Cell dynamics in the correct control of bone metabolism using natural treatments.

The present study was undertaken in order to assess the efficacy of a commercial product containing calcium and silicon (Osteosil-Calcium) on cell metabolism. MG-63 osteblast-like cells were cultured in the presence of three different drug concentrations (10, 5 and 2.5 microg/mL). Either serum-free culture and standard culture with serum were investigated. Morpho-functional tests (MTT and ALP), scanning electron microscopy (SEM), microanalysis (EDAX) and time-lapse video microscopy were performed. Cell actin cytoskeletal modification with fluorescence phalloidin staining was also tested. Our data show the in vitro functional efficacy of Osteosil-Calcium on MG63 cell viability and ALP production. This study demonstrates its positive effect on the metabolism of the single cell and suggests wider uses of this drug in health protection and or in Regenerative Medicine therapies which are currently applied to the elderly.

Tyrosine phosphorylation in type-1 diabetes by immunogold detection: an in vitro human aortic endothelial cell (HAEC) study in the presence of diabetic low density lipoproteins (LDL).

An immunomorphometric study of tyrosine phosphorylation was performed by the immunogold technique on cultured human aortic endothelial cells (HAEC) with a view to demonstrating their impaired signal transduction status, induced in vitro by incubation with low-density lipoproteins from the plasma of Type-1 diabetic patients. The results seem to sustain the hypothesis that extranuclear bioenergetic derangement induced by low-density lipoproteins from Type-1 diabetic patients may be associated with an up-regulation of the nuclear energetic machinery aimed at maintaining intracellular metabolic equilibrium. Our data demonstrate that phosphorylated tyrosine is a useful marker to monitor this metabolic condition.

Vascular endothelial growth factor expression as a prognostic index in serous ovarian cystoadenocarcinomas: relationship with MIB1 immunostaining.

OBJECTIVE: The aim of our study was to investigate the expression of vascular endothelial growth factor (VEGF) by neoplastic cells in serous ovarian cystoadenocarcinomas; the correlation between this marker of angiogenesis, histopathologic parameters, disease-free survival, and MIB1 immunostaining was also evaluated. MATERIALS AND METHODS: Thirty-two patients with serous ovarian cystoadenocarcinoma, treated at the Institute of Gynecology and Obstetrics, Ancona University (Italy), were used as study population; 10 women with serous cystoadenoma were also analyzed. The expression of VEGF was immunohistochemically evaluated by polyclonal antibody anti-VEGF (Santa Cruz, CA, dilution 1:100) on formalin-fixed paraffin-embedded tissue. RESULTS: Compared to cystoadenomas, the tissutal VEGF immunostaining was significantly higher in cystoadenocarcinomas, with the highest values in architectural grade 3 neoplasms (P < 0. 001). A direct relationship was observed between VEGF immunostaining and MIB1 index (r = 0.44, P = 0.013). A relationship was defined between VEGF expression and disease-free survival, evaluated by Cox hazards analysis (P < 0.001). CONCLUSIONS: Angiogenesis, evaluated by VEGF immunostaining, seems to be an interesting prognostic indicator in serous ovarian cystoadenocarcinoma, involved in neoplastic proliferation.

Expression of vascular endothelial growth factor related to 72-kilodalton metalloproteinase immunostaining in patients with serous ovarian tumors.

BACKGROUND: The aim of the current study was to investigate the expression of vascular endothelial growth factor (VEGF) by neoplastic cells in patients with serous ovarian tumors. The correlation between neoangiogenesis and 72-kilodalton metalloproteinase (MMP2) immunostaining also was evaluated. METHODS: Fifty-eight patients with serous ovarian tumors who were treated at the Institute of Gynecology and Obstetrics of Ancona University (Ancona, Italy) were used as the study population; 10 women had serous cystoadenoma, 16 women had a serous borderline tumor, and 32 women had invasive cystoadenocarcinoma. Expression of VEGF and MMP2 was evaluated immunohistochemically by polyclonal antibody anti-VEGF (dilution, 1:100) and affinity purified, rabbit anti-MMP2, formalin fixed, paraffin embedded tissue. Positive staining was expressed as a percentage of positive cells per 10(3) counted neoplastic cells. RESULTS: Compared with cystoadenomas and borderline tumors, the tessutal VEGF immunostaining was significantly higher in cystoadenocarcinomas, with the highest values detected in architectural International Federation of Gynecology and Obstetrics Grade 3 neoplasms (P2 < 0.001). A direct relation was observed between VEGF and MMP2 immunostaining (correlation coefficient, 0.44; P2 = 0.013). A relation was found between VEGF expression and disease free survival as evaluated by Cox hazards analysis (P2 = 0.03). CONCLUSIONS: Neoangiogenesis detected by VEGF immunostaining appears to be a promising indicator of aggressiveness in serous ovarian tumors. In cystoadenocarcinomas, VEGF expression appears to be related to MMP2 index.

