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The Italian Space Agency, within the frame of the mission “Beyond” has promoted the "YiSS—Youth ISS Science" competition, to involve secondary school students in the process of conception and execution of a space experiment. The XENOGRISS experiment was selected by the Italian Space Agency to fly onboard the ISS. The project foresees an active involvement of students into a multi-disciplinary activity aimed at studying the effect of microgravity on growth and regeneration processes, using an animal model (Xenopus laevis) that allows both processes to be observed simultaneously. The project involves the preparation of a Xenopus laevis tadpole culture within a Xenopus Experiment Unit (XEU). The XEU has been integrated into a powered metallic sealed container called Biokon. The activation on the ISS ensured the feeding of the tadpoles, the recirculation of water and the acquisition of images. The control electronics, including the acquisition system, was designed and realized by the students under teacher supervision in collaboration with engineers from Kayser Italia. The integration process of the experiment onboard the ISS was provided by the UTISS Team (Argotec/Telespazio) that supported safety evaluation, requirements verification, payload manifesting, delivery, operations and recovery. The facility has been launched with Space-X CRS-19 and recovered after 30 days. The information derived from this experiment will help to understand the mechanisms underlying the effect of microgravity upon the processes of growth, repair and regeneration of tissues. A better knowledge of these processes is important in defining protocols for the management of traumatic injuries, wounds and chronic ulcers both in Space and on Earth.

Astronauts on board the International Space Station (ISS) are exposed to the damaging effects of microgravity and cosmic radiation. One of the most critical and sensitive districts of an organism is the eye, particularly the retina, and > 50% of astronauts develop a complex of alterations designated as spaceflight-associated neuro-ocular syndrome. However, the pathogenesis of this condition is not clearly understood. In the current study, we aimed to explore the cellular and molecular effects induced in the human retinal pigment ARPE-19 cell line by their transfer to and 3-day stay on board the ISS in the context of an experiment funded by the Agenzia Spaziale Italiana. Treatment of cells on board the ISS with the well-known bioenergetic, antioxidant, and antiapoptotic coenzyme Q10 was also evaluated. In the ground control experiment, the cells were exposed to the same conditions as on the ISS, with the exception of microgravity and radiation. The transfer of ARPE-19 retinal cells to the ISS and their living on board for 3 days did not affect cell viability or apoptosis but induced cytoskeleton remodeling consisting of vimentin redistribution from the cellular boundaries to the perinuclear area, underlining the collapse of the network of intermediate vimentin filaments under unloading conditions. The morphological changes endured by ARPE-19 cells grown on board the ISS were associated with changes in the transcriptomic profile related to the cellular response to the space environment and were consistent with cell dysfunction adaptations. In addition, the results obtained from ARPE-19 cells treated with coenzyme Q10 indicated its potential to increase cell resistance to damage.

This work inserts in the research field regarding the effects of altered gravity conditions on biological plant processes. Pinus pinea seeds germination was studied in simulated microgravity (2x10−3g) and hypergravity (20g) conditions. The effects of simulated gravity were evaluated monitoring the levels of the key enzymes, involved in the main metabolic pathway during germination process of lipid–rich seeds (oilseeds): isocitrate lyase and malate synthase for glyoxylate cycle, 3-hydroxyacyl-CoA dehydrogenase for beta-oxidation, isocitrate dehydrogenase for Krebs cycle, pyruvate kinase for glycolysis and glucose 6 phosphate dehydrogenase for pentose phosphate shunt. The simulated micro and hypergravity conditions were obtained by a Random Position Machine and a Hyperfuge, respectively. Results show that the levels of some tested enzymes, at different lag times of the germination process, have the same trend of controls (g = 1), but with significant differences from quantitative point of view. They are higher in microgravity conditions and lower in hypergravity ones, suggesting that, from a biochemical point of view, the germination process results accelerated in microgravity conditions and delayed in hypergravity ones. These biochemical results show a good correlation with morphological ones, obtained with the measurement of the length of the seeds sprouting radicle. These results give promising indications regarding the possibility to grow plant with lipid-rich seeds in spatial environment, to obtain food sources for astronauts during long term space missions and to reconstitute new atmosphere.

