Michelle E Turvey

An immune paradox: how can the same chemokine axis regulate both immune tolerance and activation?: CCR6/CCL20: a chemokine axis balancing immunological tolerance and inflammation in autoimmune disease.

Chemokines (chemotactic cytokines) drive and direct leukocyte traffic. New evidence suggests that the unusual CCR6/CCL20 chemokine receptor/ligand axis provides key homing signals for recently identified cells of the adaptive immune system, recruiting both pro-inflammatory and suppressive T cell subsets. Thus CCR6 and CCL20 have been recently implicated in various human pathologies, particularly in autoimmune disease. These studies have revealed that targeting CCR6/CCL20 can enhance or inhibit autoimmune disease depending on the cellular basis of pathogenesis and the cell subtype most affected through different CCR6/CCL20 manipulations. Here, we discuss the significance of this chemokine receptor/ligand axis in immune and inflammatory functions, consider the potential for targeting CCR6/CCL20 in human autoimmunity and propose that the shared evolutionary origins of pro-inflammatory and regulatory T cells may contribute to the reason why both immune activation and regulation might be controlled through the same chemokine pathway.

Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy

IntroductionPodocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression.MethodsWe silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice.ResultsAlthough deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis.ConclusionsWe show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.

Identification of beer spoilage microorganisms using the MALDI Biotyper platform

Beer spoilage microorganisms present a major risk for the brewing industry and can lead to cost-intensive recall of contaminated products and damage to brand reputation. The applicability of molecular profiling using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) in combination with Biotyper software was investigated for the identification of beer spoilage microorganisms from routine brewery quality control samples. Reference mass spectrum profiles for three of the most common bacterial beer spoilage microorganisms (Lactobacillus lindneri, Lactobacillus brevis and Pediococcus damnosus), four commercially available brewing yeast strains (top- and bottom-fermenting) and Dekkera/Brettanomyces bruxellensis wild yeast were established, incorporated into the Biotyper reference library and validated by successful identification after inoculation into beer. Each bacterial species could be accurately identified and distinguished from one another and from over 5600 other microorganisms present in the Biotyper database. In addition, wild yeast contaminations were rapidly detected and distinguished from top- and bottom-fermenting brewing strains. The applicability and integration of mass spectrometry profiling using the Biotyper platform into existing brewery quality assurance practices within industry were assessed by analysing routine microbiology control samples from a local brewery, where contaminating microorganisms could be reliably identified. Brewery-isolated microorganisms not present in the Biotyper database were further analysed for identification using LC-MS/MS methods. This renders the Biotyper platform a promising candidate for biological quality control testing within the brewing industry as a more rapid, high-throughput and cost-effective technology that can be tailored for the detection of brewery-specific spoilage organisms from the local environment.

Dynamic Self-Referencing Approach to Whispering Gallery Mode Biosensing and Its Application to Measurement within Undiluted Serum

Biosensing within complex biological samples requires a sensor that can compensate for fluctuations in the signal due to changing environmental conditions and nonspecific binding events. To achieve this, we developed a novel self-referenced biosensor consisting of two almost identically sized dye-doped polystyrene microspheres placed on adjacent holes at the tip of a microstructured optical fiber (MOF). Here self-referenced biosensing is demonstrated with the detection of Neutravidin in undiluted, immunoglobulin-deprived human serum samples. The MOF allows remote excitation and collection of the whispering gallery modes (WGMs) of the microspheres while also providing a robust and easy to manipulate dip-sensing platform. By taking advantage of surface functionalization techniques, one microsphere acts as a dynamic reference, compensating for nonspecific binding events and changes in the environment (such as refractive index and temperature), while the other microsphere is functionalized to detect a specific interaction. The almost identical size allows the two spheres to have virtually identical refractive index sensitivity and surface area, while still having discernible WGM spectra. This ensures their responses to nonspecific binding and environmental changes are almost identical, whereby any specific changes, such as binding events, can be monitored via the relative movement between the two sets of WGM peaks.