Histomorphometric studies in rat cerebral cortex: normal aging and cell loss.

One characteristic feature of the aged central nervous system (CNS) is neuron loss. Programmed cell death (PCD) has been implicated in neuronal death during development and may be involved in a number of age-related neurodegenerative diseases of the CNS. Cell death in the aging cerebral cortex was investigated in the present morphometric and immunohistochemical study of rat frontal cortex by detection of bcl-2 as the factor preventing PCD. The results were interpreted in the light of the bioenergetic features of aged motoneuron cells. Our results showed that 1) bcl-2 does not influence neuronal survival, and ii) the presence in aging frontal cortex of minor cellular morphometric and bioenergetic modifications, confirming the difference between normal aging and neurodegenerative disease.

Advances in the understanding of malignant transformation of keratinocytes: an immunohistochemical study.

AIM: We set out to investigate the interactions between malignant transformation of keratinocytes, presence of oncoproteins and immunosurveillance in squamous cell carcinoma (SCC) and in a preneoplastic lesion, actinic keratosis (AK). METHODS: Samples of SCC, AK and normal skin (NS) were subjected to quantitative analysis using the following antibodies: anti-p53, Ki67, OKT6, OK-DR, B7/BB1, anti-CD54, anti-CD11, OKT3, OKT4, OKT8; positivity for ras-p21, EGFr and bcl-2 was evaluated by semiquantitative analysis. RESULTS: Oncoprotein alterations and increased keratinocyte proliferative activity were observed both in AK and SCC. The number of Langerhans cells (CD1a+ cells) was similar in the two lesions but lower in SCC compared to AK. The proportion of CD1a(+)-B7/BB1+ cells was slightly higher in AK and SCC than in NS. The Langerhans cells expressed the HLA-DR antigen in all groups. Values were highest in AK and NS, and quite low in SCC. Cytotoxic T lymphocytes were more numerous in SCC than in AK and NS. Interestingly, the total CD4/CD8 ratio was much lower in SCC than in AK and NS, which indicates an increase in the CD8+ subpopulation in samples of SCC. In the epithelia of SCC samples there were a considerable number of B7/BB1+ keratinocytes. CONCLUSIONS: We suggest that alterations in the immunodefence mechanisms have an important role in the transformation of AK into SCC, and that these changes affect not only lymphocytes, but also professional (i.e., Langerhans cells) and non-professional (i.e., keratinocytes) antigen presenting cells.

Prostate-specific antigen expression in a case of intracystic carcinoma of the breast: characterization of immunoreactive protein and literature surveys.

A case is presented of female breast intracystic carcinoma with prostate-specific antigen (PSA) expressed in high amounts in aspirated cystic fluid (55 micrograms/L). Tumor extract analysis revealed the presence of both estrogen and progesterone receptors (0.38 and 1.87 nmol/L, respectively) and high quantities of PSA too (19.52 micrograms/L). Chromatographic analysis of cystic fluid revealed two peaks of PSA, at the expected positions for free and bound serine protease. A major proportion of 33-kDa free from was also confirmed by Western blotting analysis. Free PSA was heat-stable at 56 degrees C and displayed no change after freezing-thawing. These findings are discussed in the context of a detailed literature survey. Our data support the contention that PSA immunoreactivity in intracystic fluid of breast carcinoma is partly the result of secretory activity by the neoplastic cells and that the steroid receptors can also modulate its expression.

Biomaterials for orthopedic surgery in osteoporotic bone: a comparative study in osteopenic rats.

