Monica Monici

Natural fluorescence of white blood cells: spectroscopic and imaging study

Autofluorescence has been proved to be an intrinsic parameter of biological substrates that may aid in both the characterization of the physiological state and the discrimination of pathological from normal conditions of cells, tissues and organs. In this work, the fluorescence properties of human white blood cells have been studied in suspension and on single cells at microscopy. The results indicate that suspensions of agranulocytes and granulocytes differ in the amplitude of the fluorescence signal on excitation at wavelengths in the range 250-370 nm. The differences are particularly enhanced when excitation is performed in the 250-265 nm range. Microspectrofluorometric analysis, performed on single cells, allows several leukocyte families to be characterized. Lymphocytes, monocytes, neutrophils and eosinophils can be distinguished according to the intensity and spectral shape of the autofluorescence emission in the visible range from 440 to 580 nm. Both the nature and extent of the differences change when the excitation wavelength is moved from 366 to 436 nm. Differences in the intrinsic metabolic engagement, rather than in the cell dimensions, seem to be responsible for the differences observed between the leukocyte populations. The results identify interesting perspectives for autofluorescence as a discriminating parameter in the differential counting of human white blood cells.

The Impact of Microgravity and Hypergravity on Endothelial Cells

The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature.

ENZYME-ASSISTED CELL PHOTOSENSITIZATION : A PROPOSAL FOR AN EFFICIENT APPROACH TO TUMOR THERAPY AND DIAGNOSIS. THE ROSE BENGAL FLUOROGENIC SUBSTRATE

Abstract— Rose bengal, a xanthene derivative among the most efficient producer of singlet oxygen, was submitted to a chemical modification consisting in the introduction of an acetate group into the aromatic ring fluorophore structure. The acetate group acts as a quencher, thus inactivating both fluorescence and photosensitization properties of the molecule. In the modified structure, rose bengal acts as a fluorogenic substrate giving rise to the cellular reaction termed fluorochromasia. The acetate group is recognized by a carboxylic esterase activity that splits it. Removal of the quencher group results in restoring the native structure of photosensitizer inside the cells. The intracellular turnover of rose bengal acetate was studied in rat glioma‐derived cultured cells, in terms of the balance of the processes of influx and enzyme hydrolysis of the fl0075 orogenic substrate, and of the efflux of the fluorescent product. A large intracellular accumulation of photosensitizer is obtained when treatments are performed with the fluorogenic substrate, even at the drug concentration at which rose bengal does not enter the cells. The intracellular localization allows rose bengal to exert a more effective photosensitization effect. Provided that the quencher group is selected according to the metabolic properties of the tumor cells, the use of fluorogenic substrates as photosensitizer precursors could improve fluorescence diagnosis and the photodynamic therapy of tumors, exploiting the biological properties that distinguish pathological from normal conditions.

Differentiating effects of the glucagon-like peptide-1 analogue exendin-4 in a human neuronal cell model

Glucagon-like peptide-1 (GLP-1) is an insulinotropic peptide with neurotrophic properties, as assessed in animal cell models. Exendin-4, a GLP-1 analogue, has been recently approved for the treatment of type 2 diabetes mellitus. The aim of this study was to morphologically, structurally, and functionally characterize the differentiating actions of exendin-4 using a human neuronal cell model (i.e., SH-SY5Y cells). We found that exendin-4 increased the number of neurites paralleled by dramatic changes in intracellular actin and tubulin distribution. Electrophysiological analyses showed an increase in cell membrane surface and in stretch-activated-channels sensitivity, an increased conductance of Na+ channels and amplitude of Ca++ currents (T- and L-type), typical of a more mature neuronal phenotype. To our knowledge, this is the first demonstration that exendin-4 promotes neuronal differentiation in human cells. Noteworthy, our data support the claimed favorable role of exendin-4 against diabetic neuropathy as well as against different neurodegenerative diseases.

Multispectral imaging autofluorescence microscopy for the analysis of lymph-node tissues.

