Expression profiling of extramedullary acute myeloid leukemia suggests involvement of epithelial-mesenchymal transition pathways.

Extramedullary (EM) colonization is a rare complication of acute myeloid leukemia (AML), occurring in about 10% of patients, but the processes underlying tissue invasion are not entirely characterized. Through the application of RNAseq technology, we examined the transcriptome profile of 13 AMLs, 9 of whom presented an EM localization. Our analysis revealed significant deregulation within the extracellular matrix (ECM)-receptor interaction and focal-adhesion pathways, specifically in the EM sites. The transcription factor TWIST1, which is known to impact on cancer invasion by dysregulating epithelial-mesenchymal-transition (EMT) processes, was significantly upregulated in EM-AML. To test the functional impact of TWIST1 overexpression, we treated OCI-AML3s with TWIST1-siRNA or metformin, a drug known to inhibit tumor progression in cancer models. After 48 h, we showed downregulation of TWIST1, and of the EMT-related genes FN1 and SNAI2. This was associated with significant impairment of migration and invasion processes by Boyden chamber assays. Our study shed light on the molecular mechanisms associated with EM tissue invasion in AML, and on the ability of metformin to interfere with key players of this process. TWIST1 may configure as candidate marker of EM-AML progression, and inhibition of EMT-pathways may represent an innovative therapeutic intervention to prevent or treat this complication.

The Anti-Leukemia Effect of Ascorbic Acid: From the Pro-Oxidant Potential to the Epigenetic Role in Acute Myeloid Leukemia.

Data derived from high-throughput sequencing technologies have allowed a deeper understanding of the molecular landscape of Acute Myeloid Leukemia (AML), paving the way for the development of novel therapeutic options, with a higher efficacy and a lower toxicity than conventional chemotherapy. In the antileukemia drug development scenario, ascorbic acid, a natural compound also known as Vitamin C, has emerged for its potential anti-proliferative and pro-apoptotic activities on leukemic cells. However, the role of ascorbic acid (vitamin C) in the treatment of AML has been debated for decades. Mechanistic insight into its role in many biological processes and, especially, in epigenetic regulation has provided the rationale for the use of this agent as a novel anti-leukemia therapy in AML. Acting as a co-factor for 2-oxoglutarate-dependent dioxygenases (2-OGDDs), ascorbic acid is involved in the epigenetic regulations through the control of TET (ten-eleven translocation) enzymes, epigenetic master regulators with a critical role in aberrant hematopoiesis and leukemogenesis. In line with this discovery, great interest has been emerging for the clinical testing of this drug targeting leukemia epigenome. Besides its role in epigenetics, ascorbic acid is also a pivotal regulator of many physiological processes in human, particularly in the antioxidant cellular response, being able to scavenge reactive oxygen species (ROS) to prevent DNA damage and other effects involved in cancer transformation. Thus, for this wide spectrum of biological activities, ascorbic acid possesses some pharmacologic properties attractive for anti-leukemia therapy. The present review outlines the evidence and mechanism of ascorbic acid in leukemogenesis and its therapeutic potential in AML. With the growing evidence derived from the literature on situations in which the use of ascorbate may be beneficial in vitro and in vivo, we will finally discuss how these insights could be included into the rational design of future clinical trials.

[Global health education in Italian medical schools: survey from 2007 to 2010]. / L'insegnamento della Salute Globale nelle Facoltà di Medicina e Chirurgia in Italia: l'offerta formativa nel triennio 2007-2010.

Global Health (GH) issues are becoming a common feature of Medical and Public Health Schools worldwide. In Italy the Network for Education on Global Health (RIISG) was created with the purpose of spreading the concept of GH. The aim of the study was to assess the availability of educational opportunities in Italian Health Faculties from 2007 to 2010. A survey was carried out using a questionnaire administered to Professors. A frequency distribution of GH elective courses, grouped by three Italian geographical areas (North, Centre, South and Islands), for each academic year was assessed. The features of the courses - consistent with the pattern of course, suggested by RIISG - were analysed through a score. From 2007 onwards, in chronological order the surveyed faculties were 40, 36, 36 and the main coverage of survey was 92%. The courses listed were 26, 22 and 40 respectively for each academic year considered. The average of the courses number highlighted an increasing trend: national mean rose from 0.65 (SD +/- 1.53) in 2007 to 1.11 (SD +/- 1.18) in 2010. Regarding the evaluation of consistency a national improvement was shown. The assessment revealed a limited educational offer and differences between macroareas. Further investigations are needed.

