Siderophore activity of myo-inositol hexakisphosphate in Pseudomonas aeruginosa

Myo-Inositol hexakisphosphate (InsP6), which is found in soil and most, if not all, plant and animal cells, has been estimated to have an affinity for Fe3+ in the range of 10(25) to 10(30) M-1. In this report, we demonstrate that the Fe-InsP6 complex has siderophore activity and is able to reverse the iron-restricted growth inhibition of Pseudomonas aeruginosa by ethylene diamine di(o-hydroxyphenyl)acetic acid. With 55Fe-InsP6 in transport studies, iron uptake is strongly iron regulated, being repressed after growth in iron-replete conditions and inhibited by treatment with potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone. The kinetics of iron transport revealed a Km of 100 nM. Self-displacement of binding of [3H]InsP6 to isolated membranes by InsP6 revealed a single class of binding sites (Kd = 143 +/- 6 nM; Hill coefficient, 1.1 +/- 0.1). The binding of [3H]InsP6 to membranes was not dependent on whether cells had been grown under conditions of high or low iron concentrations. We believe that this is the first report of inositol polyphosphate activity in prokaryotic cells.

Expression of the human cachexia-associated protein (HCAP) in prostate cancer and in a prostate cancer animal model of cachexia

Prostate cancer (CaP) patients with disseminated disease often suffer from severe cachexia, which contributes to mortality in advanced cancer. Human cachexia-associated protein (HCAP) was recently identified from a breast cancer library based on the available 20-amino acid sequence of proteolysis-inducing factor (PIF), which is a highly active cachectic factor isolated from mouse colon adenocarcinoma MAC16. Herein, we investigated the expression of HCAP in CaP and its potential involvement in CaP-associated cachexia. HCAP mRNA was detected in CaP cell lines, in primary CaP tissues and in its osseous metastases. In situ hybridization showed HCAP mRNA to be localized only in the epithelial cells in CaP tissues, in the metastatic foci in bone, liver and lymph node, but not in the stromal cells or in normal prostate tissues. HCAP protein was detected in 9 of 14 CaP metastases but not in normal prostate tissues from cadaveric donors or patients with organ-confined tumors. Our Western blot analysis revealed that HCAP was present in 9 of 19 urine specimens from cachectic CaP patients but not in 19 urine samples of noncachectic patients. HCAP mRNA and protein were also detected in LuCaP 35 and PC-3M xenografts from our cachectic animal models. Our results demonstrated that human CaP cells express HCAP and the expression of HCAP is associated with the progression of CaP and the development of CaP cachexia. © 2003 Wiley-Liss, Inc.

The development of an in vivo model of drug-induced terminal differentiation of Leukaemic cells

The technique of growing human leukaemic cells in diffusion chambers was developed to enable chemicals to be assessed for their ability to induce terminal differentiation. HL-60 promyelocytic leukaemia cell growth, in a lucite chamber with a Millipore filter, was optimised by use of a lateral incision site. Chambers were constructed using 0.45um filters and contained 150ul of serum-free HL-60 cells at a density of 1x106 cells/ml. The chambers were implanted into CBA/Ca mice and spontaneous terminal differentiation of the cells to granulocytes was prevented by the use of serum-free medium. Under these conditions there was an initial growth lag of 72 hours and a logarithmic phase of growth for 96 hours; the cell number reached a plateau after 168 hours of culture in vivo. The amount of drug in the plasma of the animal and in chambers that had been implanted for 5 days, was determined after a single ip injection of equitoxic doses of N-methylformamide, N-ethylformamide, tetramethylurea, N-dibutylformamide, N-tetramethylbutylformamide and hexamethylenebisacetamide. Concentrations of both TMU and HMBA were obtained in the plasma and in the chamber which were pharmacologically effective for the induction of differentiation of HL-60 cells in vitro, that is 12mM TMU and 5mM HMBA. A 4 day regime of treatment of animals implanted with chambers demonstrated that TMU and HMBA induced terminal differentiation of 50% and 35%, respectively, of the implanted HL-60 cells to granulocyte-like cells, assessed by measurement of functional and biochemical markers of maturity. None of the other agents attained concentrations in the plasma that were pharmacologically effective for the induction of differentiation of the cells in vitro and were unable to induce the terminal differentiation of the cells in vivo.

