Mycobacterium tuberculosis subverts negative regulatory pathways in human macrophages to drive immunopathology.

Tuberculosis remains a global pandemic and drives lung matrix destruction to transmit. Whilst pathways driving inflammatory responses in macrophages have been relatively well described, negative regulatory pathways are less well defined. We hypothesised that Mycobacterium tuberculosis (Mtb) specifically targets negative regulatory pathways to augment immunopathology. Inhibition of signalling through the PI3K/AKT/mTORC1 pathway increased matrix metalloproteinase-1 (MMP-1) gene expression and secretion, a collagenase central to TB pathogenesis, and multiple pro-inflammatory cytokines. In patients with confirmed pulmonary TB, PI3Kδ expression was absent within granulomas. Furthermore, Mtb infection suppressed PI3Kδ gene expression in macrophages. Interestingly, inhibition of the MNK pathway, downstream of pro-inflammatory p38 and ERK MAPKs, also increased MMP-1 secretion, whilst suppressing secretion of TH1 cytokines. Cross-talk between the PI3K and MNK pathways was demonstrated at the level of eIF4E phosphorylation. Mtb globally suppressed the MMP-inhibitory pathways in macrophages, reducing levels of mRNAs encoding PI3Kδ, mTORC-1 and MNK-1 via upregulation of miRNAs. Therefore, Mtb disrupts negative regulatory pathways at multiple levels in macrophages to drive a tissue-destructive phenotype that facilitates transmission.

Interactions of human endothelial cells with gold nanoparticles of different morphologies.

The interactions between noncancerous, primary endothelial cells and gold nanoparticles with different morphologies but the same ligand capping are investigated. The endothelial cells are incubated with gold nanospheres, nanorods, hollow gold spheres, and core/shell silica/gold nanocrystals, which are coated with monocarboxy (1-mercaptoundec-11-yl) hexaethylene glycol (OEG). Cell viability studies show that all types of gold particles are noncytotoxic. The number of particles taken up by the cells is estimated using inductively coupled plasma (ICP), and are found to differ depending on particle morphology. The above results are discussed with respect to heating efficiency. Using experimental data reported earlier and theoretical model calculations which take into account the physical properties and distribution of particles in the cellular microenvironment, it is found that collective heating effects of several cells loaded with nanoparticles must be included to explain the observed viability of the endothelial cells.

Laser-induced damage and recovery of plasmonically targeted human endothelial cells.

Laser-induced techniques that employ the surface plasmon resonances of nanoparticles have recently been introduced as an effective therapeutic tool for destroying tumor cells. Here, we adopt a low-intensity laser-induced technique to manipulate the damage and repair of a vital category of noncancerous cells, human endothelial cells. Endothelial cells construct the interior of blood vessels and play a pivotal role in angiogenesis. The degree of damage and repair of the cells is shown to be influenced by laser illumination in the presence of gold nanoparticles of different morphologies, which either target the cellular membrane or are endocytosed. A pronounced influence of the plasmonic nanoparticle laser treatment on the expression of critical angiogenic genes is shown. Our results show that plasmon-mediated mild laser treatment, combined with specific targeting of cellular membranes, enables new routes for controlling cell permeability and gene regulation in endothelial cells.

Foam-in-Vein: Characterisation of Blood Displacement Efficacy of Liquid Sclerosing Foams.

Sclerotherapy is among the least invasive and most commonly utilised treatment options for varicose veins. Nonetheless, it does not cure varicosities permanently and recurrence rates are of up to 64%. Although sclerosing foams have been extensively characterised with respect to their bench-top properties, such as bubble size distribution and half-life, little is known about their flow behaviour within the venous environment during treatment. Additionally, current methods of foam characterisation do not recapitulate the end-point administration conditions, hindering optimisation of therapeutic efficacy. Here, a therapeutically relevant apparatus has been used to obtain a clinically relevant rheological model of sclerosing foams. This model was then correlated with a therapeutically applicable parameter-i.e., the capability of foams to displace blood within a vein. A pipe viscometry apparatus was employed to obtain a rheological model of 1% polidocanol foams across shear rates of 6 s-1 to 400 s-1. Two different foam formulation techniques (double syringe system and Tessari) and three liquid-to-gas ratios (1:3, 1:4 and 1:5) were investigated. A power-law model was employed on the rheological data to obtain the apparent viscosity of foams. In a separate experiment, a finite volume of foam was injected into a PTFE tube to displace a blood surrogate solution (0.2% w/v carboxymethyl cellulose). The displaced blood surrogate was collected, weighed, and correlated with foam's apparent viscosity. Results showed a decreasing displacement efficacy with foam dryness and injection flowrate. Furthermore, an asymptotic model was formulated that may be used to predict the extent of blood displacement for a given foam formulation and volume. The developed model could guide clinicians in their selection of a foam formulation that exhibits the greatest blood displacement efficacy.

