Image: Flickr - Brent 2.0 Researchers from the Wyss Institute for Biologically Inspired Engineering at Harvard University, Harvard Medical School and Children’s Hospital Boston have created a lung-on-a-chip which may help to speed pharmaceutical development by reducing reliance on animal models. The device consists of a layer of human alveolar cells separated from a layer of human endothelial cells by a porous membrane and mimics the boundary between the lung’s air sacs and capillaries. The cells on the chip can also be made to ‘breathe’ by cyclically applying a vacuum to increase the width of the membrane and stretch the cells before allowing them to contract again.
The, lung-on-a-chip has the potential to model the effects of environmental toxins, the inflammatory response to inhaled pathogens and the effectiveness of new drugs. Because the chip is transparent, responses can be captured in real time using high-resolution fluorescence microscopy. When E.Coli bacteria were introduced into the air on the ‘lung’ side of the chip, white blood cells on the ‘blood’ side of the chip migrated through the porous membrane into the air chamber to destroy the bacteria. ‘Breathing’ was found to enhance absorption of nanoparticles, some of which induced an inflammatory response and overproduction of free radicals by the lung cells.
The team are now exploring whether the system can mimic gas exchange between alveolar cells and the bloodstream and believe that the device provides proof-of-principle for the concept that organs-on-chips could replace many animal studies in the future.
Image: Flickr - GanMed64 Although oestrogen replacement lowers cardiovascular risk in post-menopausal women, treatment is associated with an increased risk of uterine and breast cancer.
The increased cancer risk is linked to oestrogen’s action at nuclear receptors but researchers at UT Southwestern Medical Center have now found that a subpopulation of oestrogen receptors outside the cell nucleus mediate the beneficial cardiovascular effects. The extra-nuclear receptors in endothelial cells are important for blood vessel maintenance and repair and also regulate production of nitric oxide which has a number of beneficial cardiovascular effects. The team have found that an oestrogen-macromolecule complex which is excluded from the nucleus is highly effective in stimulating the extra-nuclear receptors. Similar dendrimer conjugates have been successfully used as drug delivery device in animal models and the oestrogen complex was shown to provide cardiovascular protection in high cholesterol ovariectomized female mice without stimulating growth of breast or uterine cancer. The team believe that such oestrogen-macromolecule complexes could provide cardiovascular protection for both men and women and are creating molecules that may be suitable for use in humans.
Cell culture experiments to screen for compounds that can inhibit cell migration – and potentially metastasis of cancer cells – are typically carried out in a 2-D environment, but researchers at Johns Hopkins University and the University of Washington suggest that results from such experiments may be, at best, misleading. The team has shown that the way in which cells move in a 3-D environment, such as the human body, is different both qualitatively and quantitatively from the way they move in a 2-D environment such as a culture dish. When cells are grown in 2-D, they develop broad fan-shaped protrusions called lamella along their leading edge which help them to move forward. Macromolecular assemblies known as focal adhesions which can last for up to several minutes are also formed. These focal adhesions mediate cell signalling, force transduction and adhesion. In 3-D, the cells take on a more spindle-like appearance, with two pointed protrusions at opposite ends and focal adhesions – if they form at all – are so small and short-lived that they cannot be resolved by microscopy. The authors suggest that the shape and movement of cells in 2-D culture experiments are artifacts of the environment and could produce misleading results in studies to test the effects of drugs on cell motility. This may explain why positive results from cell culture experiments do not always translate into efficacy in animal models.
Even in cell culture systems designed to more closely mimic a 3-D environment, the cells may be only partially embedded in a matrix and produce misleading results. Using live-cell microscopy, the team showed that, when cells are fully embedded in a 3-D matrix, focal adhesion proteins do not form aggregates, but are distributed throughout the cytoplasm. The focal adhesion proteins still modulate cell motility, but not in the same way as in a 2-D environment. Because loss of adhesion and increased motility are hallmarks of cancer cells, it is important to understand cell motility under physiological conditions and to use culture techniques that most closely mimic this.
Image: Flickr - fbobolas ApoE is a lipid transport protein with roles in transport of dietary lipids, regulation of plasma cholesterol, and protection from atherosclerosis. In humans, there are three variants of ApoE (ApoE2, ApoE3 and ApoE4) and one of these, ApoE4, has been linked to earlier onset of Alzheimer’s disease. The mechanisms underlying the increased risk of Alzheimer’s disease remain unclear but researchers at UT Southwestern Medical Center have now shown that the ApoE4 variant reduces surface expression of receptors involved in synaptic plasticity by sequestering the receptors inside the cell.
