Renato Scandroglio

The analysis of chromatin organisation allows selection of mouse antral oocytes competent for development to blastocyst.

Mouse antral oocytes can be classified in two different types termed SN or NSN oocytes, depending on the presence or absence, respectively, of a ring of Hoechst 33342-positive chromatin surrounding the nucleolus. The aim of the present study was to test the developmental competence to blastocyst of the two types of oocytes. Here we show that following isolation, classification and culture of cumulus-free antral oocytes, 14.7% and 74.5% of NSN and SN oocytes, respectively, reached the metaphase II stage. When fertilised and further cultured none of the metaphase II NSN oocytes developed beyond the 2-cell stage whilst 47.4% of the metaphase II SN oocytes reached the 4-cell stage and 18.4% developed to blastocyst. The findings reported in this paper may contribute to improved procedures of female gamete selection for in vitro fertilisation of humans and farm animals. Furthermore, the selection of oocytes with better developmental potential may be of interest for studies on nuclear/cytoplasm interaction, particularly in nuclear-transfer experiments.

Implants of heterologous demineralized bone matrix for induction of posterior spinal fusion in rats

The authors tested The osteoinductive capacity of powdered heterologous (bovine) demineralized bone matrix in rats. The first part of the study concerned a monolateral posterior spinal implant after decortication of three vertebrae, using as a control area the animals contralateral side, in which neither bone graft nor any other material were placed, In another group of rats, a comparative evaluation was made of powdered heterologous demineralized bone matrix and fresh autologous bone. In the same animal, autologous bone was implanted to realize a thoracic posterior fusion and demineralized bone matrix, to induce a posterior fusion in the lumbar area. All data obtained suggested a good osteoinductive activity of heterologous powdered demineralized bone matrix. The two posterior spinal fusions done in the same animal with heterologous demineralized bone matrix or authologous bone, respectively, had similar callus development and required the same fine for formation.

Time course assessment of methylmercury effects on C6 glioma cells: submicromolar concentrations induce oxidative DNA damage and apoptosis

Organic mercury is a well‐known neurotoxicant although its mechanism of action has not been fully clarified. In addition to a direct effect on neurons, much experimental evidence supports an involvement of the glial component. We assessed methylmercury hydroxide (MeHgOH) toxicity in a glial model, C6 glioma cells, exposed in the 10−5–10−8 M range. The time course of the effects was studied by time‐lapse confocal microscopy and supplemented with biochemical data. We have monitored cell viability and proliferation rate, reactive oxygen species (ROS), mitochondrial transmembrane potential, DNA oxidation, energetic metabolism and modalities of cell death. The earliest effect was a measurable ROS generation followed by oxidative DNA damage paralleled by a partial mitochondrial depolarization. The effect on cell viability was dose dependent. TUNEL, caspase activity and real‐time morphological observation of calcein‐loaded cells showed that apoptosis was the only detectable mode of cell death within this concentration range. N‐acetyl‐cysteine (NAC) or reduced glutathione (GSH) completely prevent the apoptotic effect of MeHgOH. The lowest effective MeHgOH concentration was 10−7 M for ROS and DNA OH‐adducts generation. The effect of submicromolar concentrations of MeHgOH on C6 cells could be relevant in the developmental neurotoxicity caused by low dose, long‐term exposures, such as those of food origin. In addition, we have shown that the same concentrations are effective in the induction of DNA oxidative damage, with further potential pathogenetic implications.

Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8 + cells in patients with hepatocellular carcinoma