Effects of benign prostatic hyperplasia and finasteride therapy on prostatic blood flow in dogs.

Benign prostatic hyperplasia (BPH) is characterized by an enlargement of the prostate accompanied by an increase in prostatic blood perfusion and vascularization. The most indicated treatment is to perform orchiectomy, however, medical treatment with finasteride can be an option for breeding dogs or elderly animals with a critical health status. In dogs, the influence of medical treatment on prostatic hemodynamics is still unknown. Therefore, this study aimed to evaluate the effects of benign prostatic hyperplasia and finasteride therapy on hemodynamic and vascular features of the canine prostate. For this purpose, twenty dogs of different breeds, body weights (10-30 kg) and ages (5-13 years) were used, assigned for: Healthy-non treated group (n = 5), BPH-non treated group (n = 5), Healthy-finasteride treated group (n = 5) and BPH-finasteride treated group (n = 5). Dogs that presented hematospermia and at least one general clinical sign (tenesmus, hematuria or dysuria) were presumptively diagnosed with BPH. Dogs were evaluated ultrasonographycally by B-mode and Doppler of the prostatic artery in a monthly interval (day 0, 30 and 60) in order to measure prostate volume (PV), expected prostate volume (EPV), prostate vascularization score (scored as minimum, intermediary and maximum) and prostatic artery blood flow parameters with the use of spectral and color Doppler ultrasound. It was possible to observe a decrease in prostate vascularization score between Day 0 (intermediary degree) and 60 (minimum degree) in finasteride treated dogs. Moreover, non-treated dogs had higher score of vascularization at Day 60 compared to animals treated with finasteride, regardless of BPH diagnosis. Healthy-non treated animals presented higher peak systolic:diastolic velocity (S/D) than BPH-non treated dogs. Furthermore, BPH-non treated dogs had lower S/D than BPH-finasteride treated dogs. In 30 and 60 days, no difference on PV was observed between BPH-finasteride treated group and Healthy-non treated group. At day 60, no difference between PV and EPV was observed for the BPH-finasteride treated group. In conclusion, finasteride treatment reduces simultaneously the volume, local vascularization and blood flow of the prostate, thus, being considered an effective and additional choice of therapy for BPH. Moreover, the course of therapy in dogs can be followed by analyzing changes in prostatic artery.

Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone.

Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm(2) show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation.

The aggregation of pig articular chondrocyte and synthesis of extracellular matrix by a lactose-modified chitosan.

A reductive amination reaction (N-alkylation) obtained exploiting the aldheyde group of lactose and the amino group of the glucosamine residues of chitosan (d.a. 89%) afforded a highly soluble engineered polysaccharide (chitlac) for a potential application in the repair of the articular cartilage. Chitosan derivatives with 9% and 64% of side chain groups introduced have been prepared and characterized by means of potentiometric titration, (1)H-NMR and intrinsic viscosity. Both polymers, with respect to the unmodified chitosan, induce cell aggregation when in contact with a primary culture of pig chondrocytes, leading to the formation of nodules of considerable dimensions (up to 0.5-1 mm in diameter). The nodules obtained from chondrocytes treated with chitlac with the higher degree of substitution have been studied by means of optical and electron microscopy (SEM, TEM) and the production of glycosaminoglycans (GAGs) and collagen has been measured by means of colorimetric assays. The chondro-specificity of GAG and collagen was determined by RT-PCR. The results show that the lactose-modified chitosan is non-toxic and stimulates the production of aggrecan and type II collagen.

Osteoprotegerin (OPG) and RANKL expression and distribution in developing human craniomandibular joint.

