s of the 1st Joint Meeting of Pathology and Laboratory Diagnostics September 12-15, 2012 Udine Congress and Exhibition Centre, Udine, Italy ADVANCES IN MOLECULAR THERAPIES AMT1. In Vitro Evaluation of Type II Ribosome-Inactivating Proteins (RIPs) for Experimental Chemoablation of Muscle Cells in Strabismus and EyeMovement Disorders D. Mercatelli1, M. Bortolotti1, L. Polito1, M. Battelli1, A. Bolognesi1 1Dipartimento Patologia Sperimentale Bologna, Bologna, Italy Background: Today the treatment with botulinum toxin (BTX) is the most used molecular surgery of strabismus and other eye-movement disorders, as alternative to traditional surgery. However the temporary effect of BTX treatment requires the research of alternative therapies. To this purpose the in vitro effects of three type II RIPs from plants (lanceolin, stenodactylin and ricin)and of the skeletal musclespecific immunotoxin saporin-mAb73 were evaluated on muscle cells. Methods: The RIPs and the immunotoxin were tested for their cytotoxicity in three cell lines: L6E9 (myoblasts), TE671 and RD/18 (rhabdomyosarcoma), both undifferentiated and differentiated. The aspecific toxicity was evaluated on conjunctival IOBA-NHC cell line. Protein synthesis inhibition, viability and apoptotic changes were assayed. Results: All substances showed a strong cytotoxic effect on protein synthesis and viability, with IC50 and LC50 ranging from 0.1 nM to 0.01 pM. Lanceolin and stenodactylin were 1-2 logs more toxic than ricin and 2-3 logs more toxic than the immunotoxin. Myoblasts were particularly susceptible to stenodactylin (IC50<0.01pM). All RIP-treated cells showed typical morphological apoptotic changes and no signs of necrosis. In further experiments miming in vivo treatment, no toxic effects were reported on conjunctival IOBA-NHC cell line. Conclusions: The strong cytotoxicity observed for stenodactylin at very low dose could be compatible with loco-regional treatments in strabismus and eye-movement disorders. It could be possible to modulate the effect on muscle fibers and to obtain a complete ablation of myoblasts, gaining more durable effects as compared to BTX treatment. Moreover, the absence of necrosis should avoid flogistic side effects.

Several studies have analyzed modulation of gene expression during physiological ageing with interesting, but often contradictory results, depending on the model used. In the present report we compare age-related metabolic and synthetic parameters in human dermal fibroblasts (HDF) isolated from young and old subjects (ex vivo ageing model) and cultured from early up to late cumulative population doublings (CPD) (in vitro ageing model) in order to distinguish changes induced in vivo by the aged environment and maintained in vitro, from those associated with cell senescence and progressive CPD. Results demonstrate that fibroblasts from aged donors, already at early CPD, exhibit an impaired redox balance, highlighting the importance of this parameter during ageing, even in the presence of standard environmental conditions, which are considered optimal for cell growth. By contrast, several proteins, as those related to heat shock response, or involved in endoplasmic reticulum and membrane trafficking, appeared differentially expressed only during in vitro ageing, suggesting that, at high CPD, the whole cell machinery becomes permanently altered. Finally, given the importance of the elastic component for a long-lasting connective tissue structural and functional compliance, this study focuses also on elastin and fibulin-5 synthesis and deposition, demonstrating a close relationship between fibulin-5 and ageing.

AIMTo evaluate the biocompatibility of a resin-based endodontic filler (RealSeal) using the indirect cytotoxicity test.METHODOLOGYHuman gingival fibroblasts were cultured ex vivo. Pellets of the materials to be tested were incubated for 24, 48, and 72 h at 37 degrees C under sterile conditions to obtain their eluates. The fibroblasts were exposed to either diluted (50%) or undiluted eluates for 24 h. A culture medium with foetal calf serum was added to the control wells. Cell viability was estimated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method. The data concerning cell viability were statistically analyzed using one-way anova test and Bonferroni multiple comparisons test.RESULTSEluates obtained after 24 h of incubation with the resin filler did not reduce cellular viability. An increase in cellular viability, as compared with control cells, was observed in the gutta-percha group. The undiluted eluate from the polyether material was cytotoxic, causing an 82 +/- 4% decrease in cellular viability. Eluates obtained after 48 h of incubation with the resin filler increased cellular viability, whereas the polyether significantly reduced viability. Gutta-percha did not cause any detectable change. After 72 h of incubation the eluate of the resin filler caused an increase in cellular viability, as did gutta-percha, whereas polyether caused a significant decrease.CONCLUSIONSRealSeal resin filler was nontoxic in this laboratory model. Further investigations are necessary to verify its usefulness in clinical applications.

