Activation of Regulatory T Cells during Inflammatory Response Is Not an Exclusive Property of Stem Cells

BACKGROUND: Sepsis and systemic-inflammatory-response-syndrome (SIRS) remain major causes for fatalities on intensive care units despite up-to-date therapy. It is well accepted that stem cells have immunomodulatory properties during inflammation and sepsis, including the activation of regulatory T cells and the attenuation of distant organ damage. Evidence from recent work suggests that these properties may not be exclusively attributed to stem cells. This study was designed to evaluate the immunomodulatory potency of cellular treatment during acute inflammation in a model of sublethal endotoxemia and to investigate the hypothesis that immunomodulations by cellular treatment during inflammatory response is not stem cell specific. METHODOLOGY/PRINCIPAL FINDINGS: Endotoxemia was induced via intra-peritoneal injection of lipopolysaccharide (LPS) in wild type mice (C3H/HeN). Mice were treated with either vital or homogenized amniotic fluid stem cells (AFS) and sacrificed for specimen collection 24 h after LPS injection. Endpoints were plasma cytokine levels (BD™ Cytometric Bead Arrays), T cell subpopulations (flow-cytometry) and pulmonary neutrophil influx (immunohistochemistry). To define stem cell specific effects, treatment with either vital or homogenized human-embryonic-kidney-cells (HEK) was investigated in a second subset of experiments. Mice treated with homogenized AFS cells showed significantly increased percentages of regulatory T cells and Interleukin-2 as well as decreased amounts of pulmonary neutrophils compared to saline-treated controls. These results could be reproduced in mice treated with vital HEK cells. No further differences were observed between plasma cytokine levels of endotoxemic mice. CONCLUSIONS/SIGNIFICANCE: The results revealed that both AFS and HEK cells modulate cellular immune response and distant organ damage during sublethal endotoxemia. The observed effects support the hypothesis, that immunomodulations are not exclusive attributes of stem cells

Clonal Characterization of Rat Muscle Satellite Cells: Proliferation, Metabolism and Differentiation Define an Intrinsic Heterogeneity

Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (∼75%) and the high proliferative clones (HPC), present instead in minor amount (∼25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (ΔΨm), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described

Pegylation of the antimicrobial peptide nisin A: problem and perspectives

Nisin is a natural antimicrobial peptide produced by Lactococcus lactis and widely employed as food preservative. Its low solubility in neutral aqueous solutions, its instability at physiological pH and its rapid breakdown by proteolytic enzymes has limited its use for processed foods (processed cheese, milk and derivatives, canned vegetables). The conjugation to poly(ethylene glycol) (PEG) could improve its solubility and protect it towards enzymes present in non optimally processed food. We report the synthesis of a PEG-nisin conjugate, and the microbiology assays against some bacterial cell lines

Anchimeric assistance effect on regloselective hydrolysis of branched PEGs: a mechanistic investigation

Branched poly(ethylene glycols) (PEG2) are nowadays widely used for protein and peptides bioconjugation, for their favourable properties (such as the ability to protect the protein surface in an 'umbrella like' fashion). The discovery that mPEG(2)-LysMetbeta AlaOEt lost one mPEG chain during standard base-catalysed ester hydrolysis conditions prompted us to investigate the hydrolytic stability of such systems and the mechanism involved in the PEG chain loss. A series of branched PEGs, substituted with different aminoacids and dipeptides, have been prepared to test the influence of steric hindrance, chain lengths, ramification and Lys-AA amide substitution on hydrolysis. Unexpected results reveal an anchimeric assistance of the Lys-AaA amide proton to the hydrolysis of the carbamoyl moiety joining mPEG to the alpha-amino group of lysine through the formation of an hydantoin system

An improved procedure for the synthesis of branched polyethylene glycols (PEGs) with the reporter dipeptide Met-betaAla for protein conjugation.

