Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm²) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm² format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.     

Discrepancies in Outcome Reporting Exist Between Protocols and Published Oral Health Cochrane Systematic Reviews

Objectives

To assess discrepancies in the analyzed outcomes between protocols and published reviews within Cochrane oral health systematic reviews (COHG) on the Cochrane Database of Systematic Reviews (CDSR).

Study Design and Setting

All COHG systematic reviews on the CDSR and the corresponding protocols were retrieved in November 2014 and information on the reported outcomes was recorded. Data was collected at the systematic review level by two reviewers independently.

Results

One hundred and fifty two reviews were included. In relation to primary outcomes, 11.2% were downgraded to secondary outcomes, 9.9% were omitted altogether in the final publication and new primary outcomes were identified in 18.4% of publications. For secondary outcomes, 2% were upgraded to primary, 12.5% were omitted and 30.9% were newly introduced in the publication. Overall, 45.4% of reviews had at least one discrepancy when compared to the protocol; these were reported in 14.5% reviews. The number of review updates appears to be associated with discrepancies between final review and protocol (OR: 3.18, 95% CI: 1.77, 5.74, p<0.001). The risk of reporting significant results was lower for both downgraded outcomes [RR: 0.52, 95% CI: 0.17, 1.58, p = 0.24] and upgraded or newly introduced outcomes [RR: 0.77, 95% CI: 0.36, 1.64, p = 0.50] compared to outcomes with no discrepancies. The risk of reporting significant results was higher for upgraded or newly introduced outcomes compared to downgraded outcomes (RR = 1.19, 95% CI: 0.65, 2.16, p = 0.57). None of the comparisons reached statistical significance.

Conclusion

While no evidence of selective outcome reporting was found in this study, based on the present analysis of SRs published within COHG systematic reviews, discrepancies between outcomes in pre-published protocols and final reviews continue to be common. Solutions such as the use of standardized outcomes to reduce the prevalence of this issue may need to be explored.     

Population density of striped hyenas in relation to habitat in a semi-arid landscape,western India

We used camera trapping in conjunction with a spatial explicit capture–recapture model to estimate striped hyena (Hyaena hyaena) density and occupancy models to investigate factors affecting striped hyena detection probabilities in Ranthambhore Tiger Reserve (RTR), Rajasthan, India. A sampling effort of 4,450 trap days/nights over 75 days yield 68 photo captures of 21 unique striped hyenas (based on individual markings and visual identification); the estimated striped hyena density was 5.49?±?1.27 individuals/100 km². Results of our occupancy model suggested that a rugged terrain is an important factor that influences striped hyena detection probability. Correlation with striped hyena detection with human settlement provides evidence of social tolerance of striped hyena towards humans, and more occurrence of resources allowed coexistence of hyena in a human-dominated landscape. This elasticity (inhabited areas close to humans) demonstrated by striped hyenas is an exception among carnivore communities living in this semi-arid habitat.     

Epidermal Growth Factor Receptor Activation Remodels the Plasma Membrane Lipid Environment To Induce Nanocluster Formation

