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Biomedical subjects

A Calligaro

Publications and source records attributed to A Calligaro.

At least 19 recordsLinked to original sources
PubMed2004

Differential distribution of elastic tissue in human natural skin and tissue-engineered skin.

Tissue-engineered skins (TES), manufactured by epidermal and dermal equivalents, are now being used in biological, pharmacotoxicological and clinical applications. It is thus interesting to know to what extent artificial organs are similar to natural counterparts. Elastic fibres are important constituents of the extracellular matrix of natural skin (NS). The aim of our study was to investigate the possible occurrence and distribution of elastic tissue in a model of human TES using different histochemical techniques, including classical Orcein and Fuchsin-Resorcin methods and immunohistochemistry, at both light and electron microscopical levels. Immunoperoxidase and high resolution immunogold methods were used. In NS, classical staining techniques and elastin-immunohistochemistry revealed a well-organized network of elastic fibres. High resolution immunocytochemistry revealed an intense labelling in the amorphous component of elastic fibres. Fibres of different diameters were immunostained. In TES, no stained elastic fibres were observed using classical staining techniques, and the interpretation of immunoperoxidase observations was not clear-cut. In contrast, immunogold staining at the electron microscopical level provided specific labelling of elastin-like immunoreactive material in the dermal equivalent. However, ultrastructural immunocytochemistry revealed that elastic tissue organization in TES was poor compared to that in NS. This study demonstrates that elastic fibres are a component of the extracellular matrix in this model of TES and suggests that fibroblasts of the dermal equivalent are engaged in matrix secretion. Nevertheless, the level of extracellular matrix organization in TES is low compared to NS. Moreover, this study also suggests that different models of bilayered TES may differ with respect to extracellular matrix organization. These aspects should be considered when TES is used in biological and pharmacotoxicological studies. A better understanding of the factors influencing extracellular matrix formation in TES is necessary to achieve further development of skin generation in vitro.

Cells, Cultured↗
PubMed2001

Cell proliferation and differentiation in a model of human skin equivalent.

Recent advances in culturing technology has permitted the production of organotypic models that may be referred to as human skin equivalents (HSE). We have studied histochemical, ultrastructural, and kinetic aspects of an HSE composed by an epidermal equivalent and a dermal equivalent separated by a basement membrane. Only keratinocytes and fibroblasts were present in the epidermal and dermal equivalents, respectively; cells of other lineages were lacking. Keratinocyte stratification and differentiation seemed similar to natural skin. Evidence is shown that such an HSE may also release growth factors such as vascular endothelial growth factor that are believed to play a role in skin grafting. The distribution of cycling cells as well as the values of the growth fraction are comparable to those observed in natural skin. Although the absence of several cells populations that reside in natural skin is a remarkable feature of this HSE, the high levels of tissue organization and cell differentiation lead us to believe that such an HSE may be considered a candidate substitute of human skin in biological, pharmacologic, and clinical applications.

Cell Differentiation↗
PubMed2001

Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes.

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.

Animals↗
PubMed2001

Giulio Bizzozero: a pioneer of cell biology.

The Italian pathologist Giulio Bizzozero began his haematological investigations more than 130 years ago. Among his outstanding achievements was the discovery of the role of platelets in haemostasis and the identification of the bone marrow as the site of production of blood cells. One hundred years after his untimely death, the significance of these, and many more of his findings, is still recognized.

Biology↗
PubMed2001

Cell kinetics in a model of artificial skin. An immunohistochemical and flow cytometric analysis.

Bioengineered organs raised in vitro are candidate substitutes for natural organs in biological, pharmacological and clinical applications. We have studied cell kinetics in a human skin equivalent (HSE) using a combined immunohistochemical and flow cytometric approach. Morphological analysis has shown that, relative to unstimulated natural skin, cell proliferation mainly occurs in the basal layer of the epidermal equivalent. Immunohistochemical and flow cytometric measurements of the growth fraction suggested a cell turnover comparable to that of natural skin. Immunohistochemical labelling indices matched well with flow cytometric data. These observations are consistent with morphological and histochemical data demonstrating normal cell differentiation and tissue architecture in HSE and suggest that such HSE may be a usefull substitute for human skin.

