Effects of plant lectin and extracts on adhesion molecules of endothelial progenitors

Promise of cell therapy has advanced the use of adult stem cells towards the development of novel approaches to promote regeneration of injured endothelium. The aim of this study was to stimulate endothelial progenitor cells (EPCs) with lectin isolated from Solanum tuberosum (potato) shoot and Calendula officinalis (marigold) extracts, in order to increase EPCs proliferation and gene expression of molecules with roles in chemotaxis and adhesion for a better attachment to injured vascular tissue. EPCs were differentiated from umbilical cord blood-derived mononuclear cells and characterized by light microscopy, flow cytometry, and vascular tube-like structures formation on Matrigel. Cell proliferation was determined by MTS assay, and gene expression of molecules involved in EPCs adhesion (VCAM-1, VE-cadherin, ICAM-1, PECAM-1, P-selectin) and chemotaxis was determined (CXCR4, Tie-2) by RT-PCR. For the assessment of cell motility, wound-healing assay was employed. Both potato shoot lectin and marigold extracts stimulated EPCs proliferation in a concentration dependent manner and were able to increase expression of adhesion and chemotactic molecules. Marigold flower extract proved to be more efficient. This study demonstrates the usefulness of potato lectin and marigold extracts to increase EPCs proliferation and modulate gene expression of chemotactic and adhesion molecules, which may facilitate EPCs attachment to injured endothelium.     

Epilepsy in kcnj10 Morphant Zebrafish Assessed with a Novel Method for Long-Term EEG Recordings

We aimed to develop and validate a reliable method for stable long-term recordings of EEG activity in zebrafish, which is less prone to artifacts than current invasive techniques. EEG activity was recorded with a blunt electrolyte-filled glass pipette placed on the zebrafish head mimicking surface EEG technology in man. In addition, paralysis of agarose-embedded fish using D-tubocurarine excluded movement artifacts associated with epileptic activity. This non-invasive recording technique allowed recordings for up to one hour and produced less artifacts than impaling the zebrafish optic tectum with a patch pipette. Paralyzed fish survived, and normal heartbeat could be monitored for over 1h. Our technique allowed the demonstration of specific epileptic activity in kcnj10a morphant fish (a model for EAST syndrome) closely resembling epileptic activity induced by pentylenetetrazol. This new method documented that seizures in the zebrafish EAST model were ameliorated by pentobarbitone, but not diazepam, validating its usefulness. In conclusion, non-invasive recordings in paralyzed EAST syndrome zebrafish proved stable, reliable and robust, showing qualitatively similar frequency spectra to those obtained from pentylenetetrazol-treated fish. This technique may prove particularly useful in zebrafish epilepsy models that show infrequent or conditional seizure activity.     

Direct contact of umbilical cord blood endothelial progenitors with living cardiac tissue is a requirement for vascular tube-like structures formation

The umbilical cord blood derived endothelial progenitor cells (EPCs) contribute to vascular regeneration in experimental models of ischaemia. However, their ability to participate in cardiovascular tissue restoration has not been elucidated yet. We employed a novel coculture system to investigate whether human EPCs have the capacity to integrate into living and ischaemic cardiac tissue, and participate to neovascularization. EPCs were cocultured with either living or ischaemic murine embryonic ventricular slices, in the presence or absence of a pro-angiogenic growth factor cocktail consisting of VEGF, IGF-1, EGF and bFGF. Tracking of EPCs within the cocultures was performed by cell transfection with green fluorescent protein or by immunostaining performed with anti-human vWF, CD31, nuclei and mitochondria antibodies. EPCs generated vascular tube-like structures in direct contact with the living ventricular slices. Furthermore, the pro-angiogenic growth factor cocktail reduced significantly tubes formation. Coculture of EPCs with the living ventricular slices in a transwell system did not lead to vascular tube-like structures formation, demonstrating that the direct contact is necessary and that the soluble factors secreted by the living slices were not sufficient for their induction. No vascular tubes were formed when EPCs were cocultured with ischaemic ventricular slices, even in the presence of the pro-angiogenic cocktail. In conclusion, EPCs form vascular tube-like structures in contact with living cardiac tissue and the direct cell-to-cell interaction is a prerequisite for their induction. Understanding the cardiac niche and micro-environmental interactions that regulate EPCs integration and neovascularization are essential for applying these cells to cardiovascular regeneration.     

Influence of salts and pH on growth and activity of a novel facultatively alkaliphilic,extremely salt-tolerant,obligately chemolithoautotrophic sufur-oxidizing Gammaproteobacterium <Emphasis Type="Italic">Thioalkalibacter halophilus</Emphasis> gen. nov., sp. nov. from South-Western Siberian soda lakes

A chemolithoautotrophic sulfur-oxidizing bacterium (SOB) strain ALCO 1 capable of growing at both near-neutral and extremely alkaline pH was isolated from hypersaline soda lakes in S-W Siberia (Altai, Russia). Strain ALCO 1 represents a novel separate branch within the halothiobacilli in the Gammaproteobacteria, which, so far, contained only neutro-halophilic SOB. On the basis of its unique phenotypic properties and distant phylogeny, strain ALCO 1 is proposed as a new genus and species Thioalkalibacter halophilus gen. nov. sp. nov. ALCO 1 was able to grow within a broad range of salinity (0.5–3.5 M of total sodium) with an optimum at around 1 M Na⁺, and pH (7.2–10.2, pH_opt at around 8.5). Na⁺ was required for sulfur-dependent respiration in ALCO 1. The neutral (NaCl)-grown chemostat culture had a much lower maximum growth rate (μ_max), respiratory activity and total cytochrome c content than its alkaline-grown counterpart. The specific concentration of osmolytes (ectoine and glycine-betaine) produced at neutral pH and 3 M NaCl was roughly two times higher than at pH 10 in soda. Altogether, strain ALCO 1 represents an interesting chemolithoautotrophic model organism for comparative investigations of bacterial adaptations to high salinity and pH. Nucleotide sequence accession number: The GenBank/EMBL accession number of the 16S rRNA gene sequence of strain ALCO1^T is EU124668.     

