Translocation Of Cells Research Articles

Allicin protects against cisplatin-induced vestibular dysfunction by inhibiting the apoptotic pathway

Cisplatin is an anticancer drug that causes the impairment of inner ear function as side effects, including hearing loss and balance dysfunction. The purpose of this study was to investigate the effects of allicin against cisplatin-induced vestibular dysfunction in mice and to make clear the mechanism underlying the protective effects of allicin on oto-vestibulotoxicity. Mice intraperitoneally injected with cisplatin exhibited vestibular dysfunction in swimming test, which agreed with impairment in vestibule. However, these impairments were significantly prevented by pre-treatment with allicin. Allicin markedly reduced cisplatin-activated expression of cleaved-caspase-3 in hair cells and vascular layer cells of utricule, saccule and ampulla, but also decreased AIF nuclear translocation of hair cells in utricule, saccule and ampulla. These results showed that allicin played an effective role in protecting vestibular dysfunction induced by cisplatin via inhibiting caspase-dependent and caspase-independent apoptotic pathways. Therefore, allicin may be useful in preventing oto-vestibulotoxicity mediated by cisplatin.

Folded gastrulation and T48 drive the evolution of coordinated mesoderm internalization in flies

Gastrulation constitutes a fundamental yet diverse morphogenetic process of metazoan development. Modes of gastrulation range from stochastic translocation of individual cells to coordinated infolding of an epithelial sheet. How such morphogenetic differences are genetically encoded and whether they have provided specific developmental advantages is unclear. Here we identify two genes, folded gastrulation and t48, which in the evolution of fly gastrulation acted as a likely switch from an ingression of individual cells to the invagination of the blastoderm epithelium. Both genes are expressed and required for mesoderm invagination in the fruit fly Drosophila melanogaster but do not appear during mesoderm ingression of the midge Chironomus riparius. We demonstrate that early expression of either or both of these genes in C.riparius is sufficient to invoke mesoderm invagination similar to D.melanogaster. The possible genetic simplicity and a measurable increase in developmental robustness might explain repeated evolution of similar transitions in animal gastrulation.

Immunosuppression, peripheral inflammation and invasive infection from endogenous gut microbiota activate retinal microglia in mouse models.

Although its actual role in the progression of degenerative processes is not fully known, the persistent activated state of retinal microglia and the concurrent secretion of inflammatory mediators may contribute to neuronal death and permanent vision loss. Our objective was to determine whether non-ocular conditions (immunosuppression and peripheral inflammation) could lead to activation of retinal microglia. Mouse models of immunosuppression induced by cyclophosphamide and/or peripheral inflammation by chemically induced sublethal colitis in C57BL/6J mice were used. Retinal microglia morphology, spatial distribution and complexity, as well as MHCII and CD11b expression levels were determined by flow cytometry and confocal immunofluorescence analysis with anti-CD11b, anti-IBA1 and anti-MHCIIRT1B antibodies. Retinas of mice with double treatment showed changes in microglial morphology, spatial distribution and expression levels of CD11b and MHCII. These effects were higher than those observed with any treatment separately. In addition, we also observed in these mice: (i) translocation of endogenous bacteria from gut to liver, and (ii) upregulation of TLR2 expression in retinal microglia. Using a mouse model of immunosuppression and gut colonization by Candida albicans, translocation of fungal cells was confirmed to occur in wild type and, to a higher extent, in TLR2 KO mice, which are more susceptible to fungal invasion; interestingly microglial changes were also higher in TLR2 KO mice. Hence, non-ocular injuries (immunosuppression, peripheral inflammation and invasive infection from endogenous gut microbiota) can activate retinal microglia and therefore could affect the progression of neurodegenerative disorders and should be taken into account to improve therapeutic options.

Early development of the root-knot nematode Meloidogyne incognita.