Signal transduction is regulated by the lateral segregation of proteins into nanodomains on the plasma membrane. However, the molecular mechanisms that regulate the lateral segregation of cell surface receptors, such as receptor tyrosine kinases, upon ligand binding are unresolved. Here we used high-resolution spatial mapping to investigate the plasma membrane nanoscale organization of the epidermal growth factor (EGF) receptor (EGFR). Our data demonstrate that in serum-starved cells, the EGFR exists in preformed, cholesterol-dependent, actin-independent nanoclusters. Following stimulation with EGF, the number and size of EGFR nanoclusters increase in a time-dependent manner. Our data show that the formation of EGFR nanoclusters requires receptor tyrosine kinase activity. Critically, we show for the first time that production of phosphatidic acid by phospholipase D2 (PLD2) is essential for ligand-induced EGFR nanocluster formation. In accordance with its crucial role in regulating EGFR nanocluster formation, we demonstrate that modulating PLD2 activity tunes the degree of EGFR nanocluster formation and mitogen-activated protein kinase signal output. Together, these data show that EGFR activation drives the formation of signaling domains by regulating the production of critical second-messenger lipids and modifying the local membrane lipid environment.The epidermal growth factor (EGF) receptor (EGFR) is a single transmembrane domain protein that possesses intrinsic tyrosine kinase (TK) activity. Ligand binding to the extracellular domain induces conformational changes that promote activation of the intracellular TK domain. The kinase domain then autophosphorylates a number of tyrosine residues in the C-terminal region of the protein, creating docking sites for adapter and effector proteins. Thus, the active form of the EGFR could reasonably be expected to be a dimer. However, recent studies using single-molecule imaging, image correlation spectroscopy (ICS), fluorescence correlation spectroscopy (FCS), and immunoelectron microscopy (immuno-EM) show that the EGFR is, in fact, nonrandomly organized into oligomers on the plasma membrane (6, 7, 16, 34, 44). ICS measurements estimate that, in the absence of ligand, there are, on average, 2.2 EGFRs per cluster, which increases to 3.7 receptors per cluster upon stimulation (7). Single-molecule tracking experiments also suggest that unliganded EGFRs continually fluctuate between monomers and dimers that are primed for activation (5). Furthermore, the organization of the EGFR is dynamic and clustering of the EGFR increases over time after EGF stimulation (7, 16). However, neither the precise role of EGFR oligomerization in signal transduction nor the mechanisms driving oligomer formation have been resolved.The organization of the EGFR into oligomers is dependent upon cellular cholesterol. Saffarian et al., using FCS, estimated that 70% of EGFRs exist as monomers, 20% as dimers, and 10% as oligomers (34). However, depletion of cholesterol decreases the percentage of monomeric receptors and increases the proportion of oligomeric receptors. Cholesterol depletion and actin depolymerization also alter the diffusion coefficient of the EGFR and the confinement area size (22). The finding that EGFR membrane organization is dependent upon cholesterol is of particular interest because a number of studies have demonstrated that EGFR activity is negatively regulated by cholesterol (4, 23, 28, 32).Phospholipase D2 (PLD2) hydrolyzes phosphatidylcholine (PC) to produce choline and phosphatidic acid (PA). PLD2 is localized to the plasma membrane (10), associates with the EGFR (39), and is rapidly activated upon EGF stimulation, leading to increased production of PA (15, 38, 39). A number of lines of evidence suggest that PA is an important mediator of EGFR action. First, exogenous PA is mitogenic when incubated with cells (17, 19, 42, 45). Second, direct interaction with membrane PA regulates the activity of a number of components downstream of the EGFR, including Sos (47) and Raf (12, 13, 30, 31).In the current study, we used high-resolution spatial analysis techniques to investigate EGFR plasma membrane organization. Using these approaches, we identified PA as the key molecular component responsible for driving EGFR nanocluster formation in response to EGF binding and demonstrated that the level of PLD2 activity regulates the duration of mitogen-activated protein kinase (MAPK) signal output.     

Virulence for chickens of Clostridium perfringens isolated from poultry and other sources

Clostridium perfringens type A is the most common cause of poultry necrotic enteritis (NE). Of the four “major” toxins, type A strains produce only alpha toxin (CPA), which has long been considered a major factor in pathogenesis of NE. We investigated the virulence for poultry of type A strains from a variety of enteric sources. Newly-hatched Cornish × Rock chicks were fed a low protein diet for one week, a high protein diet for a second week, and then challenged with log-phase cultures of C. perfringens, mixed 3:4 (v/v) with high protein feed. Strain JGS4143 [genotype A, beta2 positive (cpb2^pos), from a field case of NE] produced gross lesions compatible with NE in >85% of challenged birds. However, strains JGS1714 (enterotoxigenic genotype A, cpb2^pos, human food poisoning), JGS1936 (genotype A, cpb2^neg, bovine neonatal enteritis), JGS4142 (genotype A, cpb2^pos, bovine jejunal hemorrhage syndrome), JGS1473 (genotype A, cpb2^pos, chicken normal flora), JGS1070 (genotype C, cpb2^pos, porcine hemorrhagic enteritis), JGS1882 (genotype A, cpb2^pos, porcine neonatal enteritis), JGS1120 (ATCC 13124, genotype A, cpb2^neg, gas gangrene), JGS4151 (strain 13, genotype A, cpb2^pos, canine), and JGS4303 (SM101, enterotoxigenic genotype A, cpb2^neg, human food poisoning) failed to produce disease. In vivo passage failed to increase virulence of the non-NE strains. NE strains must have specific poultry-associated virulence attributes, such as the recently identified NetB and other factors, which allow for the development of disease.     

