Annalia Asti

Hepatocellular Mitochondrial Alterations in Patients With Chronic Hepatitis C: Ultrastructural and Biochemical Findings

OBJECTIVE:Hepatitis C virus (HCV) infection is associated with increased lipoperoxidation, which may lead to interference with mitochondrial function with possible depletion of mitochondrial DNA (mtDNA). We correlated the ultrastructural findings of liver biopsy specimens with the lipoperoxidation markers and contents of mtDNA in chronic hepatitis C (CHC) patients with a different HCV genotype.METHODS:Liver biopsy samples obtained from 75 CHC patients were processed for histological and electron microscopic examination. Twenty-two subjects without known liver disease served as controls. Hepatic glutathione in its reduced (H-GSH) and oxidized (H-GSSG) forms were determined from biopsy specimens by high-performance liquid chromatography. Plasmatic and lymphocytic GSH and erythrocytic malonyldialdehyde (MDA) were also determined, along with the ratio between mtDNA and nuclear DNA (nDNA).RESULTS:Ultrastructural alterations of the mitochondria were documented in 23 patients with genotype 1b, compared with 15 patients with genotype 2a/2c (p= 0.020) and seven patients with genotype 3a (p < 0.001). A significant depletion of H-GSH and lymphocytic GSH, an increase of H-GSSG and MDA, and a reduction of the mtDNA/nDNA ratio were documented in patients with genotype 1b, compared with patients with genotype 2a/2c and 3a and with controls.CONCLUSIONS:In patients with genotype 1b frequent ultrastructural alterations of the mitochondria may be observed, and the depletion of mtDNA in these patients may represent the expression of a greater impairment of the process of oxidative phosphorylation. An increased production of free radicals in patients with genotype 1b may influence the evolution of the liver disease by enhancement of the cytopathic effect of HCV.

Influence of glutaraldehyde on drug release and mucoadhesive properties of chitosan microspheres

Abstract Among bioadhesive drug delivery systems, chitosan microspheres can be considered useful formulations for mucosal administration of drugs. The feasibility of modulating drug release from chitosan microparticles is due to polymer cross-linking, i.e. by glutaraldehyde. The aim of this work was to develop a new simple ‘in vitro’ technique based on electron microscopy in order to study the effect of polymer crosslinking density on mucoadhesive properties of the chitosan microspheres. This technique consists of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations on morphological changes of chitosan microspheres with various cross-linking densities in contact with mucin solution. The results of SEM and TEM analyses have permitted to confirm the high affinity for mucin of uncross-linked chitosan microspheres and thus their bioadhesive properties. Moreover, bioadhesive characteristics of the microparticulate drug delivery systems were depressed for glutaraldehyde cross-linked chitosan microspheres.

Human adipose-derived stem cells (hASCs) proliferate and differentiate in osteoblast-like cells on trabecular titanium scaffolds

The use of stem cells in regenerative medicine is an appealing area of research that has received a great deal of interest in recent years. The population called human adipose tissue-derived stem cells (hASCs) share many of the characteristic of its counterpart of marrow including extensive proliferative potential and the ability to undergo multilineage differentiation along classical mesenchymal lineages: adipogenesis, chondrogenesis, osteogenesis, and myogenesis. The aim of this study was to evaluate with biochemical and morphological methods the adhesion and differentiation of hASCs grown on trabecular titanium scaffolds. The hASCs isolated from subcutaneous adipose tissue after digestion with collagenase were seeded on monolayer and on trabecular titanium scaffolds and incubated at 37 degrees C in 5% CO(2) with osteogenic medium or control medium.The results showed that hASCs were able to adhere to titanium scaffolds, to proliferate, to acquire an osteoblastic-like phenotype, and to produce a calcified extracellular matrix with protein, such as, decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type I collagen. These data suggest that this kind of scaffold/cells construct is effective to regenerate damaged tissue and to restore the function of bone tissue.