Receptor-mediated interactions between colloidal gold nanoparticles and human umbilical vein endothelial cells.

A new strategy to manipulate cell operations is demonstrated, based on membrane-receptor-specific interactions between colloidal peptide-capped gold nanoparticles and human umbilical vein endothelial cells. It is shown that colloidal gold nanoparticles of similar charge and size but capped with different peptide sequences can deliberately trigger specific cell functions related to the important biological process of blood vessel growth known as angiogenesis. Specific binding of the peptide-capped particles to two endothelial-expressed receptors (VEGFR-1, NRP-1), which control angiogenesis, is achieved. The cellular fate of the functional nanoparticles is imaged and the influence of the different peptide-coated nanoparticles on the gene expression profile of hypoxia-related and angiogenic genes is monitored. The findings open up new avenues towards the deliberate biological control of cellular functions using strategically designed nanoparticles.

Foam-in-vein: A review of rheological properties and characterization methods for optimization of sclerosing foams.

Varicose veins are chronic venous defects that affect >20% of the population in developed countries. Among potential treatments, sclerotherapy is one of the most commonly used. It involves endovenous injection of a surfactant solution (or foam) in varicose veins, inducing damage to the endothelial layer and subsequent vessel sclerosis. Treatments have proven to be effective in the short-term, however recurrence is reported at rates of up to 64% 5-year post-treatment. Thus, once diagnosed with varicosities there is a high probability of a permanently reduced quality of life. Recently, foam sclerotherapy has become increasingly popular over its liquid counterpart, since foams can treat larger and longer varicosities more effectively, they can be imaged using ultrasound, and require lower amounts of sclerosing agent. In order to minimize recurrence rates however, an investigation of current treatment methods should lead to more effective and long-lasting effects. The literature is populated with studies aimed at characterizing the fundamental physics of aqueous foams; nevertheless, there is a significant need for appropriate product development platforms. Despite successfully capturing the microstructural evolution of aqueous foams, the complexity of current models renders them inadequate for pharmaceutical development. This review article will focus on the physics of foams and the attempts at optimizing them for sclerotherapy. This takes the form of a discussion of the most recent numerical and experimental models, as well as an overview of clinically relevant parameters. This holistic approach could contribute to better foam characterization methods that patients may eventually derive long term benefit from.

In vitro and ex vivo evaluation of the biological performance of sclerosing foams.

Since the first reports on foam sclerotherapy, multiple studies have been conducted to determine the physical properties and behavior of foams, but relatively little is known about their biological effects on the endothelial cells lining the vessel wall. Moreover, a systematic comparison of the biological performance of foams produced with different methods has not been carried out yet. Herein, a 2D in vitro method was developed to compare efficacy of commercially available polidocanol injectable foam (PEM, Varithena) and physician-compounded foams (PCFs). Endothelial cell attachment upon treatment with foam was quantified as an indicator of therapeutic efficacy, and was correlated with foam physical characteristics and administration conditions. An ex vivo method was also developed to establish the disruption and permeabilisation of the endothelium caused by sclerosing agents. It relied on the quantitation of extravasated bovine serum albumin conjugated to Evans Blue, as an indicator of endothelial permeability. In our series of comparisons, PEM presented a greater overall efficacy compared to PCFs, across the different biological models, which was attributed to its drainage dynamics and gas formulation. This is consistent with earlier studies that indicated superior physical cohesiveness of PEM compared to PCFs.