Gravity influences physical and biological processes, especially during development and homeostasis of several tissues in the human body. Studies under altered gravity have been receiving great attention towards a better understanding of microgravity, hypogravity (1g) induced alterations. In the present work, the influence of simulated hypergravity over human tendon-derived cells (hTDCs) was studied at 5, 10, 15 and 20g for 4 or 16 h, using a large diameter centrifuge (LDC). Main results showed that 16 h of simulated hypergravity limited cell proliferation. Cell area was higher in hTDCs cultured at 5, 10 and 15g for 16 h, in comparison to 1g control. Actin filaments were more pronounced in hTDCs cultured at 5 and 10g for 16 h. Focal adhesion kinase (FAK) was mainly expressed in focal adhesion sites upon hypergravity stimulation, in comparison to perinuclear localization in control cells after 16 h; and FAK number/cell increased with increasing g-levels. A tendency towards an up-regulation of tenogenic markers was observed; SCX, TNC, COL3A1 and DCN were significantly up-regulated in hTDCs cultured at 15g and COL3A1 and DCN were significantly up-regulated in hTDCs cultured at 20g. Overall, simulated hypergravity affected the behavior of hTDCs, with more pronounced effects in the long-term period (16 h) of stimulation., The experiments reported here were performed in the framework of the Spin Your Thesis! 2016 programme, organized by ESA Education Office. Authors are grateful to the staff of ESA Education Office, Nigel Savage and Lukas Pfeiffer, for the insightful discussion and support; and to Alan Dowson and Elsa Silva for the technical support before and during the campaign. The authors would like to thank to Hospital da Prelada (Porto, Portugal) for providing tendon tissue samples. Authors also acknowledge Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia in the framework of FCT‐POPH‐FSE, the PhD grant SFRH/BD/96593/2013 of R.C‐A and the consolidator grant IF/00593/2015 of M.E.G.; to the project RL3‐TECT‐NORTE‐07‐0124‐FEDER‐000020 cofinanced by ON.2 under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF)., info:eu-repo/semantics/publishedVersion

Cells, tissues and organs of astronauts aboard the International Space Station (ISS) are exposed to the damaging effects of microgravity and cosmic radiation. Space Agencies are forced to find effective therapeutic countermeasures to safeguard astronauts’ health. Since retina is one of the most vulnerable target, we undertook a project entitled The Coenzyme Q10 (CoQ10) as countermeasure for retinal damage onboard the International Space Station: the CORM project, funded by the Italian Space Agency (ASI) and launched in the summer 2017. We selected CoQ10 as promising candidate drug, having previously first demonstrated its direct antiapoptotic property due to its ability to inhibit mitochondrial depolarization. Beside apoptosis prevention, the parameters we are measuring to evaluate the therapeutic effectiveness of CoQ10 are attenuation of cytoskeleton modifications, lowering of telomeric DNA damages, and exome and whole transcriptome alterations. Here, we present preliminary on-ground experiments that have been carried out in human retinal pigment epithelial ARPE-19 cells and the experimental design of the CORM experiment aboard the ISS. The results obtained by the CORM project will pave the way to countermeasures for astronauts who will face long-term missions.

Gravity has constantly influenced both physical and biological phenomena throughout Earth's history. The gravitational field has played a major role in shaping evolution when life moved from water to land, even if, for a while, it has been generally deemed to influence natural selection only by limiting the range of acceptable body sizes, according to Galilei's principle. Indeed, to counteract gravity, living organisms would need to develop systems to provide cell membrane rigidity, fluid flow regulation, and appropriate structural support for locomotion. However, gravity may influence in a more deep and subtle fashion the way the cells behave and build themselves.

Extremely low-frequency electromagnetic fields (ELF-EMFs) applied in magnetotherapy have frequency lower than 100 Hz and magnetic field intensity ranging from 0.1 to 20 mT. For many years, the use of magnetotherapy in clinics has been increasing because of its beneficial effects in many processes, e.g., skin diseases, inflammation and bone disorders. However, the understanding of the microscopic mechanisms governing such processes is still lacking and the results of the studies on the effects of ELF-EMFs are controversial because effects derive from different conditions and from intrinsic responsiveness of different cell types.In the present study, we studied the biological effects of 1.5 h exposure of human dermal fibroblasts to EMFs with frequencies of 5 and 50 Hz and intensity between 0.25 and 1.6 mT. Our data showed that the magnetic treatment did not produce changes in cell viability, but gave evidence of a sizeable decrease in proliferation at 24 h after treatment. In addition, immunofluorescence experiments displayed an increase in tubulin expression that could foreshadow changes in cell motility or morphology. The decrease in proliferation with unchanged viability and increase in tubulin expression could be consistent with the triggering of a transdifferentiation process after the exposure to ELF-EMFs.