Effects of Lipid Interactions on Model Vesicle Engulfment by Alveolar Macrophages

The engulfment function of macrophages relies on complex molecular interactions involving both lipids and proteins. In particular, the clearance of apoptotic bodies (efferocytosis) is enabled by externalization on the cell target of phosphatidylserine lipids, which activate receptors on macrophages, suggesting that (local) specific lipid-protein interactions are required at least for the initiation of efferocytosis. However, in addition to apoptotic cells, macrophages can engulf foreign bodies that vary substantially in size from a few nanometers to microns, suggesting that nonspecific interactions over a wide range of length scales could be relevant. Here, we use model lipid membranes (made of phosphatidylcholine, phosphatidylserine, and ceramide) and rat alveolar macrophages to show how lipid bilayer properties probed by small-angle x-ray scattering and solid-state ²H NMR correlate with engulfment rates measured by flow cytometry. We find that engulfment of protein-free model lipid vesicles is promoted by the presence of phosphatidylserine lipids but inhibited by ceramide, in accord with a previous study of apoptotic cells. We conclude that the roles of phosphatidylserine and ceramide in phagocytosis is based, at least in part, on lipid-mediated modification of membrane physical properties, including interactions at large length scales as well as local lipid ordering and possible domain formation.     

A polymorphism in CALHM1 influences Ca2+ homeostasis, Abeta levels, and Alzheimer's disease risk

Alzheimer's disease (AD) is a genetically heterogeneous disorder characterized by early hippocampal atrophy and cerebral amyloid-beta (Abeta) peptide deposition. Using TissueInfo to screen for genes preferentially expressed in the hippocampus and located in AD linkage regions, we identified a gene on 10q24.33 that we call CALHM1. We show that CALHM1 encodes a multipass transmembrane glycoprotein that controls cytosolic Ca(2+) concentrations and Abeta levels. CALHM1 homomultimerizes, shares strong sequence similarities with the selectivity filter of the NMDA receptor, and generates a large Ca(2+) conductance across the plasma membrane. Importantly, we determined that the CALHM1 P86L polymorphism (rs2986017) is significantly associated with AD in independent case-control studies of 3404 participants (allele-specific OR = 1.44, p = 2 x 10(-10)). We further found that the P86L polymorphism increases Abeta levels by interfering with CALHM1-mediated Ca(2+) permeability. We propose that CALHM1 encodes an essential component of a previously uncharacterized cerebral Ca(2+) channel that controls Abeta levels and susceptibility to late-onset AD.     

Light chain myeloma plasma cells induce a strong cell-mediated immune response mainly directed against the monoclonal light chain determinants in a murine experimental model

An important goal for the treatment of human B cell malignancies lies in the induction of an active immune response directed against the tumoral clone, and more particularly against antigenic determinants of the monoclonal immunoglobulin (Ig). The presence of idiotype-reactive T-cells in patients with multiple myeloma has been previously reported, and strategies to increase their responsiveness towards the malignant plasma cells are being actively explored. Light chain (LC) myeloma is often an aggressive form of the disease, regarding which antitumoral immunity has not yet been studied. Here, we investigated in an experimental murine model a secreted monoclonal LC that may behave as a strong tumor antigen after immunization of animals with mitomycin C-treated malignant plasma cells producing monoclonal Ig. Non-dividing plasma cells were utilized as a cell suspension to immunize the mice intraperitoneally (i.p.). The immunized mice produced anti-Ig LC antibodies, mounted a cell-mediated response mainly directed against the monoclonal LC determinants, and survived tumor challenge with significant frequency. These results suggest that plasma cells are capable of presenting antigenic determinants derived from a secreted monoclonal LC in an MHC class I context, and of predominantly inducing a monoclonal Ig-specific T-cell response which can contribute to tumor rejection.     

Growth kinetics of haloalkaliphilic,sulfur-oxidizing bacterium<Emphasis Type="Italic"> Thioalkalivibrio versutus</Emphasis> strain ALJ 15 in continuous culture

The chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio versutus strain ALJ 15, isolated from a soda lake in Kenya, was grown in a continuous culture, with thiosulfate or polysulfide as growth-limiting energy source and oxygen as electron acceptor, at pH 10 and at pH 0.6, 2 M and 4 M total sodium. The end product of the sulfur-compound oxidation was sulfate. Elemental sulfur and a cell-bound, polysulfide-like compound appeared as intermediates during substrate oxidation. In the thiosulfate-limited culture, the biomass yields and maximum specific growth rates decreased two and three times, respectively, with increasing sodium concentration. The apparent affinity constant measured for thiosulfate and polysulfide was in the micromolar range (K_s=6±3 M). The maintenance requirement (m_s=8±5 mmol S₂O₃²/g dry weight h^–1) was in the range of values found for other autotrophic sulfur-oxidizing bacteria. The organism had a comparable maximum specific rate of oxygen uptake with thiosulfate, polysulfide, and sulfide, while elemental sulfur was oxidized at a lower rate. Glycine betaine was the main organic compatible solute. The respiration rates with different species of polysulfides (S_n^2–) were tested. All polysulfide species were completely oxidized at high rates to sulfate. Overall data demonstrated efficient growth and sulfur compounds oxidation of haloalkaliphilic chemolithoautotrophic bacteria from soda lakes.Communicated by W.D. Grant