Biocompatible Materials↗
PubMed2000

Correction for age of anticardiolipin antibodies cut-off points.

Immunoglobulin (Ig) isotypes G, M, and A of anticardiolipin antibodies (aCL) are considered markers of antiphospholipid syndrome (APS). They were determined by ELISA in sera of 100 healthy children aged between 6 months and 16 yrs (mean 5.4 yrs +/-3.4 SD) and 100 healthy elderly subjects aged between 65 and 103 yrs (mean 84.2 yrs +/-8.1 SD). The frequency with which they were detected was compared to that in sera of 100 healthy adults aged between 21 and 47 yrs (mean 25.8 yrs +/-5.2 SD) in order to evaluate if adult aCL cut-off levels were fit for pediatric and elderly populations. The cut-off points were calculated adding 2.5 SD to the mean values and, in the adult group, the results were 11.6 GPL, 7.5 MPL, and 23.9 APL for IgG, IgM, and IgA, respectively. In the children, IgG aCL were positive in 26 cases (26%), IgM and IgA aCL in 1 case (1%) respectively. Statistical comparison of these results to those of adults showed a higher significant frequency for children IgG aCL (P = 0.0001) with the major contribution by children aged between 6 months and 5 yrs, and a lower significant frequency for children IgA aCL (P = 0.041). In elderly subjects IgG aCL were positive in 12 cases (12%), IgM aCL in 4 (4%), and IgA aCL in 37 (37%). In a comparison of these values to those of adults, only elderly IgA aCL frequency was significantly higher (P = 0.0001) with the major contribution by the oldest subgroup. In order to avoid false positive results for IgG aCL in children and for IgA aCL in elderly, as well as false negative results for IgA aCL in children, we introduced three new cut-off points: (1) 27.7 GPL for IgG aCL in children; (2) 13.8 APL for IgA aCL in children; and (3) 51.1 APL for IgA aCL in elderly subjects. These data suggest that a correction for age of aCL cut-off levels should be considered to raise specificity and sensitivity for APS in pediatric as well as in elderly populations.

Adolescent↗
PubMed1999

Zidovudine-induced experimental myopathy: dual mechanism of mitochondrial damage.

Myopathy often complicates Zidovudine (AZT) treatment in patients with acquired immunodeficiency syndrome (AIDS). The pathogenesis of the myopathy is controversial, since clinical phenomena intrinsic to AIDS may interfere per se with the onset of the myopathy. In the present work we investigated the in vivo effect of AZT in an animal model species (rat) not susceptible to HIV infection. Histochemical and electron microscopic analyses demonstrated that, under the experimental conditions used, the in vivo treatment with AZT does not cause in skeletal muscle true dystrophic lesions, but rather mitochondrial alterations confined to the fast fibers. In the same animal models, the biochemical analysis confirmed that mitochondria are the target of AZT toxicity in muscles. The effects of AZT on mitochondria energy transducing mechanisms were investigated in isolated mitochondria both in vivo and in vitro. Membrane potential abnormalities, due to a partial impairment of the respiratory chain capability observed in muscle mitochondria from AZT-treated rats, closely resemble those of control mitochondria in the presence of externally added AZT. mtDNA deletion analysis by PCR amplification and Southern blot analysis did not show any relevant deletion, while mtDNA depletion analysis demonstrated a significant decrease in mtDNA in AZT-treated rats. The present findings show that AZT causes damage to mitochondria by two mechanisms: a short-term mechanism that affects directly the respiratory chain, and a long-term mechanism that alters the mitochondrial DNA thus impairing the mitochondrial protein synthesis. In addition, the ultrastructural observations indicate that the fiber types are differently affected upon AZT treatment, which poses a number of questions as to the pathogenesis of this myopathy.

Animals↗
PubMed1999

The Museum for the History of the University of Pavia and the birth of histology.