Physical Vein Models to Quantify the Flow Performance of Sclerosing Foams.

Foam sclerotherapy is clinically employed to treat varicose veins. It involves intravenous injection of foamed surfactant agents causing endothelial wall damage and vessel shrinkage, leading to subsequent neovascularization. Foam production methods used clinically include manual techniques, such as the Double Syringe System (DSS) and Tessari (TSS) methods. Pre-clinical in-vitro studies are conducted to characterize the performance of sclerosing agents; however, the experimental models used often do not replicate physiologically relevant physical and biological conditions. In this study, physical vein models (PVMs) were developed and employed for the first time to characterize the flow behavior of sclerosing foams. PVMs were fabricated in polydimethylsiloxane (PDMS) by replica molding, and were designed to mimic qualitative geometrical characteristics of veins. Foam behavior was investigated as a function of different physical variables, namely (i) geometry of the vein model (i.e., physiological vs. varicose vein), (ii) foam production technique, and (iii) flow rate of a blood surrogate. The experimental set-up consisted of a PVM positioned on an inclined platform, a syringe pump to control the flow rate of a blood substitute, and a pressure transducer. The static pressure of the blood surrogate at the PVM inlet was measured upon foam administration. The recorded pressure-time curves were analyzed to quantify metrics of foam behavior, with a particular focus on foam expansion and degradation dynamics. Results showed that DSS and TSS foams had similar expansion rate in the physiological PVM, whilst DSS foam had lower expansion rate in the varicose PVM compared to TSS foam. The degradation rate of DSS foam was lower than TSS foam, in both model architectures. Moreover, the background flow rate had a significant effect on foam behavior, enhancing foam displacement rate in both types of PVM.

Human Endothelial Cells Modulate CD4+ T Cell Populations and Enhance Regulatory T Cell Suppressive Capacity.

Endothelial cells (ECs) line the luminal surface of blood vessels and have an active role in the recruitment of leukocytes, including immune cell activation. Regulatory T cells (Tregs) are immune suppressor cells that maintain peripheral tolerance and must interact with the endothelium as they traffic into tissue. We hypothesized that human ECs could modulate Tregs and their suppressor function. Cocultures of CD4+ T cells with human umbilical vein ECs (HUVECs) or dermal microvascular ECs (HDMECs) were conducted and analyzed for activation and proliferation after 72 and 120 h using flow cytometry. In monocyte-depleted cultures, human ECs were found to support CD4+ T cell proliferation in the presence of external mitogens phytohemagglutinin or anti-CD3/28 antibodies (aCD3/28). Activation was shown by CD25 expression in these cells that also transiently expressed the Treg transcription factor FOXP3. HUVECs supported the specific concurrent proliferation of both effector T cells and Tregs when cocultured with aCD3/28. Purified Tregs were also functionally activated by prior coculture with EC to suppress effector T (Teff) cell proliferation. Both direct coculture and indirect coculture of EC and Treg showed activation of the Treg suppressive phenotype. However, whereas HUVEC showed enhancement of suppression by both mechanisms, HDMEC only supported Treg suppressive activity via the contact-independent mechanism. In the contact-independent cultures, the soluble mediators IL-6, GM-CSF, or G-CSF released from ECs following interferon-γ activation were not responsible for the enhanced Treg suppressor function. Following direct coculture, Treg expression of inhibitory receptors PD-1 and OX40 was elevated while activated EC expressed the counter ligands programmed death ligand (PD-L)1 and PD-L2. Therefore, human ECs have a role in supporting T cell proliferation and increasing Treg suppressor function. This ability of EC to enhance Treg function could offer novel targets to boost Treg activity during inflammatory disorders.

ROS generation in endothelial hypoxia and reoxygenation stimulates MAP kinase signaling and kinase-dependent neutrophil recruitment.