ApoE interacts with members of the LDL receptor family and one of the receptors for ApoE, Apoer2, also acts as a signalling receptor for reelin, a protein that is important in the developing brain but also enhances NMDA receptor activity and increases long-term potentiation (LTP) in the adult brain. ApoE4 was found to reduce surface expression of NMDA and AMPA receptors as well as Apoer2 receptors, thereby impairing glutamatergic neurotransmission. β-Amyloid peptide, a hallmark of Alzheimer’s disease, suppresses LTP and the ability of reelin to counter the effects of β-amyloid peptide was almost completely abolished in mice expressing human ApoE4. The team are now trying to understand whether it is possible to build on their findings to develop new treatments for Alzheimer’s disease.
Image: Flickr - zaxl4 Regular use of NSAIDS has been linked to reduced incidence of certain types of cancer but the underlying protective mechanisms are unclear. Some of the anticancer effects are believed to be mediated through inhibition of COX-2, but a study led by investigators at Sanford-Burnham Medical Research Institute has now identified another mechanism by which the sulindac sulfide (the NSAID metabolite of sulindac) inhibits tumour growth. The team found that sulindac sulfide induces apoptosis by binding to retinoid X receptor-α (RXRα), a member of the nuclear hormone receptor family which had been already been identified as a potential target for cancer therapy. In cancer cells, levels of RXRα are often reduced, at least in part because of proteolytic processing to a truncated form, tRXRα. As with other nuclear receptors, RXRα regulates transcription of target genes by binding to DNA response elements but accumulating evidence suggests that RXRα may also have extranuclear activity. Both RXRα and tRXRα can exist in the cytoplasm and the study showed that cytoplasmic tRXRα can activate the PI3K/AKT survival pathway by interaction with the p85a subunit of PI3K, leading to anchorage-independent cell growth in vitro, and tumour growth in animals. Sulindac sulfide was found to inhibit the tRXRα-mediated PI3K/AKT activation, suggesting that the compound could provide a useful lead for anti-cancer drugs targeting this pathway.
The use of NSAIDs to reduce the incidence of cancer has been limited by the risk of major cardiovascular events and the Sanford-Burnham have identified an analogue of sulindac sulfide, K-80003 which has improved affinity for RXRα but lacks significant COX-2 inhibitory activity. K-80003 inhibited the growth of cancer cells in vitro and in animals and would be expected to have reduced COX-2-associated side effects.
The study is published in the journal Cancer Cell.
Image: Flickr - didier.bier Multiple sclerosis (MS) is an autoimmune disorder in which T-cells attack and damage the fatty myelin sheaths around the axons of the brain and spinal cord, disrupting the conduction of electrical signals along the nerve fibres. Although both genetic factors and viral infections have been suggested to contribute to the development of MS, no single virus has been conclusively linked to the disease and other mechanisms could also play a role. Animal models induced by CD8+ T-cells show similarities to human MS, and researchers at the University of Washington investigating the causes of MS have engineered mice that over-express CD8+ T-cells that recognise myelin basic protein (MBP), a candidate autoantigen in MS.
When infected with vaccinia virus engineered to produce MBP, the infection should activate the CD8+ T-cells to attack virally infected cells and also other cells that produce MBP. As expected, mice infected with the engineered virus developed MS-like disease but, surprisingly, symptoms were also triggered by infection with wild-type virus. This suggested that the engineered CD8+ T cells expressed a second receptor that recognised wild-type virus and subsequent cross-breeding experiments confirmed that some of the CD8+ T cells did indeed have receptors for both MBP and wild-type virus. Once activated by the virus, the dual-receptor CD8+ T cells were than able to attack cells producing MBP.
The study suggests a role for dual-receptor cells in autoimmune diseases and could explain how infection with a common virus triggers MS in genetically predisposed people, whilst having no lasting effects in most of the population. In the ‘dual-receptor model’, autoimmune activation could be triggered by a chance event leading to T-cells that recognise both MBP and a viral antigen. The prevalence of dual-receptor T cells is presently unclear and the team plan to assess whether they are more common in MS patients.
Image: Flickr - bibendum84 New research has shown that compounds that affect cellular energy status could also be used to treat hepatitis C virus (HCV) infections. Metformin, which is used to treat type II diabetes, and 5-amino-1-β-D-ribofuranosyl-1H-imidazole-4-carboxamide (AICAR), which has been shown to mimic the beneficial effects of exercise in mice, stimulate AMP-activated protein kinase (AMPK). AMPK is a key sensor of cellular energy status and regulates both lipid and glucose metabolism to maintain cellular energy balance and protect against metabolic stress. An increase in the AMP/ATP ratio initiates an AMPK-mediated switch from activities that consume ATP, such as lipid production, to activities that produce ATP, such as lipid and glucose oxidation.