The number of astronauts involved in long-lasting missions and extra-vehicular activities is going to increase in the future. Consequently, the chance of injury due to traumatic events or unexpected emergency surgery will also increase and medical evacuation times to earth will be prolonged. Hence, the need to address requirements for surgery and trauma care in non terrestrial environments will be a priority. Tissue repair in weightlessness should therefore be regarded as a major issue not enough studied to date. Wound healing is a complex multi-step process, crucial to the survival of the organism. It starts with an inflammatory phase followed by a remodelling phase. During repair, the extracellular matrix (ECM) is sequentially remodelled by the concerted action of different cell types, in order to rebuild a functional tissue. The available literature concerning wound healing with mechanical unloading presents controversial results. However, many studies indicate impairment of the healing processes. Here we present a study on the behaviour of cells involved in the remodelling phase of repair, e.g. fibroblasts and endothelial cells, in response to microgravity (μg). In particular, their adhesion/migration, cytoskeleton organization, production of ECM molecules and receptors have been investigated. Cell response to pulsed Nd:YAG laser irradiation has also been investigated in order to evaluate the possibility to use laser irradiation for counteracting the effect of μg on wound healing. In μg, we observed alterations in production/assembling of ECM molecules. Increased fibronectin (FN) and laminin (LM) could be the cause for impaired ECM rebuilding and altered cell adhesion/migration. Treatment with Nd:YAG laser pulses induced organized fibrillogenesis and favoured endothelial cell spreading and monolayer formation. These findings open the way for a better understanding of tissue repair mechanisms in space and future clinical applications on earth.

In the body, connective tissues have a major function in sustaining mechanical stresses. On the other hand, mechanical forces are important factors for connective tissue homeostasis. Connective tissues dynamically interact with mechanical and gravitational stimuli, changing their mechanical properties through the continuous modification of their composition, and thus improving their function. In connective tissues, mechanical forces are major regulators of extracellular matrix turnover, strongly affecting the production of extracellular matrix proteins. On the contrary, unloading conditions, such as bed rest or space flight, have a negative effect on these tissues, with loss of mass and impairment of mechanical properties. Here we describe the effect of photomechanical stress, supplied by a pulsed Nd:YAG laser, on extracellular matrix production by fibroblasts and chondrocytes, and compare it with the effect produced by hypergravity conditions. Cell morphology and structure, extracellular matrix production, cell adhesion, cell energy metabolism have been studied in treated human fibroblasts and chondrocytes by using immunocytochemistry, fluorescence and autofluorescence microscopy. The results show that photomechanical stress induce cytoskeleton remodelling, redistribution of membrane integrins, increase in production of ECM molecules, changes in cell energy metabolism. The effects are similar to those observed in the same cells exposed to cyclic hypergravitational stress (10×g).

It is well known that endothelial cells (ECs), which play a major role in cardiovascular system functioning, are very sensitive to mechanical stimuli. It has been demonstrated that changes in inertial conditions (i.e. microgravity and hypergravity) can affect both phenotypic and genotypic expression in ECs. In this report we describe the effects of hypergravity on ECs isolated from bovine aorta (BAECs). ECs were repeatedly exposed to discontinuous hypergravity conditions (5 × 10 min at 10×g with 10 min at 1×g between sets), simulated in a hyperfuge. Then, cell morphology and metabolism were analyzed by autofluorescence techniques. The phenotypic expression of cytoskeleton constituents (β-actin, vimentin, tubulin), adhesion and survival signals (integrins), mediators of inflammation and angiogenesis was evaluated by immunocytofluorescence. Quantitative PCR (Q-PCR) with Low Density Arrays (LDAs) was used to evaluate modifications in gene expression. After hypergravity exposure, no significant changes were observed in cell morphology and energy metabolism. Cells remained adherent to the substratum, but integrin distribution was modified. Accordingly, the cytoskeletal network reorganized, documenting cell activation. There was a reduction in expression of genes controlling vasoconstriction and inflammation. Proapoptotic signals were downregulated. On the whole, the results documented that hypergravity exposure maintained EC survival and function by activation of adaptive mechanisms.

