Ex vivo-expanded bone marrow CD34(+) for acute myocardial infarction treatment: in vitro and in vivo studies

Background aimsBone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34⁺-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI.MethodsThe need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34⁺ cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34⁺ cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells.ResultsGene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14⁺ subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively.ConclusionsOur data suggest that combining basal and expanded BM-derived CD34⁺ cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.     

Utility of a simplified ultrasound assessment to assess interstitial pulmonary fibrosis in connective tissue disorders - preliminary results

Introduction  

Interstitial pulmonary fibrosis (IPF) is a frequent manifestation in patients with connective tissue disorders (CTD). Recently the ultrasound (US) criterion validity for its assessment has been proposed; however, the US scoring systems adopted include the study of several lung intercostal spaces (LIS), which could be time-consuming in daily clinical practice. The aim of this study was to investigate the utility of a simplified US B-lines scoring system compared with both the US comprehensive assessment and the high-resolution computed tomography (HRCT) findings of IPF in CTD patients.     

Ligand- and proton-linked conformational changes of the ferrous 2/2 hemoglobin of Pseudoalteromonas haloplanktis TAC125

The spectroscopic and ligand-binding properties of a 2/2 globin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 have been studied in the ferrous state. It displays two major conformations characterized by CO-association rates that differ by a factor of 20, with relative fractions that depend on pH. A dynamic equilibrium is found between the two conformations, as indicated by an enhanced slower phase when lower CO levels were used to allow a longer time to facilitate the transition. The deoxy form, in the absence of external ligands, is a mixture of a predominant six-coordinate low spin form and a five-coordinate high-spin state; the proportion of low spin increasing at alkaline pH. In addition, at temperatures above the physiological temperature of 1 °C, an enhanced tendency of the protein to oxidize is observed.     

Structural heterogeneity and ligand gating in ferric Methanosarcina acetivorans protoglobin mutants

Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, displays peculiar structural and functional properties within members of the hemoglobin superfamily. In fact, MaPgb-specific loops and a N-terminal extension (20 amino acid residues) completely bury the heme within the protein matrix. Therefore, the access of diatomic gaseous molecules to the heme is granted by two apolar tunnels reaching the heme distal site from locations at the B/G and B/E helix interfaces. The presence of two tunnels within the protein matrix could be partly responsible for the slightly biphasic ligand binding behavior. Unusually, MaPgb oxygenation is favored with respect to carbonylation. Here, the crucial role of Tyr(B10)61 and Ile(G11)149 residues, located in the heme distal site and lining the protein matrix tunnels 1 and 2, respectively, on ligand binding to the heme-Fe-atom and on distal site structural organization is reported. In particular, tunnel 1 accessibility is modulated by a complex reorganization of the Trp(B9)60 and Phe(E11)93 side-chains, triggered by mutations of the Tyr(B10)61 and Ile(G11)149 residues, and affected by the presence and type of the distal heme-bound ligand.     

Osteoid-mimicking dense collagen/chitosan hybrid gels

Bone extracellular matrix (ECM) is a 3D network, composed of collagen type I and a number of other macromolecules, including glycosaminoglycans (GAGs), which stimulate signaling pathways that regulate osteoblast growth and differentiation. To model the ECM of bone for tissue regenerative approaches, dense collagen/chitosan (Coll/CTS) hybrid hydrogels were developed using different proportions of CTS to mimic GAG components of the ECM. MC3T3-E1 mouse calvaria preosteoblasts were seeded within plastically compressed Coll/CTS hydrogels with solid content approaching that of native bone osteoid. Dense, cellular Coll/CTS hybrids were maintained for up to 8 weeks under either basal or osteogenic conditions. Higher CTS content significantly increased gel resistance to collagenase degradation. The incorporation of CTS to collagen gels decreased the apparent tensile modulus from 1.82 to 0.33 MPa. In contrast, the compressive modulus of Coll/CTS hybrids increased in direct proportion to CTS content exhibiting an increase from 23.50 to 55.25 kPa. CTS incorporation also led to an increase in scaffold resistance to cell-induced contraction. MC3T3-E1 viability, proliferation, and matrix remodeling capability (via matrix metalloproteinase expression) were maintained. Alkaline phosphatase activity was increased up to two-fold, and quantification of phosphate mineral deposition was significantly increased with CTS incorporation. Thus, dense Coll/CTS scaffolds provide osteoid-like models for the study of osteoblast differentiation and bone tissue engineering.     

MbCO Embedded in Trehalosyldextrin Matrices: Thermal Effects and Protein?CMatrix Coupling

Saccharide-based biopreservation is widely studied because of its scientific importance and possible technological outcomes for food and pharmaceutical industries. Ternary protein/saccharide/water systems have been extensively exploited to model the characteristics of the in vivo biopreservation process. A tight, water dependent, protein–matrix coupling has been shown to occur in various simple saccharide amorphous matrices, which is stronger in trehalose. The efficiency as bioprotectant of trehalose has been ascribed to this tight coupling, since the appearance of damages on biological structures will more involve structural variations of the surrounding matrix. Here we present, as an applicative follow-up of this research line, a Fourier transform infrared study on protein–matrix coupling in commercial maltodextrins and trehalosyldextrins solid amorphous systems, with carboxymyoglobin embedded, and compare the results with analogous system containing trehalose and maltose, previously reported. Results point out that trehalosyldextrins are useful candidates as protecting agents, even though with an efficiency lower than trehalose, and could be used when the rheological properties of relative long-chain oligosaccharides are needed. However, it appears that a substantial improvement could be obtained by removal of the small fraction of glucose.     

COL4A1 mutations cause ocular dysgenesis, neuronal localization defects, and myopathy in mice and Walker-Warburg syndrome in humans

Muscle-eye-brain disease (MEB) and Walker Warburg Syndrome (WWS) belong to a spectrum of autosomal recessive diseases characterized by ocular dysgenesis, neuronal migration defects, and congenital muscular dystrophy. Until now, the pathophysiology of MEB/WWS has been attributed to alteration in dystroglycan post-translational modification. Here, we provide evidence that mutations in a gene coding for a major basement membrane protein, collagen IV alpha 1 (COL4A1), are a novel cause of MEB/WWS. Using a combination of histological, molecular, and biochemical approaches, we show that heterozygous Col4a1 mutant mice have ocular dysgenesis, neuronal localization defects, and myopathy characteristic of MEB/WWS. Importantly, we identified putative heterozygous mutations in COL4A1 in two MEB/WWS patients. Both mutations occur within conserved amino acids of the triple-helix-forming domain of the protein, and at least one mutation interferes with secretion of the mutant proteins, resulting instead in intracellular accumulation. Expression and posttranslational modification of dystroglycan is unaltered in Col4a1 mutant mice indicating that COL4A1 mutations represent a distinct pathogenic mechanism underlying MEB/WWS. These findings implicate a novel gene and a novel mechanism in the etiology of MEB/WWS and expand the clinical spectrum of COL4A1-associated disorders.