Infection with viruses might be expected to activate AMPK because of the energy demands put on the cell by viral replication, but research led by scientists at the University of Leeds has shown instead that HCV switches off AMPK so that the cell continues to produce the lipids needed to provide new viral particles with a protective outer coat. When the team treated HCV-infected cells with metformin or AICAR, AMPK activity was restored and viral replication was inhibited.
The team plan to carry out a small scale clinical trial to investigate the effects of AMPK activators in HCV infection and hope that such drugs may provide much-needed new treatments for HCV.
Image: Adapted from Flickr - Horia Varlan Cholesterol is essential for all animal life but high levels of cholesterol – when associated with low density lipoprotein (LDL) – are linked to an increased risk of atherosclerosis, heart disease and stroke. Circulating cholesterol can also be transported by high density lipoprotein (HDL); HDL is believed to be able to remove cholesterol from atheroma within arteries and cholesterol associated with HDL is considered to be beneficial for cardiovascular health. Cholesterol levels are determined by dietary intake, de novo synthesis and secretion by the liver: cholesterol absorption blockers and HMG-CoA reductase inhibitors (statins), which block cholesterol synthesis, are used clinically to reduce cholesterol levels.
A study led by researchers at the University of Cincinnati has now identified a new potential target for the control of cholesterol. The study, carried out in mice, found that circulation of cholesterol is regulated in the brain by the ‘hunger hormone’, ghrelin, which inhibits the melanocortin 4 receptor (MC4R) in the hypothalamus and is important for the regulation of food intake and energy expenditure. Increased levels of ghrelin were associated with increased levels of circulating HDL cholesterol, which the authors attribute to a reduction in the uptake of cholesterol by the liver. Genetically deleting or chemically blocking MCR4 in the CNS also led to increased levels of HDL cholesterol, suggesting that MCR4 is key to central control of cholesterol.
More studies are need to determine whether the effects observed in mice can be applied to humans but the finding that a neural circuit may be directly involved in the control of cholesterol metabolism by the liver could provide a target for new treatments to control cholesterol.
Image: Flickr - Alaskan Dude The cytoskeleton plays a key role in regulating many cellular functions; it maintains cell shape, protects the cell, enables cellular motion, and has important roles in proliferation and differentiation. Metastasising cancer cells exploit the cytoskeleton to produce protrusions that allow them to invade surrounding tissue and enter the blood system from where they can spread to distant tissues and seed new tumours.
The protrusions, known as pseudopodia, are highly specialised ‘feet’ that the cell uses to pull itself forward across the underlying surface. A team led by researchers at the University of California, San Diego has now identified a previously unknown kinase – termed pseudopodium-enriched atypical kinase one or PEAK1 – that regulates the cytoskeleton and plays a central role in the formation of pseudopodia. Preliminary studies in mice suggest that PEAK1 is important during tumour growth and the team also showed that PEAK1 levels are increased in primary and metastatic samples from human colon cancer patients. Whether PEAK1 is capable of transforming non-tumour cells into cancer cells has not yet been determined but the fact that PEAK1 has kinase activity suggests that it may be possible to design specific inhibitors which could help to elucidate its role in both normal and cancer cells. PEAK1, which is a 190-kDa non-receptor tyrosine kinase, could serve as a clinical biomarker that predicts whether a cancer is likely to metastasise and could also be a target for future cancer treatments.
Although the recent sporadic outbreaks of influenza A virus H5N1 and of a new variant of H1N1 in 2009 were less serious than initially feared, public health responses gave an indication of the potential for pandemic influenza A to wreak havoc amongst human populations. Timely development of vaccines should help to contain future outbreaks, but effective antiviral medicines will also be needed. Circulating strains of influenza A virus with resistance to existing neuraminidase inhibitors have already been discovered, and new molecular targets would provide additional protection in the event of a fresh outbreak.
Image: Wikimedia Commons - Armondo
Nucleozin Researchers led by a team at the University of Hong Kong have now identified a compound, nucleozin, which can aggregate the viral nucleoprotein and prevent its transport into the nucleus. The nucleoprotein plays critical roles in viral RNA replication and genome assembly, and nucleozin was shown to block replication of H1N1, H3N2, and H5N1 viruses in cell culture experiments and also to protect mice from lethal challenge with highly pathogenic avian influenza virus A H5N1.
The study, which is published in Nature Biotechnology, shows that the nucleoprotein is a viable drug target and could lead to the development of new treatments to control the impact of future influenza A outbreaks. Potential binding sites for nucleozin on the influenza nucleoprotein were also predicted using molecular docking models.