Podocalyxin in the diagnosis and treatment of cancer

Kelly M. McNagny, Michael R. Hughes, Marcia L. Graves, Erin J. DeBruin, Kimberly Snyder, Jane Cipollone, Michelle Turvey, Poh C. Tan, Shaun McColl and Calvin D. Roskelley

Quantitative proteome profiling of CNS-infiltrating autoreactive CD4+ cells reveals selective changes during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a murine model of multiple sclerosis, a chronic neurodegenerative and inflammatory autoimmune condition of the central nervous system (CNS). Pathology is driven by the infiltration of autoreactive CD4(+) lymphocytes into the CNS, where they attack neuronal sheaths causing ascending paralysis. We used an isotope-coded protein labeling approach to investigate the proteome of CD4(+) cells isolated from the spinal cord and brain of mice at various stages of EAE progression in two EAE disease models: PLP139-151-induced relapsing-remitting EAE and MOG35-55-induced chronic EAE, which emulate the two forms of human multiple sclerosis. A total of 1120 proteins were quantified across disease onset, peak-disease, and remission phases of disease, and of these 13 up-regulated proteins of interest were identified with functions relating to the regulation of inflammation, leukocyte adhesion and migration, tissue repair, and the regulation of transcription/translation. Proteins implicated in processes such as inflammation (S100A4 and S100A9) and tissue repair (annexin A1), which represent key events during EAE progression, were validated by quantitative PCR. This is the first targeted analysis of autoreactive cells purified from the CNS during EAE, highlighting fundamental CD4(+) cell-driven processes that occur during the initiation of relapse and remission stages of disease.

The atypical chemokine receptor CCX-CKR regulates metastasis of mammary carcinoma via an effect on EMT

Over the last decade, the significance of the homeostatic CC chemokine receptor‐7 and its ligands CC chemokine ligand‐19 (CCL19) and CCL21, in various types of cancer, particularly mammary carcinoma, has been highlighted. The chemokine receptor CCX‐CKR is a high‐affinity receptor for these chemokine ligands but rather than inducing classical downstream signalling events promoting migration, it instead sequesters and targets its ligands for degradation, and appears to function as a regulator of the bioavailability of these chemokines in vivo. Therefore, in this study, we tested the hypothesis that local regulation of chemokine levels by CCX‐CKR expressed on tumours alters tumour growth and metastasis in vivo. Expression of CCX‐CKR on 4T1.2 mouse mammary carcinoma cells inhibited orthotopic tumour growth. However, this effect could not be correlated with chemokine scavenging in vivo and was not mediated by host adaptive immunity. Conversely, expression of CCX‐CKR on 4T1.2 cells resulted in enhanced spontaneous metastasis and haematogenous metastasis in vivo. In vitro characterisation of the tumourigenicity of CCX‐CKR‐expressing 4T1.2 cells suggested accelerated epithelial–mesenchymal transition (EMT) revealed by their more invasive and motile character, lower adherence to the extracellular matrix and to each other, and greater resistance to anoikis. Further analysis of CCX‐CKR‐expressing 4T1.2 cells also revealed that transforming growth factor (TGF)‐β1 expression was increased both at mRNA and protein levels leading to enhanced autocrine phosphorylation of Smad 2/3 in these cells. Together, our data show a novel function for the chemokine receptor CCX‐CKR as a regulator of TGF‐β1 expression and the EMT in breast cancer cells.

p84 forms a negative regulatory complex with p110γ to control PI3Kγ signalling during cell migration

