Control of stem cell fate by engineering their micro and nanoenvironment

Stem cells are capable of long-term self-renewal and differentiation into specialised cell types, making them an ideal candidate for a cell source for regenerative medicine. The control of stem cell fate has become a major area of interest in the field of regenerative medicine and therapeutic intervention. Conventional methods of chemically inducing stem cells into specific lineages is being challenged by the advances in biomaterial technology, with evidence highlighting that material properties are capable of driving stem cell fate. Materials are being designed to mimic the clues stem cells receive in their in vivo stem cell niche including topographical and chemical instructions. Nanotopographical clues that mimic the extracellular matrix(ECM) in vivo have shown to regulate stem cell differentiation. The delivery of ECM components on biomaterials in the form of short peptides sequences has also proved successful in directing stem cell lineage. Growth factors responsible for controlling stem cell fate in vivo have also been delivered via biomaterials to provide clues to determine stem cell differentiation. An alternative approach to guide stem cells fate is to provide genetic clues including delivering DNA plasmids and small interfering RNAs via scaffolds. This review, aims to provide an overview of the topographical, chemical and molecular clues that biomaterials can provide to guide stem cell fate. The promising features and challenges of such approaches will be highlighted, to provide directions for future advancements in this exciting area of stem cell translation for regenerative medicine.     

Chemoattraction of macrophages, T lymphocytes, and mast cells is evolutionarily conserved within the human alpha-defensin family

Human defensins are natural peptide antibiotics. On the basis of the position and bonding of six conserved cysteine residues, they are divided into two families, designated alpha- and beta-defensins. Human alpha-defensins are expressed predominantly in neutrophils (human neutrophil peptides (HNP) 1-4) or intestinal Paneth cells (human defensins (HD) 5 and 6). Although alpha-defensins have been implicated in the pathogenesis of inflammatory bowel disease, their immunomodulatory functions are poorly understood. In the present study, HNP-1, HNP-3, and HD5 were found to be potent chemotaxins for macrophages but not dendritic cells using Galphai proteins and MAPK as signal transducers. Alpha-defensins were also chemoattractive for the human mast cell line HMC-1 but lacked, in contrast to beta-defensins, the ability to induce intracellular calcium fluxes. Furthermore, HNP-1, HNP-3, and HD5 comparably mobilized naive as well as memory T lymphocytes. Using the protein kinase C (PKC) inhibitors GF109 and G?6976, we observed a PKC-independent functional desensitization to occur between human alpha-defensins, which suggests a common receptor for HNP-1, HNP-3, and HD5 on immune cells. This alpha-defensin receptor was subject to heterologous desensitization by the PKC activator PMA and to PKC-dependent cross-desensitization by human beta-defensins. Conversely, alpha-defensins desensitized beta-defensin-mediated migration of immune cells in a PKC-dependent manner, suggesting unique receptors for both defensin families. Taken together, our observations indicate that chemoattraction of macrophages, T lymphocytes, and mast cells represents an immunomodulatory function which is evolutionarily conserved within the human alpha-defensin family and tightly regulated by beta-defensins.     

Level of daily physical activity in individuals with COPD compared with healthy controls

Background

Persons with Chronic Obstructive Pulmonary Disease (COPD), performing some level of regular physical activity, have a lower risk of both COPD-related hospital admissions and mortality. COPD patients of all stages seem to benefit from exercise training programs, thereby improving with respect to both exercise tolerance and symptoms of dyspnea and fatigue. Physical inactivity, which becomes more severe with increasing age, is a point of concern in healthy older adults. COPD might worsen this scenario, but it is unclear to what degree. This literature review aims to present the extent of the impact of COPD on objectively-measured daily physical activity (DPA). The focus is on the extent of the impact that COPD has on duration, intensity, and counts of DPA, as well as whether the severity of the disease has an additional influence on DPA.

Results

A literature review was performed in the databases PubMed [MEDLINE], Picarta, PEDRO, ISI Web of Knowledge and Google scholar. After screening, 11 studies were identified as being relevant for comparison between COPD patients and healthy controls with respect to duration, intensity, and counts of DPA. Four more studies were found to be relevant to address the subject of the influence the severity of the disease may have on DPA. The average percentage of DPA of COPD patients vs. healthy control subjects for duration was 57%, for intensity 75%, and for activity counts 56%. Correlations of DPA and severity of the disease were low and/or not significant.

Conclusions

From the results of this review, it appears that patients with COPD have a significantly reduced duration, intensity, and counts of DPA when compared to healthy control subjects. The intensity of DPA seems to be less affected by COPD than duration and counts. Judging from the results, it seems that severity of COPD is not strongly correlated with level of DPA. Future research should focus in more detail on the relation between COPD and duration, intensity, and counts of DPA, as well as the effect of disease severity on DPA, so that these relations become more understandable.     