Prostaglandin E2 synthases in neurologic homeostasis and disease

Prostaglandin E₂ synthases (PGES) currently comprise a group of three structurally and biologically distinct molecules. These enzymes are part of an important and complex paracrine signaling system involved in a wide range of biological processes. This review focuses on the normal physiological and pathological roles of these enzymes in the nervous system. Specific topics include the role of PGES(s) in fever and sickness behavior, inflammatory pain, and neural disease. Although the field is in its early stages, ongoing development of selective PGES inhibitors for possible use in people creates a significant need for more fully understanding the biological roles of these important enzymes.     

Travelling Waves of Attached and Detached Cells in a Wound-Healing Cell Migration Assay

During a wound-healing cell migration assay experiment, cells are observed to detach and undergo mitosis before reattaching as a pair of cells on the substrate. During experiments with mice 3T3 fibroblasts, cell detachment can be confined to the wavefront region or it can occur over the whole wave profile. A multi-species continuum approach to modelling a wound-healing assay is taken to account for this phenomenon. The first cell population is composed of attached motile cells, while the second population is composed of detached immotile cells undergoing mitosis and returning to the migrating population after successful division. The first model describes cell division occurring only in the wavefront region, while a second model describes cell division over the whole of the scrape wound. The first model reverts to the Fisher equation when the reattachment rate relative to the detachment rate is large, while the second case does not revert to the Fisher equation in any limit. The models yield travelling wave solutions. The minimum wave speed is slower than the minimum Fisher wave speed and its dependence on the cell detachment and reattachment rate parameters is analysed. Approximate travelling wave profiles of the two cell populations are determined asymptotically under certain parameter regimes.     

IL-21 promotes differentiation of naive CD8 T cells to a unique effector phenotype

IL-21, the most recently described member of the common gamma-chain cytokine family, is produced by activated CD4 T cells, whereas CD8 T cells express the IL-21 receptor. To investigate a possible role for IL-21 in the priming of naive CD8 T cells, we examined responses of highly purified naive OT-I CD8 T cells to artificial APCs displaying Ag and B7-1 on their surface. We found that IL-21 enhanced OT-I clonal expansion and supported development of cytotoxic effector function. High levels of IL-2 did not support development of effector functions, but IL-2 was required for optimal responses in the presence of IL-21. IL-12 and IFN-alpha have previously been shown to support naive CD8 T cell differentiation and acquisition of effector functions through a STAT4-dependent mechanism. Here, we show that IL-21 does not require STAT4 to stimulate development of cytolytic activity. Furthermore, IL-21 fails to induce IFN-gamma or IL-4 production and can partially block IL-12 induction of IFN-gamma production. CD8 T cells that differentiate in response to IL-21 have a distinct surface marker expression pattern and are characterized as CD44(high), PD-1(low), CD25(low), CD134(low), and CD137(low). Thus, IL-21 can provide a signal required by naive CD8 T cells to differentiate in response to Ag and costimulation, and the resulting effector cells represent a unique effector phenotype with highly effective cytolytic activity, but deficient capacity to secrete IFN-gamma.     

Thiocyanate transport in resting and IL-4-stimulated human bronchial epithelial cells: role of pendrin and anion channels

SCN(-) (thiocyanate) is an important physiological anion involved in innate defense of mucosal surfaces. SCN(-) is oxidized by H(2)O(2), a reaction catalyzed by lactoperoxidase, to produce OSCN(-) (hypothiocyanite), a molecule with antimicrobial activity. Given the importance of the availability of SCN(-) in the airway surface fluid, we studied transepithelial SCN(-) transport in the human bronchial epithelium. We found evidence for at least three mechanisms for basolateral to apical SCN(-) flux. cAMP and Ca(2+) regulatory pathways controlled SCN(-) transport through cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels, respectively, the latter mechanism being significantly increased by treatment with IL-4. Stimulation with IL-4 also induced the strong up-regulation of an electroneutral SCN(-)/Cl(-) exchange. Global gene expression analysis with microarrays and functional studies indicated pendrin (SLC26A4) as the protein responsible for this SCN(-) transport. Measurements of H(2)O(2) production at the apical surface of bronchial cells indicated that the extent of SCN(-) transport is important to modulate the conversion of this oxidant molecule by the lactoperoxidase system. Our studies indicate that the human bronchial epithelium expresses various SCN(-) transport mechanisms under resting and stimulated conditions. Defects in SCN(-) transport in the airways may be responsible for susceptibility to infections and/or decreased ability to scavenge oxidants.