CD151 is associated with prostate cancer cell invasion and lymphangiogenesis in vivo

CD151, a member of the tetraspanin family, is associated with regulation of migration of normal and tumour cells via cell surface microdomain formation. CD151 was found in our laboratory to have a prognostic value in prostate cancer and is a promoter of prostate cancer migration and invasion. These roles involve association with integrins on both cell-cell and cell-stroma levels. Furthermore, CD151 plays a role in endothelial cell motility. CD151 expression was examined in three commonly used prostate cancer cell lines. We investigated CD151 expression, angiogenesis (microvessel density; MVD) and lymphangiogenesis (lymphatic vessel density; LVD) in an orthotopic xenograft model of prostate cancer in matched tumours from primary and secondary sites. CD151 was found to be heterogeneously expressed across different prostate cancer cell lines and the levels of CD151 expression were significantly higher in the highly tumorigenic, androgen-insensitive cells PC-3 and DU-145 compared to the androgen-sensitive cell line LNCaP (P<0.05). The majority of in vivo xenografts developed pelvic lymph node metastases. Importantly, primary tumours that developed metastasis had significantly higher CD151 expression and MVD compared to those which did not develop metastasis (P<0.05). We identified, for the first time, that CD151 expression is associated with LVD in prostate cancer. These findings underscore the potential role of CD151 and angiogenesis in the metastatic potential of prostate cancer. CD151 has a prognostic value in this mouse model of prostate cancer and may play a role in lymphangiogenesis. CD151 is likely an important regulator of cancer cell communication with the surrounding microenvironment.

The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell-cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs.

Modeling the cumulative genetic risk for multiple sclerosis from genome-wide association data

Background: Multiple sclerosis (MS) is the most common cause of chronic neurologic disability beginning in early to middle adult life. Results from recent genome-wide association studies (GWAS) have substantially lengthened the list of disease loci and provide convincing evidence supporting a multifactorial and polygenic model of inheritance. Nevertheless, the knowledge of MS genetics remains incomplete, with many risk alleles still to be revealed. Methods: We used a discovery GWAS dataset (8,844 samples, 2,124 cases and 6,720 controls) and a multi-step logistic regression protocol to identify novel genetic associations. The emerging genetic profile included 350 independent markers and was used to calculate and estimate the cumulative genetic risk in an independent validation dataset (3,606 samples). Analysis of covariance (ANCOVA) was implemented to compare clinical characteristics of individuals with various degrees of genetic risk. Gene ontology and pathway enrichment analysis was done using the DAVID functional annotation tool, the GO Tree Machine, and the Pathway-Express profiling tool. Results: In the discovery dataset, the median cumulative genetic risk (P-Hat) was 0.903 and 0.007 in the case and control groups, respectively, together with 79.9% classification sensitivity and 95.8% specificity. The identified profile shows a significant enrichment of genes involved in the immune response, cell adhesion, cell communication/ signaling, nervous system development, and neuronal signaling, including ionotropic glutamate receptors, which have been implicated in the pathological mechanism driving neurodegeneration. In the validation dataset, the median cumulative genetic risk was 0.59 and 0.32 in the case and control groups, respectively, with classification sensitivity 62.3% and specificity 75.9%. No differences in disease progression or T2-lesion volumes were observed among four levels of predicted genetic risk groups (high, medium, low, misclassified). On the other hand, a significant difference (F = 2.75, P = 0.04) was detected for age of disease onset between the affected misclassified as controls (mean = 36 years) and the other three groups (high, 33.5 years; medium, 33.4 years; low, 33.1 years). Conclusions: The results are consistent with the polygenic model of inheritance. The cumulative genetic risk established using currently available genome-wide association data provides important insights into disease heterogeneity and completeness of current knowledge in MS genetics.

Exosomes in prostate cancer: Putting together the pieces of a puzzle

Exosomes have been shown to act as mediators for cell to cell communication and as a potential source of biomarkers for many diseases, including prostate cancer. Exosomes are nanosized vesicles secreted by cells and consist of proteins normally found in multivesicular bodies, RNA, DNA and lipids. As a potential source of biomarkers, exosomes have attracted considerable attention, as their protein content resembles that of their cells of origin, even though it is noted that the proteins, miRNAs and lipids found in the exosomes are not a reflective stoichiometric sampling of the contents from the parent cells. While the biogenesis of exosomes in dendritic cells and platelets has been extensively characterized, much less is known about the biogenesis of exosomes in cancer cells. An understanding of the processes involved in prostate cancer will help to further elucidate the role of exosomes and other extracellular vesicles in prostate cancer progression and metastasis. There are few methodologies available for general isolation of exosomes, however validation of those methodologies is necessary to study the role of exosomal-derived biomarkers in various diseases. In this review, we discuss “exosomes” as a member of the family of extracellular vesicles and their potential to provide candidate biomarkers for prostate cancer.