Functional specialization of memory Th cells revealed by expression of integrin CD49b

Infection or immunization induces heterogeneous memory T cell subsets, but their origin and protective value against infection are unclear. In this study, we report the functional characterization of two memory Th subsets, defined by expression of integrin CD49b. Stable CD49b expression is induced in up to one-half of all memory Th cells. More importantly, the CD49b- and CD49b+ subsets display distinct helper activities, typified by the production of IL-10 and TNF-alpha, respectively. Although the inflammatory properties of the CD49b+ subset are protective against intracellular bacterial infection, they are associated with immunopathology in acute viral infection. Modulation of the CD49b-defined memory Th subsets may provide infection type-specific interventions, where either enhancement of the inflammatory response or reduction of immunopathology is essential.     

Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate

While long-term fixation and storage of specimens is common and useful for many research projects, it is particularly important for space flight investigations where samples may not be returned to Earth for several months (International Space Station) or years (manned mission to Mars). We examined two critical challenges of space flight experimentation: the effect of long-term fixation on the quality of mouse bone preservation and the preservation of antigens and enzymes for both histochemical and immunohistochemical analyses, and how the animal/sample processing affects the preservation. We show that long-term fixation minimally affects standard histological staining, but that enzyme histochemistry and immunolabeling are greatly compromised. Further, we demonstrate that whole animal preservation is not as suitable as whole leg or stripped leg preservation for long-term fixation and all histological analyses. Overall, we recommend whole leg processing for long-term storage of bone specimens in fixative prior to embedding in plastic for histological examination.     

Cloning, expression and interaction of human T-cell receptors with the bacterial superantigen SSA.

Superantigens (SAgs) are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate a large number of T-cells through interaction with the Vbeta domain of T-cell receptors (TCRs). In this study, recombinant TCR beta chains were constructed with human variable domains Vbeta5.2, Vbeta1 and Vbeta2.1, expressed as inclusion bodies, refolded and purified. The Streptococcus pyogenes SAg SSA-1 was cloned and expressed as a soluble periplasmic protein. SSA-1 was obtained both as a monomer and a dimer that has an intermolecular disulfide bond. We analyzed the biological activity of the recombinant SAgs by proliferation assays. The results suggest that SSA dimerization occludes the TCR interaction site. Naturally occurring SSA dimerization was also observed in supernatants of S. pyogenes isolates. An SSA mutant [SSA(C26S)] was produced to eliminate the Cys responsible for dimerization. Affinity assays using a resonant biosensor showed that both the mutant and monomeric wild type SSA have affinity for human Vbeta5.2 and Vbeta1 with Kd of 9-11 microm with a fast kass and a moderately fast kdiss. In spite of the reported stimulation of Vbeta2.1 bearing T-cells by SSA, we observed no measurable interaction.     

Expression of a functional chimeric Ig-MHC class II protein.

We have generated a chimeric protein molecule composed of the alpha- and beta-chains of the MHC class II I-E molecule fused to antibody V regions derived from anti-human CD4 mAb MT310. Expression vectors were constructed containing the functional, rearranged gene segments coding for the V region domains of the antibody H and L chains in place of the first domains of the complete structural genes of the I-E alpha- and beta-chains, respectively. Cells transfected with both hybrid genes expressed a stable protein product on the cell surface. The chimeric molecule exhibited the idiotype of the antibody MT310 as shown by binding to the anti-idiotypic mAb 20-46. A protein of the anticipated molecular mass was immunoprecipitated with anti-mouse IgG antiserum. Furthermore, human soluble CD4 did bind to the transfected cell line, demonstrating that the chimeric protein possessed the binding capacity of the original mAb. Thus, the hybrid molecule retained: 1) the properties of a MHC class II protein with regard to correct chain assembly and transport to the cell surface; as well as 2) the Ag binding capacity of the antibody genes used. The generation of hybrid MHC class II molecules with highly specific, non-MHC-restricted binding capacities will be useful for studying MHC class II-mediated effector functions such as selection of the T cell repertoire in thymus of transgenic mice.     

Context-dependent memory: colour versus odour

An olfactory stimulus and a visual stimulus were employed in a context- dependent memory study using a prose passage as the to-be-remembered item. Ninety-five university students (aged 17-35 years) learned the passage of prose in the presence of one of the stimuli and were then asked to recall the passage with the original context either reinstated or not reinstated. The results revealed a significant context-dependent memory effect for the olfactory cue but not for the visual cue. They demonstrate support for the effectiveness of odours as context cues and it is suggested that context-dependent memory processes may underlie the formation and retrieval of odour-evoked autobiographical memories.