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.

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.

Candida spp. usually colonize ulcerative lesions of atrophic mucosa in patients with chemotherapy-induced oral mucositis inducing severe inflammation. The spread of antifungal-resistant strains strongly encouraged the search of complementary or alternative therapeutic strategies to cure inflamed mucosa. In this paper, we studied the effects of a near-infrared (NIR) laser system with dual-wavelength emission (808 nm + 904 nm) on the survival and inflammatory potential of C. albicans, C. glabrata, and C. parapsilosis. Laser treatment was performed with a Multiwave Locked System laser. Survival and apoptosis of fungal strains were evaluated by colony-forming units (CFU) counting and annexin V staining. Cytokine production was evaluated by ImmunoPlex array. Laser treatment significantly affected the survival of Candida spp. by inducing apoptosis and induced a lower production of inflammatory cytokines by dendritic cells compared to untreated fungi. No differences in the survival and inflammatory potential were recorded in treated or untreated Saccharomyces cerevisiae cells, used as the control non-pathogenic microorganism. Laser treatment altered the survival and inflammatory potential of pathogenic Candida spp. These data provide experimental support to the use of NIR laser radiation as a co-adjuvant of antifungal therapy in patients with oral mucositis (OM) complicated by Candida infections.

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.

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 x 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 x g conditions, is consistent with the firing of a differentiation process via a paracrine/autocrine mechanism.

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.

Physical factors may induce significant biological effects, therefore they can be applied in biomedical and biotechnological fields in order to drive and modulate biological processes. It is well known that both humoral and physical factors (in particular, but not limited to the mechanical ones) are necessary for maintaining tissue homeostasis. Both biochemical and physical factors can induce the cells to reprogram their functions to adapt dynamically to the environmental conditions.

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.

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.

Health hazards in astronauts are represented by cardiovascular problems and impaired bone healing. These disturbances are characterized by a common event, the loss of function by vascular endothelium, leading to impaired angiogenesis. We investigated whether the exposure of cultured endothelial cells to hypogravity condition could affect their behaviour in terms of functional activity, biochemical responses, morphology, and gene expression. Simulated hypogravity conditions for 72 h produced a reduction of cell number. Genomic analysis of endothelial cells exposed to hypogravity revealed that proapoptotic signals increased, while antiapoptotic and proliferation/survival genes were down-regulated by modelled low gravity. Activation of apoptosis was accompanied by morphological changes with mitochondrial disassembly and organelles/cytoplasmic NAD(P)H redistribution, as evidenced by autofluorescence analysis. In this condition cells were not able to respond to angiogenic stimuli in terms of migration and proliferation. Our study documents functional, morphological, and transcription alterations in vascular endothelium exposed to simulated low gravity conditions, thus providing insights on the occurrence of vascular tissue dysregulation in crewmen during prolonged space flights. Moreover, the alteration of vascular endothelium can intervene as a concause in other systemic effects, like bone remodelling, observed in weightlessness.

Cells contain molecules, which become fluorescent when excited by UV/Vis radiation of suitable wavelength. This fluorescence emission, arising from endogenous fluorophores, is an intrinsic property of cells and is called auto-fluorescence to be distinguished from fluorescent signals obtained by adding exogenous markers. The majority of cell auto-fluorescence originates from mitochondria and lysosomes. Together with aromatic amino acids and lipo-pigments, the most important endogenous fluorophores are pyridinic (NADPH) and flavin coenzymes. In tissues, the extracellular matrix often contributes to the auto-fluorescence emission more than the cellular component, because collagen and elastin have, among the endogenous fluorophores, a relatively high quantum yield. Changes occurring in the cell and tissue state during physiological and/or pathological processes result in modifications of the amount and distribution of endogenous fluorophores and chemical-physical properties of their microenvironment. Therefore, analytical techniques based on auto-fluorescence monitoring can be utilized in order to obtain information about morphological and physiological state of cells and tissues. Moreover, auto-fluorescence analysis can be performed in real time because it does not require any treatment of fixing or staining of the specimens. In the past few years spectroscopic and imaging techniques have been developed for many different applications both in basic research and diagnostics.