Bone marrow stromal cells stimulate neurite outgrowth over neural proteoglycans (CSPG), myelin associated glycoprotein and Nogo-A

In animal models, transplantation of bone marrow stromal cells (MSC) into the spinal cord following injury enhances axonal regeneration and promotes functional recovery. How these improvements come about is currently unclear. We have examined the interaction of MSC with neurons, using an established in vitro model of nerve growth, in the presence of substrate-bound extracellular molecules that are thought to inhibit axonal regeneration, i.e., neural proteoglycans (CSPG), myelin associated glycoprotein (MAG) and Nogo-A. Each of these molecules repelled neurite outgrowth from dorsal root ganglia (DRG) in a concentration-dependent manner. However, these nerve-inhibitory effects were much reduced in MSC/DRG co-cultures. Video microscopy demonstrated that MSC acted as "cellular bridges" and also "towed" neurites over the nerve-inhibitory substrates. Whereas conditioned medium from MSC cultures stimulated DRG neurite outgrowth over type I collagen, it did not promote outgrowth over CSPG, MAG or Nogo-A. These findings suggest that MSC transplantation may promote axonal regeneration both by stimulating nerve growth via secreted factors and also by reducing the nerve-inhibitory effects of the extracellular molecules present.

Spinal motor neurite outgrowth over glial scar inhibitors is enhanced by coculture with bone marrow stromal cells

Background context Transplantation of bone marrow cells into spinal cord lesions promotes functional recovery in animal models, and recent clinical trials suggest possible recovery also in humans. The mechanisms responsible for these improvements are still unclear. Purpose To characterize spinal cord motor neurite interactions with human bone marrow stromal cells (MSCs) in an in vitro model of spinal cord injury (SCI). Study design/setting Previously, we have reported that human MSCs promote the growth of extending sensory neurites from dorsal root ganglia (DRG), in the presence of some of the molecules present in the glial scar, which are attributed with inhibiting axonal regeneration after SCI. We have adapted and optimized this system replacing the DRG with a spinal cord culture to produce a central nervous system (CNS) model, which is more relevant to the SCI situation. Methods We have developed and characterized a novel spinal cord culture system. Human MSCs were cocultured with spinal motor neurites in substrate choice assays containing glial scar-associated inhibitors of nerve growth. In separate experiments, MSC-conditioned media were analyzed and added to spinal motor neurites in substrate choice assays. Results As has been reported previously with DRG, substrate-bound neurocan and Nogo-A repelled spinal neuronal adhesion and neurite outgrowth, but these inhibitory effects were abrogated in MSC/spinal cord cocultures. However, unlike DRG, spinal neuronal bodies and neurites showed no inhibition to substrates of myelin-associated glycoprotein. In addition, the MSC secretome contained numerous neurotrophic factors that stimulated spinal neurite outgrowth, but these were not sufficient stimuli to promote spinal neurite extension over inhibitory concentrations of neurocan or Nogo-A. Conclusions These findings provide novel insight into how MSC transplantation may promote regeneration and functional recovery in animal models of SCI and in the clinic, especially in the chronic situation in which glial scars (and associated neural inhibitors) are well established. In addition, we have confirmed that this CNS model predominantly comprises motor neurons via immunocytochemical characterization. We hope that this model may be used in future research to test various other potential interventions for spinal injury or disease states. © 2014 Elsevier Inc. All rights reserved.

Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process

Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100mL stirred spinner flasks at a density of 3×10⁵cells.mL^-1 in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal-free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post-harvest viability of 99.63±0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony-forming potential. The hMSCs were held in suspension post-harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum-free vehicle solution using a controlled-rate freezing process. Post-thaw viability was 75.8±1.4% with a similar 3h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component-free hMSC production process from expansion through to cryopreservation.

Extra-nuclear telomerase reverse transcriptase (TERT) regulates glucose transport in skeletal muscle cells

Telomerase reverse transcriptase (TERT) is a key component of the telomerase complex. By lengthening telomeres in DNA strands, TERT increases senescent cell lifespan. Mice that lack TERT age much faster and exhibit age-related conditions such as osteoporosis, diabetes and neurodegeneration. Accelerated telomere shortening in both human and animal models has been documented in conditions associated with insulin resistance, including T2DM. We investigated the role of TERT, in regulating cellular glucose utilisation by using the myoblastoma cell line C2C12, as well as primary mouse and human skeletal muscle cells. Inhibition of TERT expression or activity by using siRNA (100. nM) or specific inhibitors (100. nM) reduced basal 2-deoxyglucose uptake by ~. 50%, in all cell types, without altering insulin responsiveness. In contrast, TERT over-expression increased glucose uptake by 3.25-fold. In C2C12 cells TERT protein was mostly localised intracellularly and stimulation of cells with insulin induced translocation to the plasma membrane. Furthermore, co-immunoprecipitation experiments in C2C12 cells showed that TERT was constitutively associated with glucose transporters (GLUTs) 1, 4 and 12 via an insulin insensitive interaction that also did not require intact PI3-K and mTOR pathways. Collectively, these findings identified a novel extra-nuclear function of TERT that regulates an insulin-insensitive pathway involved in glucose uptake in human and mouse skeletal muscle cells. © 2014 Elsevier B.V.

Brain-derived neurotrophic factor as an indicator of chemical neurotoxicity:an animal-free CNS cell culture model

Recent changes to the legislation on chemicals and cosmetics testing call for a change in the paradigm regarding the current 'whole animal' approach for identifying chemical hazards, including the assessment of potential neurotoxins. Accordingly, since 2004, we have worked on the development of the integrated co-culture of post-mitotic, human-derived neurons and astrocytes (NT2.N/A), for use as an in vitro functional central nervous system (CNS) model. We have used it successfully to investigate indicators of neurotoxicity. For this purpose, we used NT2.N/A cells to examine the effects of acute exposure to a range of test chemicals on the cellular release of brain-derived neurotrophic factor (BDNF). It was demonstrated that the release of this protective neurotrophin into the culture medium (above that of control levels) occurred consistently in response to sub-cytotoxic levels of known neurotoxic, but not non-neurotoxic, chemicals. These increases in BDNF release were quantifiable, statistically significant, and occurred at concentrations below those at which cell death was measureable, which potentially indicates specific neurotoxicity, as opposed to general cytotoxicity. The fact that the BDNF immunoassay is non-invasive, and that NT2.N/A cells retain their functionality for a period of months, may make this system useful for repeated-dose toxicity testing, which is of particular relevance to cosmetics testing without the use of laboratory animals. In addition, the production of NT2.N/A cells without the use of animal products, such as fetal bovine serum, is being explored, to produce a fully-humanised cellular model.