Phosphoinositide 3‐kinase γ (PI3Kγ) consists of the catalytic subunit p110γ that forms a mutually exclusive heterodimer with one of the two adaptor subunits, p101 or p84. Although activation of PI3Kγ is necessary for cell migration downstream of G‐protein‐coupled receptor engagement, particularly within the immune system, aberrant PI3Kγ signalling has been associated with transformation, increased migration and the progression of multiple cancer types. Regulation of PI3Kγ signal activation and duration is critical to controlling and maintaining coordinated cellular migration; however, the mechanistic basis for this is not well understood. We have recently demonstrated that, in contrast to the tumour‐promoting potential of p110γ and p101, p84 possesses tumour‐suppressor activity, suggesting a negative regulatory role within PI3Kγ signalling. The present study investigated the role of p84 phosphorylation in the context of PI3Kγ signalling, cell migration and p84‐mediated tumour suppression. Two putative phosphorylation sites were characterised within p84, Ser358 and Thr607. Expression of wild‐type p84 reduced the oncogenic potential of MDA.MB.231 cells and inhibited metastatic lung colonisation in vivo, effects that were dependent on Thr607. Furthermore, loss of Thr607 enhanced migration of MDA.MB.231 cells in vitro and prevented the induction of p84/p110γ dimers. The dimerisation of wild‐type p84 with p110γ was not detected at the plasma membrane, indicating an inhibitory interaction preventing PI3Kγ lipid‐kinase activity. In contrast, Ser358 phosphorylation was not determined to be critical for p84 activity in the context of migration. Our findings suggest that p84 binding to p110γ may represent a novel negative feedback signal that terminates PI3Kγ activity.

Synthetic Charge-Invertible Polymer for Rapid and Complete Implantation of Layer-by-Layer Microneedle Drug Films for Enhanced Transdermal Vaccination

The utility of layer-by-layer (LbL) coated microneedle (MN) skin patches for transdermal drug delivery has proven to be a promising approach, with advantages over hypodermal injection due to painless and easy self-administration. However, the long epidermal application time required for drug implantation by existing LbL MN strategies (15-90 min) can lead to potential medication noncompliance. Here, we developed a MN platform to shorten the application time in MN therapies based on a synthetic pH-induced charge-invertible polymer poly(2-(diisopropylamino) ethyl methacrylate- b-methacrylic acid) (PDM), requiring only 1 min skin insertion time to implant LbL films in vivo. Following MN-mediated delivery of 0.5 μg model antigen chicken ovalbumin (OVA) in the skin of mice, this system achieved sustained release over 3 days and led to an elevated immune response as demonstrated by significantly higher humoral immunity compared with OVA administration via conventional routes (subcutaneously and intramuscularly). Moreover, in an ex vivo experiment on human skin, we achieved efficient immune activation through MN-delivered LbL films, demonstrated by a rapid uptake of vaccine adjuvants by the antigen presenting cells. These features, rapid administration and the ability to elicit a robust immune response, can potentially enable a broad application of microneedle-based vaccination technologies.

Using whispering gallery mode micro lasers for biosensing within undiluted serum

Although whispering gallery mode (WGM) biosensors have shown tremendous potential, they are still yet to find practical use as biomedical diagnostic tools. This is primarily due to the nature of the interrogation mechanism itself which relies on indirect measurement of the binding of a specific biomolecule onto the sensor through the associated refractive index change. Since nonspecific binding cannot be differentiated from the specific interaction of interest, this can result in a high rate of false positive readings when the detection is performed in complex biological samples. Here we show that this inherent limitation can be solved using a relatively simple approach. This approach involves the development of a self-referenced biosensor consisting of two almost identically sized dye-doped polystyrene microspheres placed on adjacent holes at the tip of a suspended core optical fiber. Here self-referenced biosensing is demonstrated with the detection of Neutravidin in undiluted human serum samples. The fiber allows remote excitation and collection of the WGMs of the microspheres in a dip sensing setting. By taking advantage of surface functionalization techniques, one microsphere acts as a dynamic reference, compensating for nonspecific binding events, while the other microsphere is functionalized to detect the specific interaction. The almost identical size allows the two spheres to have virtually identical refractive index sensitivity and surface area. This ensures their responses to nonspecific binding and environmental changes are almost identical, whereby any specific changes such as binding events, can be monitored via the relative movement between the two sets of WGM peaks.