A new and more efficient route to the synthesis of branched PEG for protein conjugation, bearing a reporter dipeptide Met-betaAla, is described, which allows better purification of the final product by ion exchange chromatography. The product has the combined advantages of an 'umbrella-like' branched structure, which allows a better coverage of the protein surface, and the presence of the dipeptide Met-betaAla which has been used to detect the position of PEGylation within the peptide sequence

Gene Transfer In Skeletal Muscle By Systemic Injection Of DODAC Lipopoliplexes

Lipid-based vectors are a promising tool for gene therapy applications. Several studies have reported their use in vivo to transfect different organs. Few data, however, are available about lipid-mediated gene transfer in skeletal muscle. Here we report the initial results obtained after systemic administration of lipopolyplexes based on the DODAC cationic lipid in an animal model of muscle regeneration. In particular, we compared three routes of administration: intravenous (i.v.), intracardiac (IC) and intra-arterial (IA). Analysis of reporter gene expression (luciferase) showed that regenerating muscle is more efficiently transfected in all cases and that IA injection is by far the best approach

Design of a stirred multiwell bioreactor for expansion of CD34(+) umbilical cord blood cells in hypoxic conditions

Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self-renewal. In fact, long-term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O(2) concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O(2) solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O(2) concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34(+) cells from umbilical cord blood. The stirring system was designed on top of a standard six-well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O(2) for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c-kit expression of CD34(+) cells at 5% O(2) , using 21%-O(2) cultures as a control. In hypoxia we found that CD34(+) cells maintained a higher expression of c-kit. Further investigation is needed to explore the dynamics of interaction between oxygen- and c-kit-dependent pathways at the molecular level

Microliter-bioreactor array with buoyancy-driven stirring for human hematopoietic stem cell culture

This work presents the development of an array of bioreactors where finely controlled stirring is provided at the microliter scale (100–300 μl). The microliter-bioreactor array is useful for performing protocol optimization in up to 96 parallel experiments of hematopoietic stem cell (HSC) cultures. Exploring a wide range of experimental conditions at the microliter scale minimizes cost and labor. Once the cell culture protocol is optimized, it can be applied to large-scale bioreactors for stem cell production at the clinical level. The controlled stirring inside the wells of a standard 96-well plate is provided by buoyancy-driven thermoconvection. The temperature and velocity fields within the culture volume are determined with numerical simulations. The numerical results are verified with experimental velocity measurements using microparticle image velocimetry (μPIV) and are used to define feasible experimental conditions for stem cell cultures. To test the bioreactor array’s functionality, human umbilical cord blood-derived CD34+ cells were cultured for 7 days at five different stirring conditions (0.24–0.58 μm∕s) in six repeated experiments. Cells were characterized in terms of proliferation, and flow cytometry measurements of viability and CD34 expression. The microliter-bioreactor array demonstrates its ability to support HSC cultures under stirred conditions without adversely affecting the cell behavior. Because of the highly controlled operative conditions, it can be used to explore culture conditions where the mass transport of endogenous and exogenous growth factors is selectively enhanced, and cell suspension provided. While the bioreactor array was developed for culturing HSCs, its application can be extended to other cell types

Expression pattern of FCRL (FREB, FcRX) in normal and neoplastic human B cells.

FCRL (also known as FREB and FcRX) is a recently described member of the family of Fc receptors for immunoglobulin G (IgG). In the present study we analysed its expression in normal and neoplastic lymphoid tissue using immunohistochemical techniques. FCRL was preferentially expressed in a proportion of germinal centre cells and, more weakly, in mantle zone B cells. In addition, strong labelling was observed in marginal zone B cells in the spleen, representing one of the few markers for this cell type. The majority of cases of small B-cell lymphoma, diffuse large B-cell lymphoma and lymphocyte predominance Hodgkin's disease were positive for FCRL. However, the number of positive cells varied widely, and in consequence we could not define a cut-off that distinguished subsets of diffuse large B-cell lymphoma. Our results also showed that FCRL tended to be negative in T-cell-rich B-cell lymphoma and in classical Hodgkin's disease. FCRL may therefore represent a novel marker for normal B cells (e.g. splenic marginal zone cells) and may also be useful as a potential marker of B-cell neoplasms