A brief overview is provided on the development of histology as an independent discipline at the University of Pavia, through documents and preparations preserved in the Museum for the History of the University. Studies on the organization of tissues and cells started blooming in the mid-1850s, when morphological observations were supported by significant technical advancements in microscopy and tissue preparation. The role of Albert Koelliker, the great father of European Histology in the second half of the 19th century, is also discussed. Koelliker had several contacts with Camillo Golgi and promoted the diffusion of Golgi's discoveries. The authors also underline the innovative role of Camillo Golgi in the histological approach to research and in the foundation of modern histology.

Histology↗
PubMed1999

Contributions of Camillo Golgi to renal histology and embryology.

Camillo Golgi was awarded the Nobel prize in 1906 for his studies on the fine anatomy of the central nervous system. His wide present popularity depends on his discovery of the cell apparatus, the tendon mechanoreceptor and the tactile corpuscles of the fingertips that are named after him. However, Golgi also made important contributions to nephrology. In 1882, he published novel observations on the mechanism of renal hypertrophy, which he understood to be due to renal cell proliferation. Two years later, he described tubular cell mitoses in the kidney of a patient with tubulointerstitial nephritis, which he interpreted as an essential part of the repair process. His most important findings, published in 1889, were obtained by studying the microscopic anatomy of the kidney with an original technique that allowed the isolation of whole intact nephrons. He discovered that the ascending limb of Henle's loop always returned to the glomerulus from which the tubule originated, and he realized that the early distal tubule passed between the glomerular arterioles. In addition, applying the same method to dissect nephrons in progressive phases of development, he identified exactly what tubular segments originated from the various parts of the metanephric early S-shaped structure.

History, 19th Century↗
PubMed1999

Development of the pituitary and its abnormalities.

The embryology of the pituitary gland and its normal development is described, including the importance of Pit-1. Congenital abnormalities of the hypothalamo-pituitary region are discussed.

DNA-Binding Proteins↗
PubMed1998

Quantitative changes in pharmacodynamic parameters of noradrenaline in different rat aorta preparations: influence of endogenous EDRF.

1. The aim of the present study was to assess the role of endothelial cells in the modulation of vasocontractile responses to noradrenaline in rat isolated aorta when cut as standard helical strips or as ring segments. 2. Noradrenaline-potency in helical strip preparations evaluated as -logEC50 was greater than that obtained in endothelium-intact ring preparations (9.45 +/- 0.28 versus 8.69 +/- 0.09, respectively) (P < 0.05). The maximum contractile response of helical strips was significantly higher than the response of ring preparations (P < 0.05). 3. Subsequent experiments were performed on helical strips and ring preparations where the endothelium was removed by rubbing the luminal surface of the aorta with filter paper. Removal of the endothelium potentiated the noradrenaline-induced contraction in ring preparations, but not in the helical strips. 4. The nitric oxide synthase inhibitors L-NAME (3 x 10(-5)-3 x 10(-4) M) or L-NNA (1 x 10(4)-3 x 10(-4) M) which were added to the tissue bath potentiated the noradrenaline-induced contraction in the endothelium-intact ring preparations, although only L-NNA induced a statistically significant potentiation. Both L-NAME and L-NNA had no effect on the noradrenaline-contraction induced in rings without endothelium, or in helical strips with or without endothelium. 5. Vascular acetylcholine-induced relaxation is dependent on endothelium derived relaxing factor (nitric oxide). Acetylcholine (10(-9)-10(-6) M) induced a concentration-dependent relaxation in noradrenaline preconstricted intact rings. The relaxant response was strongly reduced by L-NAME (3 x 10(-5)-1 x 10(-4) M). The relaxant response to acetylcholine was very weak in ring and helical strip preparations without endothelium, but also, surprisingly, in unrubbed standard helical strips. 6. The present results suggest that the endothelium of standard helical strip preparations may be greatly damaged, a view confirmed by morphological studies. The structural and functional damage of the endothelium induced very important changes in pharmacodynamic parameters such as in the potency and the maximal responses of vascular preparations to noradrenaline. Therefore, caution must be observed when the potency and intrinsic activity of agonists evaluated on different preparations are compared, even if these come from the same vascular segment.

Animals↗
PubMed1997

Endothelin and nitric oxide synthase in lymphatic endothelial cells: immunolocalization in vivo and in vitro.