Secretome and degradome profiling shows that Kallikrein-related peptidases 4, 5, 6, and 7 induce TGFβ-1 signaling in ovarian cancer cells

Kallikrein-related peptidases, in particular KLK4, 5, 6 and 7 (4-7), often have elevated expression levels in ovarian cancer. In OV-MZ-6 ovarian cancer cells, combined expression of KLK4-7 reduces cell adhesion and increases cell invasion and resistance to paclitaxel. The present work investigates how KLK4-7 shape the secreted proteome ("secretome") and proteolytic profile ("degradome") of ovarian cancer cells. The secretome comparison consistently identified >900 proteins in three replicate analyses. Expression of KLK4-7 predominantly affected the abundance of proteins involved in cell-cell communication. Among others, this includes increased levels of transforming growth factor β-1 (TGFβ-1). KLK4-7 co-transfected OV-MZ-6 cells share prominent features of elevated TGFβ-1 signaling, including increased abundance of neural cell adhesion molecule L1 (L1CAM). Augmented levels of TGFβ-1 and L1CAM upon expression of KLK4-7 were corroborated in vivo by an ovarian cancer xenograft model. The degradomic analysis showed that KLK4-7 expression mostly affected cleavage sites C-terminal to arginine, corresponding to the preference of kallikreins 4, 5 and 6. Putative kallikrein substrates include chemokines, such as growth differentiation factor 15 (GDF 15) and macrophage migration inhibitory factor (MIF). Proteolytic maturation of TGFβ-1 was also elevated. KLK4-7 have a pronounced, yet non-degrading impact on the secreted proteome, with a strong association between these proteases and TGFβ-1 signaling in tumor biology. © 2013 Federation of European Biochemical Societies.

A proteomic view into infection of greyback canegrubs (Dermolepida albohirtum) by Metarhizium anisopliae

Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.

Menstrual effluent induces epithelial-mesenchymal transitions in mesothelial cells

Background Menstrual effluent affects mesothelial cell (MC) morphology. We evaluated whether these changes were consistent with epithelial-mesenchymal transitions (EMT). Methods Monolayer cultures of MC were incubated overnight in conditioned media, prepared from cells isolated form menstrual effluent, with or without kinase and ATP inhibitors. Changes in cell morphology were monitored using time-lapse video microscopy and immunohistochemistry. Effects on the expression of EMT-associated molecules were evaluated using real-time RT-PCR and/or Western blot analysis. Results Incubation in conditioned media disrupted cell-cell contacts, and increased MC motility. The changes were reversible. During the changes the distribution of cytokeratins, fibrillar actin and α-tubulin changed. Sodium azide, an inhibitor of ATP production, and Genistein, a general tyrosine kinase inhibitor, antagonized these effects. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and SU6656, an Src tyrosine kinase inhibitor, only partially antagonized the effect. The expression of Snail and vimentin was markedly up-regulated, whereas the expression of E-cadherin was decreased and cytokeratins were altered. Conclusions In MC, menstrual effluent initiates a reversible, energy-dependent transition process from an epithelial to a mesenchymal phenotype. Involvement of the (Src) tyrosine kinase signalling pathway and the changes in the expression of cytokeratins, Snail, vimentin and E-cadherin demonstrate that the morphological changes are EMT.

A combination of local inflammation and central memory T cells potentiates immunotherapy in the skin

Adoptive T cell therapy uses the specificity of the adaptive immune system to target cancer and virally infected cells. Yet the mechanism and means by which to enhance T cell function are incompletely described, especially in the skin. In this study, we use a murine model of immunotherapy to optimize cell-mediated immunity in the skin. We show that in vitro - derived central but not effector memory-like T cells bring about rapid regression of skin-expressing cognate Ag as a transgene in keratinocytes. Local inflammation induced by the TLR7 receptor agonist imiquimod subtly yet reproducibly decreases time to skin graft rejection elicited by central but not effector memory T cells in an immunodeficient mouse model. Local CCL4, a chemokine liberated by TLR7 agonism, similarly enhances central memory T cell function. In this model, IL-2 facilitates the development in vivo of effector function from central memory but not effector memory T cells. In a model of T cell tolerogenesis, we further show that adoptively transferred central but not effector memory T cells can give rise to successful cutaneous immunity, which is dependent on a local inflammatory cue in the target tissue at the time of adoptive T cell transfer. Thus, adoptive T cell therapy efficacy can be enhanced if CD8+ T cells with a central memory T cell phenotype are transferred, and IL-2 is present with contemporaneous local inflammation. Copyright © 2012 by The American Association of Immunologists, Inc.