During embryogenesis the bone tissue of craniomandibular joint (CMJ) is formed through two pathways: intramembranous ossification and endochondral ossification. The development process is under the control of regulatory factors. The osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kappaB ligand are key regulators of osteoclastogenesis. The aim of this study is the localization of OPG and RANKL mRNA and protein in the foetal CMJ by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: OPG and RANKL mRNA and protein were co-localized in the same cell types; OPG and RANKL were specially immunolocated in osteogenic cells; immunolabeling was often seen in the nucleus and cytoplasm of otherwise negative hypertrophic chondrocytes; IHC and ISH labeling decreased from proliferative to hypertrophic chondrocytes; early osteocytes showed dual protein expression and some of the mature osteocytes were ISH-negative; periosteal osteoclasts and chondroclasts were mostly stained by IHC and variably labeled by ISH; the new bone matrix and trabecular borders showed intense immunolabeling. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism in the CMJ development and their extracellular presence in the new bone matrix and trabecular borders suggests a local regulatory role.

Morphological investigation of epiphyseal cartilage after glutaraldehyde-malachite green fixation.

The glutaraldehyde-malachite green (GlMG) fixation has been reported to allow preservation of tissue lipids, mainly acidic phospholipids, that in the absence of malachite green are lost in the glutaraldehyde solution. This method has been used for the morphological study of calcifying epiphyseal cartilage, with particular reference to the presence of lipids in chondrocytes, cartilage matrix and calcification nodules. Both light and electron microscopy show that after GlMG-osmium fixation the chondrocytes contain cytoplasmic dense bodies. These have the same morphology as those found with the same method in other cells and tissues and considered to be lipid globules. However, they are not modified by treatment of GlMG-fixed specimens with chloroform-methanol. Formic acid decalcification induces partial solubilization of these bodies, whereas EDTA decalcification leaves them unchanged. They are found also in the pericellular space and in calcifying matrix. GlMG fixation improves the preservation of the crystal-associated organic structures (crystal ghosts) when the cartilage is decalcified before embedding. When these structures are demonstrated by the post-embedding decalcification and staining method, their morphology is similar to that found in controls. Moreover, GlMG fixation demonstrates the presence of typical rod-like, beaded structures, probably corresponding to proteoglycans, on the outer surface of the chondrocyte membrane. Although composition and role in calcification of the dense bodies remain uncertain, the important physiological implications that can be drawn from their presence in chondrocytes and cartilage matrix require further investigation.

Combined use of malachite green fixation and PLA2-gold complex technique to localize phospholipids in areas of early calcification of rat epiphyseal cartilage and bone.

Two cytochemical methods (malachite green fixation and PLA2-gold complex technique) were combined in order to detect the presence and ultrastructural distribution of phospholipids in epiphyseal cartilage and metaphyseal bone of 25-day-old rats. Chondrocytes and osteoblasts showed a more intense PLA2-gold complex labeling of rough endoplasmic reticulum than mitochondria and plasma membranes. Roundish osmiophilic, electron-dense, lightly labeled structures were visible at the periphery of the cells. In areas of early mineralization of cartilage, not all the matrix vesicles were labeled by PLA2-gold complex. Calcification nodules showed intense labeling in comparison with the surrounding, lightly labeled uncalcified matrix; gold particles were chiefly found at their periphery. Calcification nodules of metaphyseal bone were deeply electron-dense after glutaraldehyde-malachite green fixation; PLA2-gold labeling was mainly found in connection with crystals, whereas the uncalcified matrix was weakly labeled. Calcified bone matrix showed a heavy labeling with randomly scattered gold particles. When pre-embedding decalcification was carried out in the presence of malachite green, a good preservation of organic component was obtained. The PLA2-gold positivity of decalcified areas of cartilage and bone confirmed the presence of phospholipids in mineralized matrix.

Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after high-dose treatment with corticosterone.