We have monitored viscoelasticity variation of a cell population during the cell cycle by a Quartz Crystal Microbalance (QCM). Balb 3T3 fibroblasts were synchronized in the G0/G1 phase and seeded in a QCM chamber placed in a cell incubator. After cell sedimentation, the frequency signal was characterized by an amplitude modulation attributed to the viscoelasticity variation of the cells proliferating in phase. A control experiment with nonsynchronized cells showed a similar signal trend, but without significant modulation. Interestingly, the system resulted also to perform as a device sensitive to the effect of drugs affecting the cell cycle, such as colchicine.

Pseudoxanthoma elasticum (PXE) is a genetic disease characterized by calcification and fragmentation of elastic fibres of the skin, cardiovascular system and eye, caused by mutations of the ABCC6 gene, which encodes the membrane transporter MRP6. The pathogenesis of the lesions is unknown. Based on studies of similar clinical and histopathological damage present in haemolytic disorders, our working hypothesis is that PXE lesions may result from chronic oxidative stress occurring in PXE cells as a consequence of MRP6 deficiency. Our results show that PXE fibroblasts suffer from mild chronic oxidative stress due to the imbalance between production and degradation of oxidant species. The findings also show that this imbalance results, at least in part, from the loss of mitochondrial membrane potential (ΔΨm) with overproduction of H2O2. Whether mitochondrial dysfunction is the main factor responsible for the oxidative stress in PXE cells remains to be elucidated. However, mild chronic generalized oxidative stress could explain the great majority of structural and biochemical alterations already reported in PXE. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cultured fibroblasts from the dermis of normal subjects and of Pseudoxanthoma elasticum (PXE) patients were analysed for enzyme activity, protein and mRNA expression of metalloproteases (MMP-2, MMP-3, MMP-9, MT1-MMP) and of their specific inhibitors (TIMP-1, TIMP-2 and TIMP-3). MMP-3, MMP-9 and TIMP-3 mRNAs and proteins failed to be detected in both the medium and the cell layer of both controls and PXE patients. MMP-2 mRNA was significantly more expressed in PXE than in control cell lines, whereas MT1-MMP, TIMP-1 and TIMP-2 mRNAs appeared unchanged. MMP-2 was significantly higher in the cell extracts from PXE fibroblasts than in control cells, whereas differences were negligible in the cell medium. Data suggest that PXE fibroblasts have an increased proteolytic potential, and that MMP-2 may actively contribute to connective tissue alterations in this genetic disorder.

The purpose of this study was to evaluate adhesion and growth of human dermal fibroblasts on a 0.150 mm-thick matrix of reconstituted collagen isolated from horse tendon. Collagen was extracted and polymerized according to the standard procedures (Opocrin, Corlo, Modena, Italy). By light microscopy, the bottom surface of the matrix appeared linear and compact, whereas the superficial one was indented and less homogeneous. By scanning electron microscopy, the collagen fibrils had different diameters and the great majority of them was oriented parallel to the surface of the gel. By transmission electron microscopy, collagen fibrils showed the typical banding. Human dermal fibroblasts were seeded on the collagen matrix, previously equilibrated in growth medium. Fibroblast proliferation stopped in the second week and was always significantly lower than that of the same cell strain seeded on plastic and cultured in parallel. By light microscopy, after six days culture, cells formed a confluent multilayer on the surface of the gel. By scanning and transmission electron microscopy, fibroblasts appeared flat and adherent to the matrix. Contacts of cells among themselves and with the collagen fibrils were observed. Fibroblasts never moved into the collagen gel. In conclusion, human dermal fibroblasts can be grown in a three-dimensional matrix made by horse tendon that, on the other hand, seems to condition their proliferation rate.