Altered cell interactions of subchondral bone osteoblasts and articular chondrocytes in osteoarthritis is through the mediation of ERK1/2 phosphorylation

Introduction: Osteoarthritis (OA) is the most common musculoskeletal disorder and represents a major health burden to society. In the course of the pathological development of OA, articular cartilage chondrocytes (ACCs) undergo a typical phenotype changes characterized by the expression of hypertrophic differentiation markers. Also, the adjacent subchondral bone shows signs of abnormal mineral density and enhanced production of bone turnover markers, indicative of osteoblast dysfunction. However, the mechanism(s) by which these changes occur during the OA development are not completely understood. Materials and Methods: ACCs and subchondral bone osteoblasts (SBOs) were harvested from OA and healthy patients for the cross-talk studies between normal and OA ACCs and SBOs. The involvement of mitogen activated protein kinase (MAPK) signalling pathway during the cell-cell interactions was analysed by zymography, ELISA and western blotting methods. Results: The direct and in-direct co-culture studies showed that OA (ACCs and SBOs) cells induced osteoarthritic changes of normal (ACC and SBOs) cells. This altered cell interaction induced by OA cells significantly aggravated the proteolytic activity, which resulted cartilage degeneration. The altered cell interaction appeared to significantly activate ERK 1/2 phosphorylation and inhibition of MAPK-ERK 1/2 pathway reversed the osteoarthrtitic phenotypic changes. Discussion and Conclusion: Our study has demonstrated that the altered bi-directional communication of SBOs and ACCs are critical for initiation and progression of OA related changes and that this process is mediated by MAPK signalling pathways. Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA related disorders.

Stochastic dynamics of interacting haematopoietic stem cell niche lineages

Since we still know very little about stem cells in their natural environment, it is useful to explore their dynamics through modelling and simulation, as well as experimentally. Most models of stem cell systems are based on deterministic differential equations that ignore the natural heterogeneity of stem cell populations. This is not appropriate at the level of individual cells and niches, when randomness is more likely to affect dynamics. In this paper, we introduce a fast stochastic method for simulating a metapopulation of stem cell niche lineages, that is, many sub-populations that together form a heterogeneous metapopulation, over time. By selecting the common limiting timestep, our method ensures that the entire metapopulation is simulated synchronously. This is important, as it allows us to introduce interactions between separate niche lineages, which would otherwise be impossible. We expand our method to enable the coupling of many lineages into niche groups, where differentiated cells are pooled within each niche group. Using this method, we explore the dynamics of the haematopoietic system from a demand control system perspective. We find that coupling together niche lineages allows the organism to regulate blood cell numbers as closely as possible to the homeostatic optimum. Furthermore, coupled lineages respond better than uncoupled ones to random perturbations, here the loss of some myeloid cells. This could imply that it is advantageous for an organism to connect together its niche lineages into groups. Our results suggest that a potential fruitful empirical direction will be to understand how stem cell descendants communicate with the niche and how cancer may arise as a result of a failure of such communication.

Effective Cell-Centred Time-Domain Maxwell's Equations Numerical Solvers

This research work analyses techniques for implementing a cell-centred finite-volume time-domain (ccFV-TD) computational methodology for the purpose of studying microwave heating. Various state-of-the-art spatial and temporal discretisation methods employed to solve Maxwell's equations on multidimensional structured grid networks are investigated, and the dispersive and dissipative errors inherent in those techniques examined. Both staggered and unstaggered grid approaches are considered. Upwind schemes using a Riemann solver and intensity vector splitting are studied and evaluated. Staggered and unstaggered Leapfrog and Runge-Kutta time integration methods are analysed in terms of phase and amplitude error to identify which method is the most accurate and efficient for simulating microwave heating processes. The implementation and migration of typical electromagnetic boundary conditions. from staggered in space to cell-centred approaches also is deliberated. In particular, an existing perfectly matched layer absorbing boundary methodology is adapted to formulate a new cell-centred boundary implementation for the ccFV-TD solvers. Finally for microwave heating purposes, a comparison of analytical and numerical results for standard case studies in rectangular waveguides allows the accuracy of the developed methods to be assessed.