Reactive oxygen species (ROS)-induced injury has been shown to occur during the reperfusion phase of ischemia-reperfusion and ROS are known to induce signaling events. We hypothesized that oxygen sensing in endothelial cells is also dependent on internal redox changes during hypoxia and that endothelial cells respond to changing oxygen environments via signaling, switching to an inflammatory phenotype. Endothelial cells exposed to relative hypoxia or the mitochondrial inhibitors rotenone, antimycin A, or FCCP show loss of mitochondrial membrane potential. During hypoxia, an increase in cytoplasmic ROS and glutathione S-transferase activity occurred, suggesting changes in intracellular redox state, mimicked with rotenone or FCCP but inhibited by antimycin A. Phosphorylation of stress-responsive mitogen-activated protein kinases occurred in hypoxia and was rapid and prolonged. Phosphorylation was inhibited by vitamin C, N-acetyl cysteine, or antimycin A. Chelation of intracellular calcium inhibits phosphorylation but the mitochondrial transition pore inhibitor cyclosporin A had no effect. Reoxygenation caused a further round of signaling, which was rapid but transient. Functionally, adhesion of neutrophils after hypoxia-reoxygenation under flow is ROS, P-selectin, and MAPK dependent. Therefore, changes in cellular signaling and phenotype are abrogated by ROS scavengers and suggest their use as therapeutic agents in ischemia-reperfusion.

Graphene Oxide-Upconversion Nanoparticle Based Optical Sensors for Targeted Detection of mRNA Biomarkers Present in Alzheimer's Disease and Prostate Cancer.

The development of new sensors for the accurate detection of biomarkers in biological fluids is of utmost importance for the early diagnosis of diseases. Next to advanced laboratory techniques, there is a need for relatively simple methods which can significantly broaden the availability of diagnostic capability. Here, we demonstrate the successful application of a sensor platform based on graphene oxide and upconversion nanoparticles (NPs) for the specific detection of mRNA-related oligonucleotide markers in complex biological fluids. The combination of near-infrared light upconversion with low-background photon counting readout enables reliable detection of low quantities of small oligonucleotide sequences in the femtomolar range. We demonstrate the successful detection of analytes relevant to mRNAs present in Alzheimer's disease as well as prostate cancer in human blood serum. The high performance and relative simplicity of the upconversion NP-graphene sensor platform enables new opportunities in early diagnosis based on specific detection of oligonucleotide sequences in complex environments.

Cellular crosstalk between airway epithelial and endothelial cells regulates barrier functions during exposure to double-stranded RNA.

INTRODUCTION: The epithelial and endothelial barriers of the airway mucosa are critical for regulation of tissue homeostasis and protection against pathogens or other tissue damaging agents. In response to a viral infection, epithelial cells must signal to the endothelium to initiate immune cell recruitment. This is a highly temporal regulated process; however, the mechanisms of this cross-talk are not fully understood. METHODS: In a close-contact co-culture model of human airway epithelial and endothelial cells, cellular crosstalk was analyzed using transepithelial electrical resistance (TER) measurements, immunofluorescence, electron microscopy, and ELISA. Viral infections were simulated by exposing airway epithelial cells apically to double-stranded RNA (Poly(I:C)). Using a microfluidic culture system, the temporal release of mediators was analyzed in the co-culture model. RESULTS: Within 4 h of challenge, double-stranded RNA induced the release of TNF-α by epithelial cells. This activated endothelial cells by triggering the release of the chemoattractant CX3CL1 (fractalkine) by 8 h post-challenge and expression of adhesion molecules E-selectin and ICAM-1. These responses were significantly reduced by neutralising TNF-α. CONCLUSION: By facilitating kinetic profiling, the microfluidic co-culture system has enabled identification of a key signaling mechanism between the epithelial and endothelial barriers. Better understanding of cell-cell cross-talk and its regulatory mechanisms has the potential to identify new therapeutic strategies to control airway inflammation.

Nanoparticles for inhibition of in vitro tumour angiogenesis: synergistic actions of ligand function and laser irradiation.

Careful design of nanoparticles plays a crucial role in their biomedical applications. It not only defines the stability of nanoparticles in a biological medium but also programs their biological functionality and specific interactions with cells. Here, an inorganic nanoparticulate system engineered to have a dual role as anti-angiogenic and hyperthermic agent is presented. The inorganic rod-shaped core is designed to strongly absorb near-infrared laser irradiation through the surface plasmon resonance and convert it into localized heat, while a peptide coating acts as an anti-angiogenic drug, altogether inhibiting vascular growth. The synergistic dual action provides an improved inhibition of the in vitro tumour angiogenesis, offering new possibilities for the development of nano-engineered anti-angiogenic drugs for therapies.

Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics.

The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL-8 release is detectable within the first 2h and peaks at 4-6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms.

Peroxynitrite formation from the simultaneous reduction of nitrite and oxygen by xanthine oxidase.

One electron reductions of oxygen and nitrite by xanthine oxidase form peroxynitrite. The nitrite and oxygen reducing activities of xanthine oxidase are regulated by oxygen with K(oxygen) 26 and 100 microM and K(nitrite) 1.0 and 1.1 mM with xanthine and NADH as donor substrates. Optimal peroxynitrite formation occurs at 70 microM oxygen with purine substrates. Kinetic parameters: V(max) approximately 50 nmol/min/mg and K(m) of 22, 36 and 70 microM for hypoxanthine, pterin and nitrite respectively. Peroxynitrite generation is inhibited by allopurinol, superoxide dismutase and diphenylene iodonium. A role for this enzyme activity can be found in the antibacterial activity of milk and circulating xanthine oxidase activity.

Xanthine oxidase mediates cytokine-induced, but not hormone-induced bone resorption.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) have been implicated as mediators of osteoclastic bone resorption. Xanthine oxidase (XO) a ubiquitous enzyme is widely known for its production of these ROS. We therefore evaluated the potential of XO as a source of ROS in cytokine-and hormone-induced bone resorption. XO activity in rat calvarial osteoblasts was found to be significantly elevated upon stimulation by the cytokines, TNFalpha and IL-1beta. These cytokines also caused a dose related increase in bone resorption of mouse calvariae, which was significantly inhibited by catalase (10 IU/ml). Allopurinol, the competitive inhibitor of XO, also caused a dose related (1-50 microM) inhibition of TNFalpha (20 ng/ml) and (0.01-10 microM) IL-1beta (50 IU/ml)-induced bone resorption, respectively. PTH- and 1,25-(OH)2 Vitamin D3-induced bone resorption could also be inhibited by catalase (100 IU/ml) but was unaffected by allopurinol, indicating that another mediator, other than XO, is required for hormone-induced bone resorption. These results demonstrate, that modulation of the redox balance in the bone microenvironment, which contains XO, can affect the bone resorbing process. Therefore, XO may play a pivotal role in cytokine-induced bone resorption and, if manipulated appropriately, could show a therapeutic benefit in inflammatory bone disorders such as RA.

Vascular physiology and pathology of circulating xanthine oxidoreductase: from nucleotide sequence to functional enzyme.

The evolutionarily conserved, cofactor-dependent, enzyme xanthine oxidoreductase exists in both cell-associated and circulatory forms. The exact role of the circulating form is not known; however, several putative physiological and pathological functions have been suggested that range from purine catabolism to a mediator of acute respiratory distress syndrome. Regulation of gene expression, cofactor synthesis and insertion, post-translational conversion, entry into the circulation, and putative physiological and pathological roles for human circulating xanthine oxidoreductase are discussed.

Manipulation of in vitro angiogenesis using peptide-coated gold nanoparticles.

We demonstrate the deliberate activation or inhibition of invitro angiogenesis using functional peptide coated gold nanoparticles. The peptides, anchored to oligo-ethylene glycol capped gold nanospheres, were designed to selectively interact with cell receptors responsible for activation or inhibition of angiogenesis. The functional particles are shown to influence significantly the extent and morphology of vascular structures, without causing toxicity. Mechanistic studies show that the nanoparticles have the ability to alter the balance between naturally secreted pro- and anti-angiogenic factors, under various biological conditions. Nanoparticle-induced control over angiogenesis opens up new directions in targeted drug delivery and therapy.

Exocytosis of peptide functionalized gold nanoparticles in endothelial cells.

We present the exocytosis profile of two types of peptide-coated nanoparticles, which have similar charge and size but different functionality. While one kind of particles appears to progressively exocytose, the other one has a more complex profile, suggesting that some of the particles are re-uptaken by the cells. Both types of particles retain their colloidal stability after exocytosis.