-edenite (FE) fibres, an asbestos-like amphibole, was evaluated in lung alveolar epithelial A549, mesothelial MeT-5A and monocyte-macrophage J774 cell lines. The mineral has been found in the vicinity of the town of Biancavilla (Catania, Sicily), where an abnormal incidence of mesothelioma has been documented. Cell motility, distribution of polymerized actin, and synthesis of vascular endothelial growth factor (VEGF) and of beta-catenin, critical parameters for tumour development, progression and survival, were investigated in A549 and MeT-5A cells exposed to 50 microg/ml FE fibres for 24 hr and 48 hr. The levels of cyclooxygenase (COX-2) and prostaglandin (PGE2), two molecules involved in cancer pathogenesis by affecting mitogenesis, cell adhesion, immune surveillance and apoptosis, were measured in J774 cells treated with FE fibres under the same experimental conditions. Finally, FE fibres were studied by SEM and EDS analysis to investigate their chemical composition. Exposure of A549 and MeT-5A cells to FE fibres affected differentially phalloidin-stained cytoplasmic F-actin networks, cell motility and VEGF and beta-catenin expression according to the different sensitivity of the two cell lines. In J774 cells it induced a significant increase in COX-2 expression, as assessed by Western blot analysis, and in the concentration of PGE2, measured in culture media by ELISA. SEM-EDS investigations demonstrated two types of FE fibres, edenite and fluoro-edenite, differing in chemical composition and both recognizable as calcic amphiboles. Fibre width ranged from less than 1 microm (prevalently 0.5 microm) to 2-3 microm (edenite) up to several microm (fluoro-edenite); length ranged from about 6 to 80 microm (edenite) up to some hundred microm (fluoro-edenite). Results provide convincing evidence that FE fibres are capable of inducing in vitro functional modifications in a number of parameters with crucial roles in cancer development and progression. Inhaled FE fibres have the potential to induce mesothelioma, even though their ability to penetrate lung alveoli depends on their aerodynamic diameter.

Rapid prototyping, automatic image processing (computer-aided design (CAD)) and computer-aided manufacturing techniques are opening new and interesting prospects for medical devices and tissue engineering, especially for hard tissues such as bone. The development of a bone high-resolution scaffold prototype using these techniques is described. The results testify to the fidelity existing between microtomographic reconstruction and CAD. Furthermore, stereolithographic manufacturing of this scaffold, which possesses a high degree of similarity to the starting model as monitored by morphological evaluations (mean diameter 569 +/- 147 microm), represents a promising result for regenerative medicine applications.

The aim of the present study was to evaluate experimentally whether administration of recombinant (rh) vascular endothelial growth factor (VEGF) can protect skin flaps from necrosis and to study the optimum mode of rh-VEGF administration. We used rats to study the effects of local or systemic administration of rh-VEGF on skin flap during surgery; we also tested preoperative systemic administration of rh-VEGF to assess whether it may prepare the tissue to respond to the hypoxic injury better than previously tested methods. The animals were 30 male Sprague-Dawley rats. Group I rats received multiple systemic injections of rh-VEGF in the tail artery prior to flap dissection. Group II rats were injected with rh-VEGF in the clamped left epigastric artery during flap dissection; in this group, the left flaps thus received rh-VEGF locally (via incubation for 10 min during hypoxia) and the right flaps systemically, after blood flow restoration. Group III received saline solution instead of VEGF in the same way as group II. Skin samples from the distal portion of the flaps were collected on day 7 for morphological and immunohistochemical analysis. The flaps exhibiting the least necrosis were those treated with local rh-VEGF, followed by those treated with systemic rh-VEGF. The flaps that received rh-VEGF locally showed a strong VEGF expression on keratinocytes and endothelial cells, the greatest amount of mature and newly formed vessels and strong survivin expression in endothelial cells. Local rh-VEGF administration should thus be considered as an effective therapeutic option to enhance the survival of a tissue at risk for perfusion.

The morpho-functional and energy condition of NCTC 2544 cells exposed for 1 hr to a high concentration of H2O2 (500 microM) was studied at 4 and 24 hr to investigate the short- and medium-term biomolecular mechanisms affecting energetic mitochondrial capability. Morphometric data obtained from ultrastructural investigations clearly showed significant modifications of the different mitochondrial parameters--numerical density (Nv), volume density (Vv) and Vv/Nv ratio, in interkinetic, apoptotic and mitotic cells after H2O2 exposure (from 4 to 24 hr). These results were confirmed by the detection at 24 hr of mitochondrial cytochrome c release in the cytosol, indicating impairment in mitochondrial membrane permeability. Data supporting these observations were obtained from the MTT test which showed reduced cell viability in H2O2 treated cultures at 4 hr and an even greater decrement at 24 hr. In conclusion our data imply that significant cause-effect relationships exist between the toxicity of reactive oxygen species (i.e. 500 microM H2O2) and morpho-structural mitochondrial damage in interkinetic, apoptotic and mitotic cells, respectively. They support previous results present both in the literature and also in one of our earlier papers which show that early nuclear DNA damage could initiate mitochondrial or intrinsic apoptotic pathway after H2O2 exposure.

