Venere Basile

Hypergravity affects morphology and function in microvascular endothelial cells

Cardiovascular diseases are major health problems in astronauts and pilots. The basic problem in cardiovascular diseases is the loss of function by vascular endothelium. It has been demonstrated that changes in inertial conditions (i.e. hypo- and hypergravity) can affect both phenotypic and genotypic expression in endothelial cells. This report describes the effects observed in endothelial cells from coronary post-capillary venules after repeated exposures to hypergravity conditions, alternating with recovery periods. The results showed changes in gene expression, cell energy metabolism, morphology and cytoskeleton organization.

Fibroblast autofluorescence in connective tissue disorders: a future tool for clinical and differential diagnosis?

Marfan syndrome (MFS) is an inherited disorder of connective tissue due to mutations in FBN1 (90%) and TGFBR1 and TGFBR2 (5 to 10%) genes. Clinical and differential diagnosis is difficult because of the inter- and intrafamiliar marked heterogeneity and the variable onset age of clinical manifestations. Among the disorders, in differential diagnosis, thoracic aortic aneurysm (TAA) and Ullrich scleroatonic muscular dystrophy (UCMD) are reported. We evaluate the possibility of utilizing autofluorescence (AF) analysis as a diagnostic tool in the clinical and/or differential diagnosis of MFS and related disorders and in the investigation of the molecular mechanisms involved. Both multispectral imaging autofluorescence microscopy (MIAM) and autofluorescence microspectroscopy (AMS) have been used to characterize AF emission of fibroblasts from patients affected by inherited connective tissue disorders. Our preliminary results show significant differences in AF emission between normal and pathological fibroblasts, suggesting possible improvement in diagnostics of connective tissue disorders by AF analysis.

Dendritic cells with lymphocyte stimulating activity differentiate from human CD133 positive precursors

CD133 is a hallmark of primitive myeloid progenitors. We have addressed whether human cord blood cells selected for CD133 can generate dendritic cells, and Langerhans cells in particular, in conditions that promote that generation from CD34(+) progenitors. Transforming growth factor-β1 (TGF-β1) and anti-TGF-β1 antibody, respectively, were added in some experiments. With TGF-β, monocytoid cells were recognized after 7 days. Immunophenotypically immature dendritic cells were present at day 14. After 4 more days, the cells expressed CD54, CD80, CD83, and CD86 and were potent stimulators in mixed lymphocyte reaction; part of the cells expressed CD1a and langerin, but not Birbeck granules. Without TGF-β, only a small fraction of cells acquired a dendritic shape and expressed the maturation-related antigens, and lymphocytes were poorly stimulated. With anti-TGF-β, the cell growth was greatly hampered, CD54 and langerin were never expressed, and lymphocytes were stimulated weakly. In conclusion, CD133(+) progenitors can give rise in vitro, through definite steps, to mature, immunostimulatory dendritic cells with molecular features of Langerhans cells, although without Birbeck granules. Addition of TGF-β1 helps to stimulate cell growth and promotes the acquisition of mature immunophenotypical and functional features. Neither langerin nor Birbeck granules proved indispensable for lymphocyte stimulation.

Irradiation by pulsed Nd:YAG laser induces the production of extracellular matrix molecules by cells of the connective tissues: a tool for tissue repair

Many studies demonstrated that mechanical stress is a key factor for tissue homeostasis, while unloading induce loss of mass and impairment of function. Because of their physiological function, muscle, connective tissue, bone and cartilage dynamically interact with mechanical and gravitational stress, modifying their properties through the continuous modification of their composition. Indeed, it is known that mechanical stress increases the production of extracellular matrix (ECM) components by cells, but the mechanotransduction mechanisms and the optimal loading conditions required for an optimal tissue homeostasis are still unknown. Considering the importance of cell activation and ECM production in tissue regeneration, a proper use of mechanical stimulation could be a powerful tool in tissue repair and tissue engineering. Studies exploring advanced modalities for supplying mechanical stimuli are needed to increase our knowledge on mechanobiology and to develop effective clinical applications. Here we describe the effect of photomechanical stress, supplied by a pulsed Nd:YAG laser on ECM production by cells of connective tissues. Cell morphology, production of ECM molecules (collagens, fibronectin, mucopolysaccharides), cell adhesion and cell energy metabolism have been studied by using immunofluorescence and autofluorescence microscopy. The results show that photomechanical stress induces cytoskeleton remodelling, redistribution of membrane integrins, increase in production of ECM molecules. These results could be of consequence for developing clinical protocols for the treatment of connective tissue dideases by pulsed Nd:YAG laser.