A connection has been suggested between glucocorticoid-induced osteopenia and an increase in the apoptosis of bone cells, and between the dimerization of the glucocorticoid receptor (GR) and the development of apoptosis. On this basis, a study has been carried out on the relationships between the occurrence of apoptotic cells and their detectable GR content, and between apoptosis frequency and changes in histomorphometric variables, in the growth plate and secondary spongiosa of rat long bones after the high-dose (10 mg/day) administration of corticosterone (CORT) and after recovery. The main results of the CORT treatment were: a significant increase in apoptotic osteoblasts, and a concomitant decrease in the histomorphometric variables of bone formation, with a reversal of both values during recovery; a nonsignificant increase in the apoptosis of osteoclasts, without changes in the histomorphometric variables of bone resorption; a significant increase in apoptotic terminal hypertrophic chondrocytes; the presence of GR in all types of skeletal cells in control rats, with different (cytoplasmic and/or nuclear) immunohistochemical detection in the same type of cell; a decrease in GR detection in proliferative chondrocytes and osteocytes in CORT and recovery groups, and in the maturative/hypertrophic chondrocytes of the recovery group; a fall in growth cartilage width, possibly due to the reduced proliferation of proliferative chondrocytes and increased apoptosis in terminal hypertrophic chondrocytes. In conclusion, pharmacological doses of CORT reduce bone formation by increasing osteoblast apoptosis; they reduce growth cartilage width, probably by inhibiting chondrocyte proliferation and increasing the apoptosis of terminal hypertrophic chondrocytes, and they reduce osteocyte GR. Although these effects appear to be mediated by the presence of GR in all skeletal cells, no precise correlation between GR immunohistochemical detection and apoptosis induction has been found.

A histomorphometric, structural, and immunocytochemical study of the effects of diet-induced hypocalcemia on bone in growing rats.

Despite several studies on the effect of calcium deficiency on bone status, there is relatively little information on the ensuing histological alterations. To investigate bone changes during chronic hypocalcemia, weanling rats were kept on a calcium-free diet and deionized water for 28 days while control animals were fed normal chow. The epiphyseal-metaphyseal region of the tibiae were processed for histomorphometric, histochemical, and structural analyses. The distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN), three noncollagenous bone matrix proteins implicated in cell-matrix interactions and regulation of mineral deposition, was examined using postembedding colloidal gold immunocytochemistry. The experimental regimen resulted in serum calcium levels almost half those of control rats. Trabecular bone volume showed no change but osteoid exhibited a significant increase in all its variables. There were a multitude of mineralization foci in the widened osteoid seam, and intact matrix vesicles were observed in the forming bone. Many of the osteoblasts apposed to osteoid were tartrate-resistant acid phosphatase (TRAP)- and alkaline phosphatase-positive, whereas controls showed few such TRAP-reactive cells. Osteoclasts in hypocalcemic rats generally exhibited poorly developed ruffled borders and were inconsistently apposed to bony surfaces showing a lamina limitans. Sometimes osteoclasts were in contact with osteoid, suggesting that they may resorb uncalcified matrix. Cement lines at the bone-calcified cartilage interface in some cases were thickened but generally did not appear affected at bone-bone interfaces. As in controls, electron-dense portions of the mineralized matrix showed labeling for BSP, OC, and OPN but, in contrast, there was an abundance of immunoreactive mineralization foci in osteoid of hypocalcemic rats. These data suggest that chronic hypocalcemia affects both bone formation and resorption.

Localization of bone sialoprotein (BSP) to Golgi and post-Golgi secretory structures in osteoblasts and to discrete sites in early bone matrix.

Bone sialoprotein (BSP), a bone matrix-enriched glycoprotein containing the Arg-Gly-Asp (RGD) motif and endowed with cell binding properties, was localized in osteoblasts and early bone matrix of developing rat bone at the ultrastructural level. Preliminary light microscopic observations indicated that intracellular labelling was restricted to a paranuclear dot corresponding to the "negative Golgi image" of classical histology. The same pattern was observed whether antisera against the fully glycosylated protein or a peptide antiserum to a stretch of amino acids in human BSP sequence were employed. At the EM level, we obtained labeling over the Golgi area of osteoblasts but not over the rER. The labeling was concentrated over distensions of the trans Golgi and over pro-secretory granules. In the matrix, BSP was distributed in a non-random manner. The label was concentrated over spherical aggregates of finely fibrillar material corresponding to the sites of early mineral deposition (so-called "mineralization nodules"). Such BSP-positive foci were seen both close to and away from the cell surface. The predominant association of BSP with Golgi and post-Golgi secretory structures and its absence from rER, as well as the reproducibility of the same pattern of localization with different antisera, might indicate a slow transit of the protein through the Golgi, not necessarily associated with protein glycosylation.