Multidrug resistance protein-6 (MRP6) is a membrane transporter whose deficiency leads to the connective tissue disorder Pseudoxanthoma elasticum (PXE). In vitro dermal fibroblasts from normal and PXE subjects, homozygous for the R1141X mutation, were compared for their ability to accumulate and to release fluorescent calcein, in the absence and in the presence of inhibitors and competitors of the MDR-multidrug resistance protein (MRP) systems, such as 3-(3-(2-(7-choro-2 quinolinyl) ethenyl)phenyl ((3-dimethyl amino-3-oxo-propyl)thio) methyl) propanoic acid (MK571), verapamil (VPL), vinblastine (VBL), chlorambucil (CHB), benzbromarone (BNZ) and indomethacin (IDM). In the absence of chemicals, calcein accumulation was significantly higher and the release significantly slower in PXE cells compared to controls. VBL and CHB reduced calcein release in both cell strains, without affecting the differences between PXE and control fibroblasts. VPL, BNZ and IDM consistently delayed calcein release from both control and PXE cells; moreover, they abolished the differences between normal and MRP6-deficient fibroblasts observed in the absence of chemicals. These findings suggest that VPL, BNZ and IDM interfere with MRP6-dependent calcein extrusion in in vitro human normal fibroblasts. Interestingly, MK571 almost completely abolished calcein release from PXE cells, whereas it induced a strong but less complete inhibition in control fibroblasts, suggesting that MRP6 is not inhibited by MK571. Data show that MRP6 is active in human fibroblasts, and that its sensitivity to inhibitors and competitors of MDR-MRPs' membrane transporters is different from that of other translocators, namely, MRP1. It could be suggested that MRP1 and MRP6 transport different physiological substances and that MRP6 deficiency cannot be overcome by other membrane transporters, at least in fibroblasts. These data further support the hypothesis that MRP6 deficiency may be relevant for fibroblast metabolism and responsible for the metabolic alterations of these cells at the basis of connective tissue clinical manifestations of PXE.

Proteins present within the cell layer and those released in the cell medium from in vitro cultured normal human dermal fibroblasts were separated and characterized in terms of their isoelectric point and molecular weight, by two-dimensional (2-D) gel electrophoresis. All spots in the synthetic gel were firstly analyzed by the Melanie 3 software and compared with those of breast cancer cells, colorectal epithelial cells, HL60, lymphoma cells, and platelets, already available on-line. From the identification of 144 spots from both the cell layer and the medium, we were able to recognize 89 different proteins, since a certain number of spots represented different isoforms of the same molecule. Identifications were performed by matching with on-line 2-D databases, and by matrix assisted laser-desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), in order to confirm the identification by matching, or to identify new proteins. The procedure we used allows (i) to design a highly reproducible reference map of the proteome of adult human normal fibroblasts in culture, (ii) to evaluate protein species produced in the cell layer as well as those released in the culture medium, and (iii) to compare data from gel matching with those obtained by MS. This work represents an essential step for a better knowledge of mesenchymal cells, given the widespread use of this cell type in both clinical and experimental investigations.

Aging is a complex multifactorial process still far from being completely understood. The aim of the present study was to compare the proteome of in vitro cultured dermal fibroblasts from healthy subjects of different ages (i.e. 15 +/- 2, 41 +/- 4 and 82 +/- 3 years old). Proteins of the cell layer were separated by two-dimensional electrophoresis and protein identification was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry; moreover, synthetic gels were qualitatively and quantitatively analyzed by Melanie 3 software. Our study did not reveal any protein typical of any one age group. On the other hand, we observed 38 proteins exhibiting more than three-fold reproducible variations with aging, some (45%) being reduced such as F-actin capping protein alpha1, proteasome subunit alpha type 3, heat shock protein 27, ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial thioredoxin-dependent peroxide reductase, cathepsin B, glutathione S-transferase P, cyclophilin A and calgizzarin. In contrast, T-complex protein 1, probable protein disulfide isomerase ER60, phosphoglycerate kinase 1, Ran-specific GTPase-activating protein, proteasome subunit alpha type 5, triosephosphate isomerase and superoxide dismutase (Mn) increased with age. Furthermore, annexin 1, elongation factor 1beta, proteasome activator complex subunit 1, phosphoglycerate mutase, superoxide dismutase (Cu-Zn) and cofilin, exhibited the highest levels in adult cells; whereas, septin 2 homolog, RNA-binding protein regulatory subunit and ATP synthase D chain revealed the lowest values in adults. The present investigation, underlining the complexity of the aging process, highlights the role of synthetic and degradative pathways in modulating the whole cell machinery and emphasizes that metabolic impairment with age could depend partly on different expression of a number of genes and leading to an imbalance among functional proteins.