Many cell and tissue components exposed to light of appropriate wavelength exhibit UV-visible emission. This is a spontaneous fluorescence, defined autofluorescence in contrast with that produced by highly fluorescent exogenous probes. This communication deals with the Multispectral Imaging Autofluorescence Microscopy (MIAM) technique, that allows living cell and tissue analysis without use of any reagent or treatment modifying the native conditions of the samples. The technique is suitable for a wide range of applications, practically in all the fields where samples containing fluorescent substances occur. Examples of application in biology and medicine are given, showing the power of the technique in both basic research and diagnostics.

Multispectral imaging autofluorescence microscopy (MIAM) is used here for the analysis of lymphatic tissues. Lymph node biopsies, from patients with lympthoadenopathy of different origin have been examined. Natural fluorescence (NF) images of 3 micrometers sections were obtained using three filters peaked at 450, 550 and 680 nm with 50 nm bandpass. Monochrome images were combined together in a single RGB image. NF images of lymph node tissue sections show intense blue-green fluorescence of the connective stroma. Normal tissue shows follicles with faintly fluorescent lymphocytes, as expected fro the morphologic and functional characteristics of these cells. Other more fluorescent cells (e.g., plasma cells and macrophages) are evidenced. Intense green fluorescence if localized in the inner wall of the vessels. Tissues coming from patients affected by Hodgkin's lymphoma show spread fluorescence due to connective infiltration and no evidence of follicle organization. Brightly fluorescent large cells, presumably Hodgkin cells, are also observed. These results indicate that MIAM can discriminate between normal and pathological tissues on the basis of their natural fluorescence pattern, and, therefore, represent a potentially useful technique for diagnostic applications. Analysis of the fluorescence spectra of both normal and malignant lymphoid tissues resulted much less discriminatory than MIAM.

Cells exposed to light of appropriate wavelength show fluorescence emission in the UV-visible spectral range. This phenomenon, called natural fluorescence (NF), is related to cell compounds, i.e. proteins (1), nicotinic coenzymes (2), flavins (3), etc., engaged in structural and metabolic processes. Thus a correlation does exist between NF emission and morpho-functional state of cells.

Laser therapy is used in physical medicine and rehabilitation to accelerate muscle recovery and in sports medicine to prevent damages produced by metabolic disturbances and inflammatory reactions after heavy exercise. The aim of this research was to get insight into possible benefits deriving from the application of an advanced IR laser system to counteract deficits of muscle energy metabolism and stimulate the recovery of hypotrophic tissue. We studied the effect of IR laser treatment on proliferation, differentiation, cytoskeleton organization and global protein expression in C2C12 myoblasts. We found that laser treatment induced a decrease in the cell proliferation rate without affecting cell viability, while leading to cytoskeletal rearrangement and expression of the early differentiation marker MyoD. The differential proteome analysis revealed the up-regulation and/or modulation of many proteins known to be involved in cell cycle regulation, cytoskeleton organization and differentiation.

Haematic cells, excited with radiation of suitable wavelength, give rise to a natural fluorescence (NF) emission. This paper investigates NF to develop new techniques for applications in both basic research and medical diagnostics. Results show that the cell populations examined exhibit peculiar emission bands. The intracellular fluorescence pattern reveals that flouresence is mainly located at cytoplasmic-level, thus related to the metabolic processes of the cells. The photophysical properties of cells appear different among the normal populations and between normal and leukaemic ones. Therefore the recognition of the various cellular elements, according to their fluorescence emission, is possible.

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 determination of blood cell composition has been a valuable tool in diagnoses. In particular, both total and differential counts are considered the basic parameters that characterize the leukocyte population. Since 100 years ago, manual techniques were introduced that allow a morphological examination of blood smears. At present, the automated analysis has been proved to be particularly difficult to standardize. In fact, the identification and count of the five leukocyte populations are not completely solved problems in routine methods for hematological analysis. Optoelectronics could have a decisive role in the development of new techniques that can ensure characteristics of automation, reliability, accuracy and rapidity of execution. Fluorescence spectroscopy techniques could represent a valid approach. Recently, the evaluation of tissue and cell autofluorescence has been applied to the diagnosis of solid tissue neoplasies. In this work, we have considered the possibility to develop a reliable method of leukocyte analysis based on their intrinsic fluorescence emission properties. The study has been performed by applying both spectrofluorometric techniques to enriched suspensions of cells and microspectrofluorometric techniques to single leukocytes. The results obtained have shown the possibility to recognize some cell populations on the grounds of the intrinsic fluorescence characteristics.