UNLABELLED: To evaluate orthopedic devices in pathological bone, an experimental study was performed by implanting Titanium (Ti) and Hydroxyapatite (HA) rods in normal and osteopenic bone. Twenty-four rats were used: 12 were left intact ( CONTROL: C) while the other 12 were ovariectomized (OVX). After 4 months all the animals were submitted to the implant of Ti or HA in the left femoral condyle (Ti-C, HA-C, Ti-OVX, HA-OVX). Two months later the animals were sacrificed for histomorphometric, ultrastructural and microanalytic studies. Our results show a significant difference between the Affinity Index (A.I.) of HA-C and Ti-C (77.0 +/- 7.4 vs 61.2 +/- 9.7) (p < 0.05). No significant differences were observed between the osteointegration of Ti-C and Ti-OVX (61.2 +/- 9.7 vs 48.2 +/- 6.7). Significant differences also exist between the osteointegration of HA-C and HA-OVX (77.0 +/- 7.4 vs 57.6 +/- 11.5) (p < 0.01). Microanalysis shows some modifications in Sulphur (S) concentration at the bone/biomaterial interface of the Ti-OVX group. Therefore our results confirmed the importance of biomaterials characteristics and of bone quality in osteointegration processes.

Conjugated bile acids in breast cyst fluids: relationship to cation-related cyst subpopulations.

Gross cystic breast disease is a benign lesion occurring in 7% of adult women. Apocrine changes of epithelium lining the breast cysts cause a higher risk of developing breast cancer. According to the possible role of bile acids in the pathogenesis of cancer, we analysed breast cyst fluids aspirated from 96 women for distribution of conjugated bile acid concentrations in the two subsets of breast cysts. Bile acid levels were correlated to K+ concentrations (P < 0.0001) and mean value was higher in Na/K < 3 metabolically active apocrine cyst as compared with Na/K > 3 flattened cyst (P < 0.001). Because bile acids could play an important role in the pathogenesis and growth of breast cancer, the significantly higher intracystic concentrations of these carcinogen compounds in apocrine Type I cysts might provide a further biological explanation as to why women with apocrine changes may be at higher breast cancer risk and could be useful for the biochemical knowledge occurring in the different functional stages of the gross breast cysts.

Chronic obstructive pulmonary disease and associated patterns of memory decline.

The relationship between chronic obstructive pulmonary disease (COPD) and cognitive functioning was analyzed in a study with 50 aging patients. A complex pattern of interactions was identified between emotional and cognitive functioning and chronic respiratory disease when the effects of age, sex, type and severity of disease were controlled. These patients did not show any global and diffuse cognitive impairment. Only a portion of COPD patients (about 30%) evidenced memory impairment which was confined to immediate memory. Memory impairment found in these patients did not appear to be associated with those changes present in the aging process but was mainly related to those specific clinical and instrumental parameters which are considered valid indicators of respiratory efficiency. Two types of cognitive and emotional problems were identified. A progressive stage-dependent set of characteristics was associated with the course of the disease and a fluctuating, probably reversible state-dependent set of characteristics was associated with the temporary condition of the patients during the period of examination. Patients who had received more recent medical treatment or who were under protection of vaccination for influenza showed a better cognitive and emotional efficiency.

Promoter elements involved in environmental and developmental control of potato proteinase inhibitor II expression.

The proteinase inhibitor II (pin2) gene family exhibits two different modes of expression. It is, on the one hand, constitutively expressed in flowers of potato and tomato plants. and in potato tubers. On the other hand, its expression is induced in the plant foliage by mechanical wounding. To define cis-regulatory elements involved in pin2 promoter activity, deletion analysis of a potato pin2 promoter has been performed in stably and transiently transformed potato and tobacco plants. Two different elements, a quantitative enhancer and a regulatory element, are required for promoter activity. While functional promoter elements required for pin2 activity in tubers and wounded leaves could not be separated, its expression in flowers is mediated by different cis-acting sequences. Induction of pin2 expression in leaves by treatment with the plant growth regulators abscisic acid and jasmonic acid, and the general metabolite sucrose, depends on the presence of the regulatory element involved in expression in tubers and wounded leaves. Thus, pin2 expression in tubers and wounded leaves apparently results from the action of similar hormonal signals on closely linked promoter elements, while a different signal pathway leads to its constitutive expression in flowers.

[Reclaiming the feeling of shame]. / Die Rückgewinnung des Schamgefühls.

The author resorts to Bleger's conception of regression to ambiguity in order to achieve an understanding of patients who have suffered from extreme situations such as torture, disappearance, concentration camp. The regression to ambiguity that helped these patients to survive and that elicits extreme feelings of shame must be gradually diminished so that older intact ego components as well as the original feelings of shame can be reclaimed.

S-100b protein regulates aggregation and fusion of cardiolipin vesicles.