Features of excessive alcohol drinking in older adults distinctively captured by behavioral and biological screening instruments. An epidemiological study.

The entire > or =65-year-old population living in a small Italian town, where alcohol use is almost ubiquitous, was assessed with a frequency-quantity questionnaire for alcohol intake and with two screening instruments for alcohol problems, the CAGE questionnaire and the MCV-gammaGT test. Aim of the study was to assess whether these instruments identify different subsets of subjects with alcohol problems. Of the 649 participants, 19.1% were at-risk drinkers (average intake > 40 g/day in men and > 20 g/day in women). Both the screening instruments were positive in only a minority of participants. Of the 377 drinkers, 53 gave > or =1 affirmative response to the CAGE questionnaire, whereas 24 had a positive MCV-gammaGT test. The concordance between positive CAGE questionnaire and MCV-gammaGT test was limited to seven subjects (kappa = 0.10), and these tests identified subjects who differed for several health and psychosocial characteristics. Participants aged > or =75 years drank less, but had similar prevalence of CAGE and MCV-gammaGT positive markers as compared to younger participants. In conclusion, excessive drinking is common in the elderly. Screening tests based on behavioral and biological markers identify two different sets of subjects with possible alcohol problems. This might indicate the opportunity to use these instruments in conjunction.

In vivo incidence of apoptosis evaluated with the TdT FragEL DNA fragmentation detection kit in cartilage and bone cells of the rat tibia.

Previous studies have shown the occurrence of cell death by apoptosis in cartilage and bone cells, and have suggested a functional relationship between bone growth and remodelling on one hand, and numbers of apoptotic cells on the other. At present, no in vivo studies are available on the frequency of the apoptotic process measured at one time and in one place using the cartilage and bone cells of single specimens. The aim of the present investigation was to measure the in vivo incidence of apoptosis in cartilage and bone cells of the upper epiphysis and secondary ossification metaphyseal bone of the tibia in normal young adult rats. Apoptotic cells were visualized with the terminal deoxynucleotidyl transferase (TdT) FragEL DNA fragmentation detection kit, which is analogous to the TdT-mediated nick end-labelling (TUNEL) method. In the growth cartilage, only a few TUNEL-positive terminal hypertrophic chondrocytes were found; they were 1.32 +/- 0.70% of the total hypertrophic chondrocytes counted along the chondro-osseous junction. There were only a few apoptotic osteoblastic cells and osteocytes (0.22 +/- 0.22% and 0.15 +/- 0.16% of total osteoblasts and osteocytes respectively). TUNEL-positive osteoclasts were 1.03 +/- 0.57% of the total of osteoclastic cells; they usually showed only one or two apoptotic nuclei. The total number of TUNEL-positive bone marrow cells were also counted (56.78 +/- 10.29/mm2 of bone marrow spaces). Our results confirm that apoptosis does occur in hypertrophic chondrocytes and bone cells, and show that its frequency is very low. However, chiefly because of its short lifespan, the frequency of apoptosis in cartilage and bone may be higher than that shown by the TUNEL method. The static estimate that can be obtained with this method might lead to misleading conclusions on the physiological significance of such a dynamic, rapid and asynchronous process, whose precise importance in bone growth and remodelling remains to be determined.

Bcl-2 and Bax expression in cartilage and bone cells after high-dose corticosterone treatment in rats.