BACKGROUND/AIMS Members of the melanoma antigen encoding gene family are expressed in tumors of different histological types but not in normal tissue. For this reason, they are attractive targets for cancer immunotherapy. METHODS In the present study, we analyzed the expression of MAGE-1 and -3 genes in the hepatocellular carcinoma (HCC) tissue as well as frequency, phenotype and function of circulating and tumor infiltrating CD8+ cells specific for HLA-A1 and -A2 restricted epitopes of MAGE-1 and -3. RESULTS Our study shows for the first time the presence of MAGE/tetramer+ CD8 cells in the tumor tissue of patients with HCC. These cells are able to recognize the MAGE-1 sequence 161-169 and the MAGE-3 sequence 271-279. In a patient with a particularly high frequency of MAGE-1 sequence 161-169-specific T cells, phenotypic and functional analysis was performed showing a phenotype of recently-primed CD8 cells (CD28+CD27+CD45RA-CCR7). CONCLUSIONS The observation of a spontaneous in vivo priming of a MAGE-specific T cell response in patients with HCC and the high frequency of MAGE antigens expression in this tumor, makes this antigen a potential candidate for a MAGE-specific immunotherapy in hepatocellular carcinoma.

Calcein-AM is a detector of intracellular oxidative activity

Calcein-acetoxymethylester (calcein-AM) is a non-fluorescent, cell permeant compound, which is converted by intracellular esterases into calcein, an anionic fluorescent form. It is used in microscopy and fluorometry and provides both morphological and functional information of viable cells. In this study we have tested the response of calcein-AM to oxidation. In cell-free fluorometric assays, H2O2 and xanthine–xanthine oxidase induced a dose-dependent emission of the AM form but had no effects on calcein. Fluorometric and confocal microscopy tests on human fibroblasts confirmed that the cell permeant AM form is the actual sensor since its removal from culture medium, and its consequent back-diffusion, made the system insensitive to oxidative stimuli. In time-lapse confocal microscopy, calcein-AM detected changes in the intracellular redox state following direct oxidation (H2O2, xanthine–xanthine oxidase) and phorbol ester treatment. Comparative tests showed that calcein-AM sensitivity to oxidation is about one order of magnitude higher than other fluorescein derivatives. The absence of leakage, due to the presence of the probe in the extracellular compartment, and its low toxicity allow to perform experiments for prolonged times following the response to the same or different stimuli repeatedly applied. We propose calcein-AM as a sensitive tool for intracellular ROS generation in living cells with useful applications for real-time imaging in confocal microscopy.

Pulsed electromagnetic field stimulation on posterior spinal fusions: A histological study in rats

This study reports the histological data relative to the effect of pulsed electromagnetic fields (PEMFs) on the evolution of posterior arthrodesis induced in the lumbar vertebrae of 12 adult male Sprague-Dawley rats. After the operation, one group of six rats was stimulated with PEMFs for 18 h per day, by means of a pair of coils fixed to the outside of the cage. A control group of six rats was given no stimulation after surgery. In the groups stimulated with PEMFs an acceleration of the process of bone callus organization was already observed after 4 weeks, and even more so after 8: An early replacement was in fact observed of the newly formed cartilage tissue with primary bone (at 4 weeks) and subsequently with secondary bone (after 8 weeks).

Adhesion of human osteoblasts to titanium: A morpho-functional analysis with confocal microscopy