A new method based on contact surface profilometry for quantitative measurement of resorbed bone volume.

Bone is a dynamic tissue. Its continuous remodeling depends on the balance between bone formation and bone resorption. These two processes are carried out by specialized cells called osteoblast and ostreoclast respectively. The osteoclastic bone resorption consists in degradation of the mineral and collagen components of bone. The study of bone turnover requires accurate assessment of osteoclastic bone resorption, that becomes even more important in pathologic bone loss due to the uncoupling between bone formation and bone resorption. Osteoclastic activity is diffucult to measure. Many techniques, generally based on the detection of the resorbing lacunae (lacunae (pits) due to the bone degradation, allow to estimate bone resorption, but none of them quantitatively and directly measures the volume of resorbed bone. We propose a reliable and relatively simple method, based on contact surfact profilometry, to evaluate directly and quantitatively the volume of resorbed bone. The method has the following advantages:

Does the exposure to microgravity affect dendritic cell maturation from monocytes

The exposure to microgravity conditions results in a significant impairment of the immune function. Many reports describe morphological and functional changes in T-lymphocytes, monocytes and neutrophil granulocytes cultured in microgravity, both real and modeled, but very few studies have been made on the effect of microgravity on dendritic cells (DCs) and DC differentiation. DCs are able to process antigens and are the most efficient cells in presenting them to T-lymphocytes, thus giving a crucial contribution to the rise of an effective immune response. The aim of this study was to investigate whether the maturation of DCs from monocytes of astronauts was altered postflight. Blood samples from a crew-member of the Eneide mission were collected before the flight, soon after the return to earth and one year after the mission. In order to generate DCs, monocytes were used as precursors. They were separated, cultured for 6 days in medium supplemented with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4), then furtherly stimulated for 24 hours with a cocktail of cytokines. Differentiation was assessed by flow cytometry and immunofluorescence, assaying the expression of typical DC markers. Gene expression was analysed by RT-PCR. Morphological and functional characteristics were studied by autofluorescence microscopy. The findings showed that the maturation of DCs from monocytes collected from the astronaut immediately postflight was altered. In comparison with controls, significant differences were found in expression of DC markers, expression of genes involved in DC maturation, morphological and functional characteristics.

Healing process study of laser-welded corneal tissue by multispectral imaging autofluorescence microscopy (MIAM)

Laser welding of corneal tissue is an alternative technique to conventional suturing procedures in ophthalmic surgery. The welding effect is achieved after staining the wound with a chromophore (Indocyanine Green, shortly: ICG) and then irradiating it with a low power diode laser. We present a study on the healing process of corneal wounds using Multispectral Imaging Autofluorescence Microscopy (MIAM). This technique is based on the characterization of fluorescence arising from tissue components (autofluorescence): it is particularly useful in studying corneal tissue, because it is mainly composed of type I collagen, one of the most important endogenous fluorophores. Laser welding tests of the cornea were carried out on rabbits in which full thickness corneal cuts of about 5 mm were sutured using a diode laser emitting at 810 nm, with a power of 80 mW. Bioptic sections of rabbit corneas were examined in a follow up study of 90 days after surgery, and the results were complementary to histological analysis performed in previous studies. Autofluorescence images showed a faster healing process and a better reorganization of the architecture of stromal fibers, in comparison with conventional suturing procedures. MIAM technique can represent a new tool to study the morphology of corneal tissue, offering some real advantages with respect to standard histological analysis. In fact, it does not require any chemical manipulation of the samples, providing information on the biological structure by directly monitoring distribution and emission intensity of endogenous fluorophores.