BACKGROUND: Endothelin (ET) is an endothelium-derived multifunctional peptide that produces a potent, long-lasting vasoconstriction. Nitric oxide (NO), besides being the most important endothelium-derived relaxant factor in blood vessels, is supposed to be involved in regulating the interactions among endothelium, adhesive molecules, and leukocytes. METHODS: We investigated the possible occurrence and distribution of ET and constitutive nitric oxide synthase (NOs), the enzyme that generates NO from L-arginine, in bovine lymphatic vessels and primary culture of lymphatic endothelium by using immunocytochemistry. RESULTS: Specific immunostaining with both ET and endothelial constitutive NOs antisera was detectable at light and electron microscopic levels in the endothelial cell layer of lymphatic vessels, whereas no immunostaining could be observed in the muscular and adventitial layers. The immunoreaction showed a diffuse pattern throughout the cytoplasm of endothelial cells. Primary cultures of endothelial cells isolated from lymphatic vessels also displayed cytoplasmic ET- and NOs-like immunoreactivities. The endothelial nature of cell monolayers was confirmed by the positive reaction to the von Willebrand factor, a reliable marker of endothelial cells, and by ultrastructural features of cultured cells. CONCLUSIONS: These findings suggest that the endothelium is a major source of ET and NO in lymphatic vessels. Interestingly, the lymphatic endothelium maintains the capability of producing such vasoactive sustances also in vitro, thus suggesting that lymphatic endothelial cells in culture may be used in studies concerning the role of the endothelium in the generation of vasoactive molecules. According to previous functional studies, the occurrence of ET and NOs immunoreactivities in lymphatic vessel endothelium supports the view that lymphatic endothelium may play an important role in the regulation of lymphatic vascular tone and in the production of vascular contractile activity promoting lymph flow.

Animals↗
PubMed1997

Immunohistochemical localization of lipoperoxidation products in normal human placenta.

4-Hydroxynonenal (4-HNE) is a major propagation product of lipid peroxidation that is supposed to be responsible for some of the effects associated with oxidative stress in tissues. We have investigated the possible occurrence and distribution of 4-HNE-immunoreactivity in human normal placenta using immunocytochemistry. Specific immunostaining was observed in cytotrophoblast cells, syncytiotrophoblast, some cells of the villous mesenchyme and some endothelial cells of first trimester and term placentae. The detection of 4-HNE-immunoreactivity in placenta raises the question whether lipoperoxidation products are produced locally in placental cells or represent exogenous products that derive from maternal blood flow. Since trophoblastic cells and villous macrophages are provided by a scavenger receptor, it is conceivable that these cells may play a protective role with regard to the diffusion of lipoperoxidation products from the mother to the embryo. However, since a significant degree of lipid oxidative modification does not take place in plasma, it is presumed that 4-HNE is a local product of placental metabolism. In line with this hypothesis, it is proposed that maternal low density lipoproteins, which are the major source of cholesterol for placental steroid synthesis, might be oxidized by villous cells during their traversal through the villous wall.

Aldehydes↗
PubMed1997

Cell proliferation in developing human dental pulp. A combined flow cytometric and immunohistochemical study.

Information concerning cell proliferation and differentiation in dental pulp may be important to understand tooth response to exogenous stimuli. Since few data concerning human dental pulp are available, we have investigated the growth fraction and the localization of proliferating cells in pulp tissue of third molars of young adult human males and females, using flow cytometry and immunohistochemistry. Flow cytometric analysis demonstrates a low proliferative activity of pulp tissue that appears to be confined to radicular pulp, as revealed by immunohistochemical detection of proliferating cells. No polyploid or aneuploid cell populations could be identified, and G2-blocked cells, if any, represented a negligible cell population. Odontoblasts, cells of the sub-odontoblastic layer, and cells of coronal pulp were found to be not proliferating under normal conditions. These data provide the basis for future investigations on proliferative activity and regenerative potentiality of human pulp cells in experimental and clinical situations.

Adolescent↗
PubMed1997

[Surfactants of the airways. Critical review and personal research].