Low and high molecular weight hyaluronan (HA) was added to adult human fibroblasts grown in monolayer to assess its influence on CD44 expression, its internalisation and effect on cell growth. CD44 expression on the surface of in vitro fibroblasts was not modified by different concentrations of FCS, whereas it was sensitive to cell cycle, being higher in the growing than in the resting phase. Independently from molecular weight, upon addition of exogenous HA (from 0.1 up to 1 mg/mL) to fibroblasts in the growing phase, a slight but constant decrease of the expression of CD44 on the surface of fibroblasts was observed; moreover, HA induced a rearrangement of CD44 into patches in close relationship with the terminal regions of stress fibers, which became thicker and more rigid after a few hours from the addition of HA to the medium. Fluorescent HA, added to the culture medium, rapidly attached to the plasma membrane and in less than two minutes was observed within cells, partly in association with its receptor CD44. By the contemporary use of neutral red, which accumulates into functional lysosomes, the great majority of internalised HA was found within lysosomes. HA receptor RHAMM-IHABP was rather homogeneously localised within the cytoplasm of normal growing fibroblasts. Upon addition of HA, the RHAMM-IHABP distribution became discontinuous around the nucleus. Addition of HA to fibroblasts induced a significant inhibition of cell growth, which was dependent on HA concentration and irrespective of HA molecular weight, at least in the ranges tested. Results show that extra-cellular HA is rapidly taken up by human dermal fibroblasts together with its CD44 receptor, and transported mostly to the lysosomes. Both low and high molecular weight HA induced down-regulation of cell proliferation, which would seem to be mediated by HA catabolism.

Human sterol 27-hydroxylase catalyses the first step in the alternative pathway of bile acids biosynthesis in hepatocytes. However the gene encoding this enzyme (CYP27 gene) is expressed in every tissue and some evidence suggests that this enzyme plays a role in cholesterol homeostasis. Although modulation of CYP27 expression has been reported, the mechanisms underlying the regulation of this gene in human tissues is still poorly understood. To elucidate the mechanism governing CYP27 expression we cloned a 4.3 kb fragment of the 5' flanking region of the human CYP27 gene and constructed deletion mutants which were transfected into HepG2 cells. Functional assays showed that the -217/-10 nucleotide region from the translation start site (minimal promoter), devoid of TATA and CAAT boxes, contains all the elements for basal transcription. Foot-printing analysis of minimal promoter showed four protected regions (A-D). Regions A, B and D each contain one Sp1 binding site, and region C contains a HNF4 site. Electrophoretic mobility shift assays demonstrated that Sp1, Sp3 and HNF4 transcription factors bind these sites. Mutagenesis of any of these sites resulted in the loss of promoter activity. Co-transfection of the minimal promoter with Sp1 and Sp3 expression vectors transactivated CYP27 gene promoter in Drosophila SL2 cells, which lack endogenous Sp proteins. Transactivation of the minimal promoter was also observed in HeLa cells co-transfected with HNF4 expression vector. Therefore, Sp1, Sp3 and HNF4 co-operate in the expression of the human CYP27 gene in HepG2 cells.

Given the importance of hyaluronan (HA) for the homeostasis of connective tissues during embryogenesis and aging and its role in tissue repair, the aim of the present study was to examine the effect of exogenous HA on the synthesis of total protein, collagen and HA by in vitro human dermal fibroblasts. With differences between different cell strains, HA, at concentrations between 0.5 and 1μM, induced a significant decrease in total protein synthesised and secreted into the medium compared to controls (P< 0.05), and particularly in collagen (− 40%;P< 0.05). The ratios between collagen types I and III and between collagen types V and I were normal. Pulse and chase experiments showed that protein degradation was normal. The presence of exogenous HA did not affect HA synthesis. Data strongly indicate that a relatively high concentration of HA in the extracellular space, such as during development and in the first phases of tissue repair, would partially limit the deposition of the extracellular matrix, and of collagen in particular. This would suggest a role for HA in delaying tissue differentiation during embryogenesis and in preventing fibrosis and scar formation in fetus and in the early phases of wound healing.

