Serous cutaneous glands substructural organization in anurans as a model of peptide/protein aggregation

A combined Transmission Electron Microscope (TEM) and Fourier Transformation (FT) analysis has been performed on the secretory granules storing active peptides/ proteins in cutaneous glands of n. 12 anuran species. Previous TEM investigation showed that the granules are provided with remarkable repeating substructures based on discrete subunits, arranged into a consistent framework. Furthermore, TEM analysis revealed that this recurrent arrangement is acquired during a prolonged post-Golgian (or maturational) processing that affects the secretory product. Maturation leads to a variety of patterns that depends on the degree of subunit clustering. Since the ultrastructural features of these biological samples were found to be suitable for FT analysis, this variety of recurrent patterns has been plotted into a range of FT frequency spectra. Through this quantitative approach we found that the variable granule substructure can be reduced to a main mechanism of peptides/proteins aggregation

Morphological analysis of the Hippocampal region of aged rats, role of Clasmatodendrosis

Clasmatodendrosis is a phenomenon first described by Alzheimer in 1910, which was observed in aged nervous system and in the course of neurodegenerative diseases. It consists in the loss of astrocytic distal processes. The occurrence of clasmatodendrosis is frequently associated with an increase of autofluorescent aggregates in different cell types of nervous tissue. In this study we designed a calibrated excitation/emission method of spectral unmixing aimed to discriminate the fluorescence emitted by commercial fluorochrome-conjugated antibodies from the autofluorescent signal, by using confocal microscopy and multiphoton fluorescence lifetime imaging techniques. By this method, the immunolabeled GFAP localization in the CA1 Hippocampal region of aged rats was analyzed. Autofluorescent debris showed a strong positivity to GFAP labeling, suggesting that the detached fragments of clasmatodendrotic astrocytes might take part in the generation of these structures. By 3D confocal analysis we found that these aggregates, were located on neuronal cell surfaces, as well as inside the soma and that, in addition, the presence of autofluorescent aggregates seemed to be related with increased adhesion phenomena among neurons. These data were compared with those obtained in control adult rats and in rats infused with lipopolysaccharide (LPS) in the 4th ventricle to induce a chronic inflammatory state. The presence of autofluorescent aggregates was detected in LPS rats and also in control rats, even if they appeared smaller and with a lesser intensity as compared with the aged rats. These findings suggest that clasmatodendrosis is a process involving the interaction of neurons and astrocytes in a prolonged timespan of life. Its severity increases with aging or under inflammatory and/or neurodegenerative diseases. In conclusion, our results seem to suggest that clasmatodendrosis can affect neuron functionality not only due to a decreased astrocyte activity, but also by direct interaction of the detached astrocytic fragments with neuron somata

Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

AbstractRecent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7+ satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration

Bone marrow mesenchymal stromal cells stimulate skeletal myoblast proliferation through the paracrine release of VEGF.

Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies

Confocal reflectance microscopy for determination of microbubble resonator thickness

Optical Micro Bubble Resonators (OMBR) are emerging as new type of sensors characterized by high Q-factor and embedded micro-fluidic. Sensitivity is related to cavity field penetration and, therefore, to the resonator thickness. At the state of the art, methods for OMBR's wall thickness evaluation rely only on a theoretical approach. The purpose of this study is to create a non-destructive method for measuring the shell thickness of a microbubble using reflectance confocal microscopy. The method was validated through measurements on etched capillaries with different thickness and finally it was applied on microbubble resonators

Mesenchymal stromal cells and their paracrine factors regulate MMP-2 and MMP-9/ TIMP-2 balance in skeletal myoblasts and fibroblasts: new insights into the potential role of MSC-cell therapy in muscle regenerative medicine

Recent studies showed that mesenchymal stromal cell (MSCs) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are still poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMPs) expression and activity in different cells of the muscle tissue. It was found that MSCs in co-culture with C2C12 cells up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-2. Similar results were obtained upon incubation of conditioned medium from MSCs (MSC-CM) or in experiments where MSCs were separated from myoblasts by polycarbonate membranes, enabling diffusion of soluble factors while preventing the physical contact between the two cell types, suggesting that MSCs regulated MMP/TIMP balance in skeletal myoblasts by paracrine factors. In the single muscle fibre experiments, MSC-CM administration increased MMP-2 and MMP-9 expression in Pax-7+ satellite cells and stimulated their mobilization, differentiation and fusion into multinucleated myotubes. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts, as detected by reduced expression of α-smooth actin and type-I collagen in the fibroblasts incubated with MSC-CM. These findings add novel information on the effects of MSCs on the skeletal muscle healing, suggesting that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration

GENE EXPRESSION TIME-SERIES ANALYSIS OF CAMPTHOTECIN EFFECTS IN U-87-MG AND DBTRG-05 GLIOBLASTOMA CELL LINES

The clinical efficacy of camptothecin (CPT), a drug specifically targetingtopoisomerase I (TopoI), is under evaluation for the treatment of malignant gliomas. Due to thehigh unresponsiveness of these tumours to chemotherapy, it would be very important to study thesignalling network that drives camptothecin outcome in this type of cancer cells. To address thisissue, we had previously compared the expression profile of human U87-MG glioblastoma cellswith that of a CPT-resistant counterpart, giving evidence that the development of a robustinflammatory response was the main transcriptional effect associated with CPT resistance.Here we report time-related changes and cell line specific patterns of gene expression after CPTtreatment by using two p53 wild-type glioblastoma cell lines, U87-MG and DBTRG-05, withdifferent sensitivities to TopoI inhibition