The literature proving the presence of a surface tension lowering substance (STLS) on the lining layer of mammalian Eustachian tube (ET) is critically reviewed. A further review of the chemical studies on tubal washings based on chromatographic analysis methods (TLC and HPLC) is performed, and is concluded that ET epithelium is coated by a mixture of phospholipids, similar but not identical to the pulmonary surfactant and with similar but less powerful surface activity. In both cases, and with minor differences between the different mammalian species, phosphatidylcholine (PC), and in particular its disaturated fraction, dipalmitoilphosphatidylcholine (DPPC), is the predominating and the most active compound. ET surfactant is synthesized by ET epithelium and secreted in form of osmiophilic multilamellar bodies into the tubal lumen. The exact function of the ET surfactant is not fully understood: it may play an important role in ET physiology by facilitating the tubal opening to allow for aeration of the middle ear and adequate drainage or could act as a release agent, preventing solid-to-solid adhesion of the tubal walls and contrasting the adhesive action of the glycoproteins of the mucous blanket. On the other hand a phospholipidic surfactant seems to be produced by the mucosa of the other parts of the upper airways, i.e. nose and trachea. In this case a surface active agent could act in preventing the transudation of serum into the lumen, in enhancing the phagocytosis or in facilitating the mucociliary transport. Recent data on humans, suggesting that a relative deficiency or an alterated production of tubal surfactant could play a role in the pathogenesis of secretory otitis media (SOM) or middle ear effusion (MEE), are reviewed. Administration of exogenous surfactant or pharmacological stimulation of the production of tubal surfactant could improve ET function and be of value in some cases of SOM. Personal data, suggesting than ambroxol (a drug stimulating the production of pulmonary surfactant by the alveolar type II pneumocytes) exerts a similar activating effect on the tubotympanal secretory cells, are reported. These data support the results of clinical studies on the treatment of SOM with ambroxol.

Ambroxol↗
PubMed1995

A microscopic and immunocytochemical study of structural changes in dysfunctional human temporomandibular joint discs.

The central part of 12 articular discs from patients with serious alterations in function of the temporomandibular joint were investigated. The control discs were removed at autopsy from individuals who did not have any such functional defects. The anomalous discs had an increased cellular component; fibrocytes, fibroblasts, numerous myofibroblasts and, less frequently, smooth muscle cells and mast cells were present. The myofibroblasts, which had numerous bundles of thin filaments in the cytoplasm, were positive to the immunocytochemical reaction with antibody to alpha-smooth muscle actin. In one disc only a large part had changed into a mostly fatty tissue. In the majority of the altered discs, the part examined, which usually is neither vascularized nor innervated, was characterized by the presence of numerous blood vessels. Besides the capillary network several larger vessels were present. In one disc, several myelinated and unmyelinated fibres, isolated or in nerve bundles, were also seen. These observations show that the disc fibrous tissue may undergo deep structural modifications that appear to indicate not only a capacity for repair but also an ability to adapt to new functional conditions.

Actin Cytoskeleton↗
PubMed1995

Ultrastructural immunolocalization of enamel matrix proteins during early stages of ameloblast differentiation.

Enamel matrix proteins (EMP) represent specific molecular markers of ameloblast secretion. In order to study early differentiation stages of the cells of the inner enamel epithelium, we have investigated the ultrastructural localization of EMP-immunoreactivity in rat tooth germ. Pre-secretory stages of ameloblast differentiation were identified by the absence of EMP-immunoreactivity within epithelial cells as well as adjoining extra-cellular matrix. During subsequent secretory stages EMP-like immunoreactive material could be detected both within epithelial cells as well as within the adjoining extra-cellular matrix. The intensity of the immunoreactivity increased while advancing with the differentiation of epithelial cells. Intracellularly, EMP-immunoreactivity was detectable in cytoplasmic compartments involved in exocrine secretion pathway. During the early secretory stage, EMP-immunoreactive material was also detectable in the basement membrane of the epithelial-mesenchymal interface and within the pre-dentine, close to odontoblast plasma membranes and processes. It is thus suggested that EMP may cross the basement membrane between epithelial and mesenchymal cells. Our study suggests that this aspect might be important in molecular mechanisms that regulate epithelial-mesenchymal interactions during odontogenesis.

Ameloblasts↗