The development of functional astrocyte-neuron networks derived from stem cells

There is currently great scientific and medical interest in the potential of tissue grown from stem cells. These cells present opportunities for generating model systems for drug screening and toxicological testing which would be expected to be more relevant to human outcomes than animal based tissue preparations. Newly realised astrocytic roles in the brain have fundamental implications within the context of stem cell derived neuronal networks. If the aim of stem cell neuroscience is to generate functional neuronal networks that behave as networks do in the brain, then it becomes clear that we must include and understand all the cellular components that comprise that network, and which are important to support synaptic integrity and cell to cell signalling. We have shown that stem cell derived neurons exhibit spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling (1). Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, astrocytes exhibit morphology and functional properties consistent with this glial cell type. Astrocytes also respond to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. Astroctyes also generate propagating calcium waves that are gap junction and purinergic signalling dependent. Our results show that stem cell derived astrocytes exhibit appropriate functionality and that stem cell neuronal networks interact with astrocytic networks in co-culture. Using mixed cultures of stem cell derived neurons and astrocytes, we have also shown both cell types also modulate their glucose uptake, glycogen turnover and lactate production in response to glutamate as well as increased neuronal activity (2). This finding is consistent with their neuron-astrocyte metabolic coupling thus demonstrating a tractable human model, which will facilitate the study of the metabolic coupling between neurons and astrocytes and its relationship with CNS functional issues ranging from plasticity to neurodegeneration. Indeed, cultures treated with oligomers of amyloid beta 1-42 (Aβ1-42) also display a clear hypometabolism, particularly with regard to utilization of substrates such as glucose (3). Both co-cultures of neurons and astrocytes and purified cultures of astrocytes showed a significant decrease in glucose uptake after treatment with 2 and 0.2 μmol/L Aβ at all time points investigated (p <0.01). In addition, a significant increase in the glycogen content of cells was also measured. Mixed neuron and astrocyte co-cultures as well as pure astrocyte cultures showed an initial decrease in glycogen levels at 6 hours compared with control at 0.2 μmol/L and 2 μmol/L P <0.01. These changes were accompanied by changes in NAD+/NADH (P<0.05), ATP (P<0.05), and glutathione levels (P<0.05), suggesting a disruption in the energy-redox axis within these cultures. The high energy demands associated with neuronal functions such as memory formation and protection from oxidative stress put these cells at particular risk from Aβ-induced hypometabolism. As numerous cell types interact in the brain it is important that any in vitro model developed reflects this arrangement. Our findings indicate that stem cell derived neuron and astrocyte networks can communicate, and so have the potential to interact in a tripartite manner as is seen in vivo. This study therefore lays the foundation for further development of stem cell derived neurons and astrocytes into therapeutic cell replacement and human toxicology/disease models. More recently our data provides evidence for a detrimental effect of Aβ on carbohydrate metabolism in both neurons and astrocytes. As a purely in vitro system, human stem cell models can be readily manipulated and maintained in culture for a period of months without the use of animals. In our laboratory cultures can be maintained in culture for up to 12 months months thus providing the opportunity to study the consequences of these changes over extended periods of time relevant to aspects of the disease progression time frame in vivo. In addition, their human origin provides a more realistic in vitro model as well as informing other human in vitro models such as patient-derived iPSC.

Nicotine exposure reduces cell viability but induces anti-inflammatory effects in human cystic fibrosis bronchial epithelial cells

Background: Mouse models of cystic fibrosis (CF) fail to truly represent the respiratory pathology. We have consequently developed human airways cell culture models to address this. The impact of cigarette smoke within the CF population is well documented, with exposure being known to worsen lung function. As nicotine is often perceived to be a less harmful component of tobacco smoke, this research aimed to identify its effects upon viability and inflammatory responses of CF (IB3-1) and CF phenotype corrected (C38) bronchial epithelial cells. Methods: IB3-1 and C38 cell lines were exposed to increasing concentrations of nicotine (0.55-75μM) for 24 hours. Cell viability was assessed via Cell Titre Blue and the inflammatory response with IL-6 and IL-8 ELISA. Results: CF cells were more sensitive; nicotine significantly (P<0.05) reduced cell viability at all concentrations tested, but failed to have a marked effect on C38 viability. Whilst nicotine induced anti-inflammatory effects in CF cells with a significant reduction in IL-6 and IL-8 release, it had no effect on chemokine release by C38 cells. Conclusion: CF cells may be more vulnerable to inhaled toxicants than non-CF cells. As mice lack a number of human nicotinic receptor subunits and fail to mimic the characteristic pathology of CF, these data emphasise the importance of employing relevant human cell lines to study a human-specific disease.