Can a Bacterial Endotoxin be a Key Factor in the Kinetics of Amyloid Fibril Formation

Data found in literature have reported that bacterial endotoxins may be involved in the inflammatory and pathological processes associated with amyloidosis and Alzheimers disease (AD). In fact, it has been observed that the chronic infusion of the bacterial lipopolysaccharide, the outer cell wall component of Gram negative bacteria, into the fourth ventricle of rats reproduces many of the inflammatory and pathological features seen in the brain of AD patients. In this context, a key player in the pathogenesis of AD is the amyloid-β peptide (Aβ) that is capable of aggregating in fibrils that represent the main component of amyloid plaques. These deposits that accumulate among brain cells are indeed one of the hallmarks of AD. This aggregation in fibrils seems to correlate with Aβ toxic effects. However, recent data have shown that amyloid fibril formation not only results in toxic aggregates but also provides biologically functional molecules; such amyloids have been identified on the surface of fungi and bacteria. The aim of this work was to gain insight into the influence of bacterial endotoxins on Aβ fibrillogenesis; factors that influence fibril formation may be important for Aβ toxic potential. Following three days of incubation at 37°C, Aβ was organized in compact fibrils and the in vitro Aβ fibrillogenesis was potentiated by the Escherichia coli endotoxin. This suggests the importance of infectious events in the pathogenesis of AD and proposes a new aspect related to the putative pathological factors that can be implicated in the mechanisms involved in Aβ25-35 fibrillogenesis.

Stem Cells Grown in Osteogenic Medium on PLGA, PLGA/HA, and Titanium Scaffolds for Surgical Applications

Pluripotent adipose tissue-derived stem cells (hASCs) can differentiate into various mesodermal cell types such as osteoblasts, chondroblasts, and myoblasts. We isolated hASCs from subcutaneous adipose tissue during orthopaedic surgery and induced the osteogenic differentiation for 28 days on three different synthetic scaffolds such as polylactide-co-glycolide (PLGA), polylactide-co-glycolide/hydroxyapatite (PLGA/HA), and trabecular titanium scaffolds (Ti6Al4V). Pore size can influence certain criteria such as cell attachment, infiltration, and vascularization. The aim of this study was to investigate the performance of PLGA and PLGA/HA scaffolds with a higher porosity, ranging between 75% and 84%, with respect to Ti scaffolds but with smaller pore size, seeded with hASCs to develop a model that could be used in the treatment of bone defects and fractures. Osteogenesis was assessed by ELISA quantitation of extracellular matrix protein expression, von Kossa staining, X-ray microanalysis, and scanning electron microscopy. The higher amount of protein matrix on the Ti scaffold with respect to PLGA and PLGA/HA leads to the conclusion that not only the type of material but the structure significantly affects cell proliferation.

Unexpected finding of barium sulphate on the surface of a microspinal catheter

During a study with a scanning electron microscope to evaluate the structure of microspinal catheter after its removal from subarachnoid space, we found an unusual case. The observation with the microscope of the tip of a catheter removed at the end of an operation for hip replacement in a old female showed the presence of grounded particles with a crystal shape covering the outer surface. Further analysis of this material with an Energy-Dispersive Spectrometer (EDS) showed that it was barium. The patient performed a large bowel barium enema 8 months earlier for a painful syndrome to the lower abdomen. Authors rule out the contamination from the skin and suggest two possible mechanisms of passage of barium from blood to cerebrospinal fluid (CSF) and so to the surface of the catheter.

Cellule staminali mesenchimali di tessuto adiposo (hASCs) differenziano in cellule osteoblasto-simili su scaffold di titanio trabecolare

The adipose tissue has been taken during an hip prosthesis surgery. The hASCs, as staminal cells, can differentiate in more cellular tipes. After been taken they are coltivated on plates till appear a cellular colture that express specific staminal markers. In this work the hASCs are seed on titanium trabecular scaffolds that allowed cellular adhesion and cellular growing. This complex should be used than to replace the lost bone tissue. To stimulate cells growing and differentiation it been added specific proliferative and osteogenic media that allowed to have an osteogenic population. To prove osteoblatic origin of the cells it been used specific laboratory methods that allowed to demonstrate the presence of specific bone proteins in the ECM, as alcalin fosfatasi, collagen I, osteopontin, osteocalcin and a calcific ECM that is deposited in the extracellular space by the cells.