Asbestos fibers, such as chrysotile and crocidolite, are known to have cytotoxic effects on different cell types. in vivo exposure to asbestos fibers can induce both fibrotic and malignant lung diseases , however, the mechanisms linking exposure to the subsequent development of the diseases are unknown. Numerous investigations suggest the involvement of reactive oxygen species (ROS). ROS are known to damage biological macromolecules including proteins, cell membrane lipids and nucleic acids; alterations of these essential cellular components can alter cell function and can drive the cell to neoplastic transformation or to cell death. Because the mitochondrial respiratory chain is an important source of ROS and RNS (reactive nitogen species) in the cells, we have investigated the effects of aqueous extracts of asbestos (natural and synthetic) fibers on some mitochondrial activities. Our data show that crocidolite fibers release substances in solution that may interfere directly with the mitochondrial cytochrome oxidase complex. Moreover, the calcium ions released from these fibers induce opening of the permeability transition pore of the inner membrane leading to a possible cytotoxic effect due to the release of apoptotic factors normally localized in the mitochondrial intermembrane space. In addition, crocidolite extracts enhance the mitochondrial production of ROS. No significant biochemical effects are exerted by chrysotile, either natural or synthetic, on isolated mitochondria. Nevertheless, all asbestos fibers tested induce morphological alterations visualized by transmission electron microscopy and morphometric analysis.

Biocompatibility relies essentially on surface phenomena, represented by cell-cell, cell-material and material (polymer)-protein interactions. An in vivo and in vitro experimental investigation was carried out on the biomaterials of two different classes with a good potential for in situ utilisation. Non-resorbable (Polypyrrole, Polyaniline, Polyimide) and resorbable (PLLA-PDXO-PLLA) materials for tissue engineering were studied for their overall tissue tolerance and cellular interactions. These non-resorbable polymers conceived for biosensor applications and implantable drug-delivery systems are intrinsically conductive. The PLLA-PDXO-PLLA triblock copolymer showed interesting tensile properties for bone and cartilage tissue engineering due to the presence of 1,5-dioxepan-2-one. In vitro and in vivo parallel studies showed an interesting correspondence: a) the cells in contact with the resorbable material that appeared to be capable of migratory-regenerative aspects in vitro exhibited good compatibility in vivo; whereas b) the non-resorbable materials, which are designed to remain in situ in vivo, were seen to have the potential to represent an adverse factor (inflammation, fibrotic reactions) that correlated with some aspects of cell behaviour in vitro.

Owing to their possible carcinogenic effect, asbestos and other silica derivatives have been identified as priority substances for risk reduction and prevention of pollution. Neutralisation procedures have thus become a topical research subject in many European and American countries. In the present study, silica derivatives (asbestos-containing and asbestos substitutes like slag wool, rock wool, cement asbestos) were fully impregnated with an epoxy resin according to the procedure used for the in situ impregnation with viscous polymeric media, which penetrate and cement the fibres in place and reduce the risk of their dispersion in air. Untreated and treated samples were used to investigate their in vitro interaction with a human continuous epithelial cell line (NCTC 2544 keratinocytes) and test the resin's efficiency in passivating the surface activity of the fibrous particulate. SEM and morpho-quantitative data evidenced that impregnation with the epoxy resin modifies the mineral fibres' bioactivity (reduction of cell adhesion and decreased spread/round cell ratio) and demonstrated the value of in vitro cell testing after passivation as a risk-assessment procedure. These tests could be used for the rapid determination of the level of passivation of new synthetic mineral fibrous materials subjected to resin impregnation.

Surface topography is important in establishing tissue organisation adjacent to implants, smooth surfaces generally being associated with fibrous encapsulation. By virtue of its large hydrated molecular volume and its capacity to form molecular matrix, hyaluronic acid can expand the interfibrillar collagen spaces to allow the movement of cells, although it can also hamper their locomotion. Low molecular-weight hyaluronan can also stimulate cell proliferation, especially at low concentrations. The aim of the present work was to evaluate in vitro the growth and migratory behaviour of NCTC 2544 keratinocytes cultured on different materials microstructured with hyaluronic acid or sulfated hyaluronic acid to assess the possibility of using these devices in the repair process of soft tissues. Ultrastructural morphological analyses, morphometric evaluations and detection of cytoskeletal elements were performed. Our observations provide evidence that micrometer-size parallel grooves of hyaluronic acid can influence cell growth behaviour since cells seeded onto the microstructured substrate arranged themselves according to a shape and an orientation that clearly reflected the chemotropism exerted on them by the two forms of acid. These data also highlight the importance of accurate microtexture fabrication. We intend to follow up these in vitro studies with in vivo experimental applications using PET and gelatin substrates structured with HyalS to evaluate wound healing responses, and to extend our investigations of the cytoskeletal modifications induced by different microstructures.