The fibrogenic or carcinogenic response to the inhalation of crystalline silica dusts is strictly related to the physicochemical properties of the particles, which, in turn, are mostly determined by the "origin" and the history of the dust. Several physicochemical properties have been reported to modulate silica pathogenicity. None of them simply correlate with the reported toxicity in all the systems used to study silica pathogenicity. This confirms, on the one hand, that several properties are implicated at the same time, and on the other that pathogenicity is the result of a multistage process. There is a general consensus on the key role played by alveolar macrophages in silica-related diseases. For this article the cytotoxicity of a large variety of silicas, including rather unusual forms, with controlled micromorphology and surface properties, has been studied on a mouse monocyte-machrophage tumor cell line successfully employed in previous studies on cristobalite (Fubini et al., 1999). When compared on a per unit surface basis, crystalline silicas were more cytotoxic than amorphous ones, with the notable exception of stishovite, the nonpathogenic crystalline polymorph, with octahedrally coordinated silicon atoms. Among the amorphous ones, a diatomaceous earth and a powdered silica glass exhibited an intermediate toxicity, higher than what was elicited by a pyrogenic silica. In this study a new class of crystalline silicas have been considered, pure-silica zeolites, which constitute a new morphological entity with which cells may be confronted. The cytotoxicity of these samples varies from inert to highly cytotoxic, covering all the range of toxicity covered by the traditional silica dusts. We discuss the influence of morphological properties and surface reactivity on the cytotoxicity of several pure-silica zeolites. The extent of exposed surface and the shape of the particles correlate with cell toxicity. The lower cytotoxicity of one "non-pathogenic quartz" and of an aluminum-coated Min-U-Sil quartz, compared with the original pathogenic Min-U-Sil quartz, suggest a depressive effect of the aluminum ions present at the surface of both quartzes. The extreme variability in the biological response to crystalline silicas is confirmed and a new class of materials is brought to the study of the mechanisms of silica pathogenicity.

The interaction between inhaled particles and alveolar macrophages plays a key role in silica-related diseases. It has been previously shown [Fubini, B., et al. (1999) Chem. Res. Toxicol. 12, 737-745] that a monocyte-macrophage cell line (J774) may be employed in the evaluation of the degree of cytotoxicity to alveolar macrophages of various silica dusts. In this paper, pure-silica zeolites (porosils) in microcrystalline form have been employed as "model solids" in an effort to show which physicochemical properties of the silica particle are playing a major role in the toxicity to macrophages. The samples employed covered four different porosil crystal structures (MFI, FAU, TON, and MTT) and also include a synthetic rodlike cristobalite (CRIS-rd). When compared at equal weight, the samples cover a wide range of cytotoxicity from inert to toxic as unheated mineral cristobalite [Fubini, B., et al. (1999) Chem. Res. Toxicol. 12, 737-745]. Mild grinding did not affect cytotoxicity. Calcined (open pores) and uncalcined (pore filled with template) TON exhibited the same cytotoxicity, indicating that only the outer surface is implied. The hydrophobic and/or hydrophilic character of TON, evaluated by adsorption calorimetry, is close to what has been previously found for silicalite and is consistent with a hydrophilic outer surface and hydrophobic pore walls. The potential for generating hydroxyl radicals from hydrogen peroxide varies among the various porosils that have been studied. A model is proposed for the correlation between inhibition of growth on proliferating cells and physicochemical properties varying from one to the other sample. The extent of external surface and the aspect ratio were related to the intensity of the cytotoxic effect, while the level of radical release was not. This suggests, on one hand, that comparison of toxicity among various dusts should be made at equal particle surface and, on the other, that in the model studied, free radical release does not play a crucial role in the primary event of toxicity to alveolar macrophages.

Pseudoxanthoma elasticum (PXE) is a genetic connective tissue disease, whose gene and pathogenesis are still unknown. Dermal fibroblasts from patients affected by PXE have been compared in vitro with fibroblasts taken from sex and age-matched normal individuals. Cells were grown and investigated in monolayer, into three-dimensional collagen gels and in suspension. Compared with normal cells, PXE fibroblasts cultured in monolayer entered more rapidly within the S phase and exhibited an increased proliferation index; on the contrary, similarly to normal fibroblasts, PXE cells did not grow in suspension. Furthermore, compared with normal fibroblasts, PXE cells exhibited lower efficiency in retracting collagen type I lattices and lower adhesion properties to collagen type I and to plasma fibronectin. This behavior was associated with higher expression of integrin subunits α2, α5, αv, whereas β1 subunit as well as α2β1 and α5β1 integrin expression was lower than in controls. Compared to controls, PXE fibroblasts had higher CAM protein expression in accordance with their high tendency to form cellular aggregates, when kept in suspension. The demonstration that PXE fibroblasts have altered cell-cell and cell-matrix interactions, associated with modified proliferation capabilities, is consistent with the hypothesis that the gene responsible for PXE might have a broad regulatory role on the cellular machinery.