Properties of surface affect the interactions between the implant and osteoblasts and direct the clinical osteointegrative outcome. The aim of this in vitro study was to describe the adhesion of living human osteoblasts to titanium disks with differently prepared surfaces: sand blasted with ZrO(2) particles and acid-etched (Soft-SLA, S-SLA) or with Al(2)O(3) particles and acid-etched (Hard-SLA, H-SLA), smooth surface (SS). Confocal microscopy was exploited to follow cell morpho-functional features either on living cells (cell shape with calcein-acethoxymethylester and mitochondria with tetramethylrhodamine methyl ester) or on fixed cells (immunocytochemistry of beta1-integrin and of actin) 6h or 24h after seeding. The underlying surface was visualized simultaneously on the same field. No cytotoxic effect was detected at any time and on any surface. At 6h after seeding, osteoblasts showed either a rounded or polygonal shape on both rough surfaces. Several features suggested that adhesion was faster with a higher level of organization on S-SLA than on H-SLA. Indeed osteoblasts grown on S-SLA were wider and with more extended protrusions than those on H-SLA. Active mitochondria on S-SLA occupied perinuclear areas and cellular prolongations, whereas on H-SLA they were mainly focused around nucleus. Organization of integrin beta1-subunit and actin, confirmed different kinetics of cell adhesion. At 6h integrin beta1-subunit was distributed along the periphery on the cell-biomaterial focal complexes in cells grown on S-SLA, whereas it was unevenly dispersed in membrane of cells cultured on H-SLA. Stress actin fibers were well defined in cells cultured on S-SLA, whereas they were scarcely evident on H-SLA. Osteoblasts seeded on smooth surface for 6h had morpho-functional features typical of adhesion, with some elements characterized by an elongated shape with an evident main longitudinal axis. At 24h osteoblasts were spread-out onto all surfaces. Nonetheless, different morphologies were shown in response to the different surfaces tested: polygonal cells prevailed on SLA surfaces, whereas almost all the cells on SS were long with two principal prolongations. At 24h number of cells adhered to the three kind of surfaces was similar, but during the following three days, cells seeded on S-SLA and on SS proliferated to a greater extent than those cultured on H-SLA. Analysis of morpho-functional parameters performed in living cells, and in particular the study of mitochondria organization, proved to be a valuable tool to follow cell-biomaterial adhesion. A higher level of spreading occurring in osteoblasts grown on S-SLA and SS at early times accounted for a faster subsequent cell proliferation. Nonetheless, these comparable activities were exerted by cells showing polygonal or elongated shapes when grown respectively on S-SLA or on SS. The former is typical of osteogenic cells, whereas the latter resumes a fibroblast-like morphology, that would result in an ineffective in vivo osteointegrative process.

Low-temperature heat-deproteinated compact bone to heal large bone defects.

The potential of low-temperature (400 degrees C), heat-treated bone matrix in osteorepair has been evaluated in vivo by implantation into defects artificially created in rodent tibia. Histological and ultrastructural analysis of the bone--implant interface has been carried out on samples obtained at 1 to 6 weeks from operation. The obtained data showed that calcined bone is well tolerated and does not cause acute or chronic inflammatory reactions. Osteoid tissue, tightly adhered to the implant, appears within 2 weeks of the operation, while after 6 weeks newly formed bone surrounds and infiltrates the implant. Of greater note, the detection of good adhesion between bone and implant ultrastructurally is demonstrated by the absence of fibrillar connective tissue at the interface. For these reasons, our preliminary observations suggest that low-temperature calcined bone (biological apatite or heat-deproteinated bone) may have a rightful place among the osteointegrators.

Three-dimensional localization and dynamics of centromeres in mouse oocytes during folliculogenesis

Very little is known about oocyte nuclear architecture during folliculogenesis. Using antibodies to reveal centromeres, Hoechst-staining to detect the AT-rich pericentromeric heterochromatin (chromocenters), combined with confocal microscopy for the three-dimensional analysis of the nucleus, we demonstrate that during mouse folliculogenesis the oocyte nuclear architecture undergoes dynamic changes. In oocytes isolated from primordial and primary follicles, centromeres and chromocenters were preferentially located at the periphery of the nucleus. During oocyte growth, centromeres and chromocenters were initially found spread within the nucleus and then progressively clustered around the periphery of the nucleolus. Our results indicate that the oocyte nuclear achitecture is developmentally regulated and they contribute to a further understanding of the role of nuclear organization in the regulation of genome functioning during differentiation and development.

The fine structure of HNK-1 (Leu7) positive cells

SummaryThe ultrastructural characteristics of HNK-1 (Leu7) positive cells, visualized with a peroxidase labelled anti-mouse IgM serum, were analysed. Our investigation demonstrates: 1) the majority of Leu7 positive cells has a low nuclear/cytoplasmic ratio (N/C), an irregular outline, a well developed smooth endoplasmic reticulum, parallel tubular arrays (PTA) and electron dense granules; 2) the minority of Leu7 positive cells has a high N/C, regular profiles and lacks electron dense granules. The presence of two distinct ultrastructural patterns within Leu7 positive cells may represent: 1) the expression of subsequent stages of cell differentiation; 2) two distinct Leu7 positive cell subpopulations.