We have recently shown that S-100b protein interacts with the polar surface of cardiolipin vesicles [6]. This interaction produces changes in the secondary structure of S-100b as well as changes in the structural organization of cardiolipin vesicles. We report here on the effects of S-100b on cardiolipin vesicles as investigated by turbidity, terbium-dipicolinate fluorescence and freeze-fracture. Experiments were carried out in the absence and in the presence of Ca2+. In the absence of Ca2+ (0.1 mM EDTA), S-100b favors the aggregation and fusion of vesicles to some extent. Under these conditions, electron microscope analyses reveal the presence of fused vesicles along with particles similar to those observed in protein reconstituted systems or to lipid particles observed during fusional processes. In the presence of Ca2+, S-100b counteracts the Ca2(+)-dependent tendency of vesicles to aggregate and fuse. Under these conditions, bilayer phases along with hexagonal phases can be observed by electron microscopy. The latter effects of S-100b are not due to chelation of Ca2+ because of the relative concentrations of S-100b and Ca2+ under our experimental conditions and since much larger concentrations of EDTA are required to produce the S-100b effects. We propose that the dimeric nature of S-100b plays a major role in these events. In the absence of Ca2+, the S-100b molecules probably cross-link adjacent vesicles, one subunit contacting one vesicle and the other subunit contacting another vesicle through electrostatic bonds. In the presence of Ca2+, due to the large changes occurring in the conformation of the protein (which loses about 52% of its alpha-helical content), S-100b associates strongly with the polar surface of individual vesicles, thus generating some kind of physical barrier to aggregation and fusion of vesicles.

Particular ultrastructural aspects of cryptorchid testis in adult and advanced age.

The authors have observed with E.M. Philips 301 some biopsies obtained from testes of cryptorchid patients aged between 38 and 72 to study the ultrastructural aspects of seminiferous tubules in this age. Tissues were fixed in 2% phosphate-buffered solution of glutaraldehyde and then postfixed in 1% osmium tetroxide, processed for routine electron microscopy. The seminiferous tubules appear strongly damaged and we have observed numerous Sertoli cells and only few dark spermatogonia. The Sertoli cells appear either normal and mature or with a cytoplasm filled with big lipid droplets and large amounts of glycogen. The basal membrane of the seminiferous tubules is thickened. Leydig cells are characterized by dilated smooth endoplasmic reticulum and numerous nuclear bodies.

Association between centriole and nuclear matrix in human lymphocytes.

Nuclear matrices, purified from normal and chronic lymphocytic leukemia lymphocytes, exhibit a close association with the centriole. This finding suggests that the nuclear and cytoplasmic skeletal systems are linked by transmembrane connections represented by nuclear matrix constituents. This could account for the observed synchrony between transformations of the centriole and particular nuclear events which take place during the cell cycle and suggests that the nuclear matrix, besides being involved in DNA replication and chromosome condensation, should affect the centriole cycle which controls the cytoskeleton organization.

Electron microscopy applied to fine-needle aspiration. A report of six cases from various sites.

The authors report the results obtained from the application of electron microscopy techniques to the cytology of fine-needle-aspirated samples of neoplastic lesions from various body sites. These results show that the tissue structure, which is usually lost during the squashing necessary for light microscopy cytology, is preserved when the samples are processed for ultrastructural analysis. Electron microscopy also allows a highly detailed study of the cell's inner structures. Thus, when this technique is applied, fine needle-aspirated samples can be regarded as actual microbiopsies. However, because of the high cost of ultrastructural techniques, we suggest that actual analysis be performed only in selected cases, whereas fixation and inclusion for electron microscopy could be done routinely.

The normal human parathyroid gland. A histochemical and ultrastructural study with particular reference to follicular structures.

Bioptic specimens of 10 normal human parathyroid glands taken during thyroidectomies were studied both by light microscopy, using a histochemical technique, and electron microscopy. All the glands exhibited regions of different sizes in which follicles made up the tissue architecture. The follicles contained a homogeneous colloid-like substance. Our results seem to exclude the amyloid nature of the follicular content suggested by some Authors, though no further clarification as to its real make-up is provided. The cells lining the follicles belong to a type not as yet described in the normal human parathyroid. These cells appear to be large and clear and are also found in the non-follicular parenchyma. On the basis of their morphologic features we have classified the large clear cells as a particular, deviated stage of the actively secreting chief-cell. The origin and significance of the follicles are also discussed.