The expression of Bcl-2 and Bax has been evaluated by immunohistochemistry in normal rats, and in rats after treatment with high-dose corticosterone (CORT). Proliferative (PC) and maturative/hypertrophic (MaHC) chondrocytes of the growth plate have been examined, as well as osteoblasts (Obs), osteocytes (Ots) and osteoclasts (Ocs) of the metaphyseal secondary spongiosa. For each cell type, the Bcl-2 and Bax immunopositive cells were expressed as a percentage of the total number of cells. Bcl-2 and Bax expression was considered to be enhanced when the percentage of positive cells rose. Bcl-2 and Bax were expressed in all cell types, and two main kinds of labeling distribution, both suggestive of association with intracellular organelles, were observed in the cytoplasm: scarce and spotty labeling (type 1) or abundant, granular and diffuse labeling (type 2). In some cases, nuclear membranes could also be seen to be positive. Positive PCs and Obs generally showed a labeling of type 1, MaHCs and Ocs of type 2, while Ots varied with labeling of type 1 or type 2. CORT administration induced a fall in the percentage of Bcl-2 immunopositive cells, and a rise in that of Bax immunopositive cells, in PCs and Ots. The same trend was observed in MaHCs, although the Bcl-2 decrease was not significant. The percentage of Bcl-2 and Bax immunopositive Obs rose, and their labeling distribution shifted from type 1- to type 2-labeled cells. Ocs showed the highest immunopositivity for both Bcl-2 and Bax, which did not change after CORT administration. These data suggest that CORT treatment, by lowering Bcl-2, and raising Bax expression, may promote the apoptotic process in PCs, MaHCs and Ots. Obs, however, do not undergo the same variations. This finding, together with the results of a previous study showing that CORT administration raises the frequency of apoptotic Obs, does not support a direct relationship between apoptosis and Bax overexpression, at least in Obs. The CORT effect might be related to cell types and their state of differentiation, so that Bcl-2 and Bax might regulate not only the machinery of cell death, but also cell proliferation and differentiation.

Immunohistochemical investigation on the presence of chondroitin sulfate in calcification nodules of epiphyseal cartilage.

Chondroitin sulfate localization in mouse epiphyseal cartilage was studied using CS-56 monoclonal antibody immunospecific for the glycosaminoglycan portion of the molecule. For light and fluorescence microscopy, decalcified specimens were embedded in paraffin, Lowicryl, or were frozen and cryostat-sectioned, and the antigen-antibody reaction was demonstrated by treating sections with IgM-peroxidase, IgM-alkaline phosphatase, or IgM-fluorescein conjugates. For electron microscopy, decalcified and undecalcified specimens were embedded in Lowicryl; ultrathin sections from undecalcified specimens were decalcified by flotation on EDTA; sections from both types of specimens were treated with IgM-immunogold conjugate for demonstration of CS-56 reaction. Before immunoreaction, part of all decalcified sections were digested with Streptomyces or testicular hyaluronidase. Control sections were treated with either mouse and goat non-immune serum, or mouse monoclonal antiserum to human dendritic reticulum cells. Both light and electron microscopy show CS-56 reaction with cytoplasmic components of maturing and hypertrophic chondrocytes. Under the light microscope, immunoreaction was not visible in calcified matrix, and was visible in uncalcified matrix only after hyaluronidase digestion. Under the electron microscope, it was evident both in uncalcified and calcified matrix, although the latter showed few immunogold particles, usually placed on areas which appeared incompletely calcified. Gold particles were chiefly distributed at the periphery of calcification nodules and fully calcified matrix. These results show that CS-56, besides reacting with cytoplasm of maturing and hypertrophic chondrocytes, binds to crystal ghosts and other components of cartilage matrix, immunoreactivity decreasing as calcification increases. This suggests that chondroitin sulfate molecules are either degraded during calcification, or segregated into macromolecular complexes, or both degraded and segregated. The second possibility is supported by the increase of immunosensitivity induced by hyaluronidase digestion.

MC22-33F monoclonal antibody shows unmasked polar head groups of choline-containing phospholipids in cartilage and bone.

The importance of the role lipids may have in biological calcification, and the paucity and poor specificity of the methods available for studying their ultrastructural localization, call for further investigations involving new techniques. The monoclonal antibody MC22-33F displays a specific reaction with phosphatidylcholine, sphingomyelin and dimethylphosphatidylethanolamine, as confirmed by its reaction with synthetic lyposomes of pure phosphatidylcholine. For this reason, it was used to demonstrate the presence and ultrastructural localization of choline-containing phospholipids in calcifying cartilage and bone. The MC22-33F MAb immunoreaction was found in the cytoplasm of maturing and hypertrophic and, to a lesser extent, proliferating and degenerating chondrocytes; it was strongly positive in matrix vesicles, at the periphery of calcification nodules, and at the periphery of calcified matrix. In bone, the immunoreaction was especially strong along the peripheral membrane of the osteoblasts, in the interfibrillary spaces of the osteoid border, in matrix vesicles and in the peripheral zone of the calcification nodules. The central zone of the nodules, the fully calcified matrix and most of the intracellular membranes were negative in both cartilage and bone. These results confirm the presence of choline-containing phospholipids in early areas of calcification, including matrix vesicles. They also show that the intracellular membrane phospholipids are not accessible to the antibody, possibly because they are masked by other substances. These are not proteoglycans, because their enzymatic digestion does not increase or modify the reactivity of MC22-33F MAb. In spite of this limitation, MC22-33F MAb offers considerable advantages in the study of the calcification process, because the positive immunoreaction of the calcifying bone matrix, and the negativity of calcified matrix, confirm without doubt that phospholipids have an active role in biological calcification.