Abstract Although histochemical and immunohistochemical methods are the standard procedures in diagnosis of lymphoproliferative disorders, useful improvements in evidencing histopathologic manifestations can be obtained with the introduction of tissue autofluorescence analyses. We used microspectrofluorometry and a Multispectral Imaging Autofluorescence Microscopy (MIAM) technique to analyze lymph-node biopsies from patients with lymphoadenopathy of different origins. Images of tissue autofluorescence were obtained by excitation at 365 nm of lymph-node sections and sequential detection with interference filters (50 nm bandwidth) peaked at 450, 550 and 658 nm. Monochrome images were combined together in a single red–green–blue color image. Most of the fluorescence was observed within the blue spectral band because of large contributions from extracellular collagen and elastin fibers as well as from reduced form of intracellular nicotinamide adenine dinucleotide (phosphate). Autofluorescence imaging shows morphological differences between neoplastic and non-neoplastic tissues. The reactive hyperplasia samples show the typical lymph-node organization with weak fluorescent follicles separated by high fluorescent connective trabeculae. In the neoplastic lymph nodes the loss of follicle organization is observed. Consequently, MIAM permits to discriminate between non-neoplastic and neoplastic tissues on the basis of their autofluorescence pattern. Multispectral imaging of tissue autofluorescence may present some advantages with respect to standard histochemical microscopy since it (1) does not require any chemical manipulation of samples; (2) gives real-time results performing the analysis immediately upon specimen resection; and (3) supplies a representation of the biological structure organization linked to endogenous fluorophores.

Modeled gravitational unloading triggers differentiation and apoptosis in preosteoclastic cells

Gravity acts permanently on organisms as either static or dynamic stimulation. Understanding the influence of gravitational and mechanical stimuli on biological systems is an intriguing scientific problem. More than two decades of life science studies in low g, either real or modeled by clinostats, as well as experimentation with devices simulating different types of controlled mechanical stimuli, have shown that important biological functions are altered at the single cell level. Here, we show that the human leukemic line FLG 29.1, characterized as an osteoclastic precursor model, is directly sensitive to gravitational unloading, modeled by a random positioning machine (RPM). The phenotypic expression of cytoskeletal proteins, osteoclastic markers, and factors regulating apoptosis was investigated using histochemical and immunohistochemical methods, while the expression of the corresponding genes was analyzed using RT‐PCR. A quantitative bone resorption assay was performed. Autofluorescence spectroscopy and imaging were applied to gain information on cell metabolism. The results show that modeled hypogravity may trigger both differentiation and apoptosis in FLG 29.1 cells. Indeed, when comparing RPM versus 1 × g cultures, in the former we found cytoskeletal alterations and a marked increase in apoptosis, but the surviving cells showed an osteoclastic‐like morphology, overexpression of osteoclastic markers and the ability to resorb bone. In particular, the overexpression of both RANK and its ligand RANKL, maintained even after return to 1 × g conditions, is consistent with the firing of a differentiation process via a paracrine/autocrine mechanism. J. Cell. Biochem. 98: 65–80, 2006.

Dependence of leukemic cell autofluorescence patterns on the degree of differentiation

The characterisation of leukemic cell autofluorescence during differentiation, induced by 12-O-tetradecanoylphorbol 13-acetate and all-trans retinoic acid, was performed by autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy. We have found that a dependence exists between the cell autofluorescence pattern and the degree of cell differentiation. When cells differentiate, their autofluorescence emission changes, following the morphological and functional rearrangement of cell structures. A decrease in emission intensity and a different distribution of endogenous fluorophores are observed. Thus, autofluorescence monitoring on living cells is a potentially useful tool for in vitro study of the differentiation processes. Furthermore, different maturation steps can be distinguished on the basis of the cell fluorescence pattern, leading the way to future application of the technique in diagnostics.

Hypergravity Stimulation Enhances PC12 Neuron-Like Cell Differentiation

Altered gravity is a strong physical cue able to elicit different cellular responses, representing a largely uninvestigated opportunity for tissue engineering/regenerative medicine applications. Our recent studies have shown that both proliferation and differentiation of C2C12 skeletal muscle cells can be enhanced by hypergravity treatment; given these results, PC12 neuron-like cells were chosen to test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells, in particular promoting an enhanced differentiated phenotype. PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h before being stimulated with different values of hypergravity (50 g and 150 g). Effects of hypergravity were evaluated at transcriptional level 1 h and 48 h after the stimulation, and at protein level 48 h from hypergravity exposure, to assess its influence on neurite development over increasing differentiation times. PC12 differentiation resulted strongly affected by the hypergravity treatments; in particular, neurite length was significantly enhanced after exposure to high acceleration values. The achieved results suggest that hypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential of hypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes.

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.

Hypergravity effects on myoblast proliferation and differentiation.

This study aimed at the investigation of behavior of myoblasts in conditions of altered gravity. C2C12 cells underwent stimulations by different hypergravity intensities (2 h at 5 g, 10 g, and 20 g) in the Large Diameter Centrifuge of the European Space Agency (ESA), highlighting positive effects on both proliferation and differentiation.