Morphological and histochemical study of supragingival human calculus and dental plaque using ruthenium hexammine trichloride and acridine orange.

Ruthenium hexammine trichloride (RHT) and acridine orange were used to preserve and visualize anionic groups in human plaque and dental calculus. RHT-reacting material was present on the membrane of micro-organisms and in intermicrobial spaces of the calcifying areas, and seems to correspond to, and derive from, acidic glyco- and phospholipids of the plasma membrane of the micro-organisms. However, the presence of acidic salivary peptidoglycans cannot be ruled out. Two types of calcification were found: extramicrobial and intramicrobial. The former consisted of calcified deposits irregularly scattered in the intermicrobial matrix. They were in close relationship with RHT-reacting material, or were placed inside vesicular structures delimited by a membrane. Intramicrobial calcification consisted of small aggregates of needle-shaped crystals and/or of granular deposits; in both cases, they either masked the whole cytoplasm of the micro-organisms, or were located only over the plasma membrane. These results suggest that mineral deposition occurs in connection with acidic components of intermicrobial matrix, microbial plasma membranes, and cytoplasms. The addition of RHT and acridine orange to fixing and decalcifying solutions yields satisfactory preservation of dental calculus and plaque, and apparently reduces loss of their anionic organic components and increases their electron density. However, these substances are not sufficient to preserve all ultrastructural details in decalcified areas, probably because the inorganic substance prevents reaction of acridine orange and RHT with the organic components of the calcified matrix.

Localization of the glucocorticoid receptor mRNA in cartilage and bone cells of the rat. An in situ hybridization study.

The in vivo localization of glucocorticoid receptor (GR) mRNA expression was studied in the cartilage and bone cells of the femur of young adult rats to compare its distribution with that of the GR protein, which had previously been shown histochemically in the same areas. To achieve this, we used a synthetic oligodeoxynucleotide as a probe, in line with the published human GR (hGR) cDNA sequence. The probe was coupled to fluorescein (FL), applying a rapid Fast-Tag TM FL nucleic acid labeling method. Negative controls were achieved by using sense sequences of the hGR oligoprobe, similarly coupled by using the Fast-Tag TM FL labeling kit. Dewaxed sections were treated for in situ hybridization (ISH) histochemistry with the antisense and sense oligoprobes. The ISH reaction product was more intense in the cytoplasm of proliferative and maturative chondrocytes of the growth plate cartilage than in that shown in the hypertrophic ones. In the metaphyseal secondary ossification zone, osteoblasts (OBs) and osteocytes (OCs) were variably labeled, whereas osteoclasts (OCLs) were always intensely stained. The labeling was also visible in some bone marrow cells, in articular chondrocytes, in the cells of tendon-bone junctions, and in the perichondrium and periosteal cells. Our results confirm a cellular co-location of GR protein and mRNA. In agreement with GR immunolocalization, the variability of labeling appeared to be related to the cell cycle, the stage of differentiation and cell-type differences.

[Ultrastructural morphological study of supragingival tartar]. / Studio morfologico ultrastrutturale di tartaro sopragengivale.

Specimens of old supragingival calculus, collected from 5 patients with periodontitis, were examined electron-microscopically. Both intracellular and extracellular calcification were found. Intracellular calcification began as needle-shaped crystals or minute amorphous deposits within microorganisms. Extracellular calcification originated within the interbacterial matrix.