BackgroundDetailed descriptions of the early development of parasitic nematodes are seldom available. The embryonic development of the plant-parasitic nematode Meloidogyne incognita was studied, focusing on the early events.ResultsA fixed pattern of repeated cell cleavages was observed, resulting in the appearance of the six founder cells 3 days after the first cell division. Gastrulation, characterized by the translocation of cells from the ventral side to the center of the embryo, was seen 1 day later. Approximately 10 days after the first cell division a rapidly elongating two-fold stage was reached. The fully developed second stage juvenile hatched approximately 21 days after the first cell division.ConclusionsWhen compared to the development of the free-living nematode Caenorhabditis elegans, the development of M. incognita occurs approximately 35 times more slowly. Furthermore, M. incognita differs from C. elegans in the order of cell divisions, and the early cleavage patterns of the germ line cells. However, cytoplasmic ruffling and nuclear migration prior to the first cell division as well as the localization of microtubules are similar between C. elegans and M. incognita.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0109-x) contains supplementary material, which is available to authorized users.

PS1/γ-Secretase-Mediated Cadherin Cleavage Induces β-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells.

Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

Abstract 3177: In vitro cellular inflammation caused by Caco2BBE and HepG2 cells secreted exosomes

Abstract Background: In colon cancer, the majority of metastatic cells end up in the liver. In order to elucidate metastasis migration, a possible road of investigation may lie in exosomes, a biological nanoparticle. Usually, exosomes range from 80-150 nm and medicate cell-to-cell communication. Additionally, exosomes house up to hundred proteins, oligonucleotides, lipids, and receptors that may contribute to affecting the inflammatory response in cancer cell lines. The aim of the study is to show preferential uptake of Caco2BBE exosomes by HepG2 cells under an inflammatory stimulus. Whereas Caco2BBE cells prohibits HepG2 exosomes uptake under an inflammatory stimulus. Methods: Exosomes was isolated by differential centrifugation from Caco2BBE, HepG2, and Raw 264.7 cell culture media stimulated with and without 10 ug/mL of LPS. Once collected, exosomes were characterized for size and charge by dynamic light scatter (DLS), atomic force microscopy (AFM), and transmission electron microcopy (TEM). Additional characterization of exosomes included toxicity as well as fluorescent microscopy to explore the specificity of Coumarin-6 labelled exosomes (C-6 Exo). Finally, the biological effects of exosomes on inflammatory markers in vitro were examined by Western blot and RT-qPCR. Results: The different measurements of exosomes size indicates an average diameter of 70-100 nm. Additionally, the exosomes extracted from each cells were not toxic to any cells. Interestingly, the uptake of C-6 Exo showed a striking find that Caco2BBE cells uptake HepG2 extracted exosomes under normal conditions. Whereas LPS stimulation of Caco2BBE cells prohibits the uptake of HepG2 exosomes. Then conversely, HepG2 cells were taking up Caco2BBE extracted exosomes when stimulated with LPS. These findings correlate with Western blot and RT-qPCR data that shows up-regulation of IL-6, TNFα, and MMP9 by Caco2BBE exosomes on HepG2 cells. Again, the LPS stimulation on HepG2 cells treated with Caco2BBE exosomes causes decrease of IL-6, TNFα, MMP9, and NF-κB. Conclusions: The characterization of these exosomes showed about 100 nm non-toxic particles that effect IL-6, TNF-α, MMP9, and NFκB on HepG2 cells treated with Caco2BBE exosomes. These effects on inflammatory markers mediate the uptake of these exosomes and especially with an inflammatory stimulation such as LPS, which can either prohibit or promote this process. Together, our data could lead to understanding the role of exosomes on the translocation of metastatic cells to the liver and could open a new therapeutics option. Citation Format: Jenniffer L. Stetler, Brandon SB Canup, Russell P. Puckett, XiangXiao Meng, Ma Qiang, Hamed Laroui. In vitro cellular inflammation caused by Caco2BBE and HepG2 cells secreted exosomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3177. doi:10.1158/1538-7445.AM2015-3177

The Response of wnt/ß-Catenin Signaling Pathway in Osteocytes Under Simulated Microgravity

Osteocytes were considered as potential sensors of mechanical loading and orchestrate the bone remodeling adapted to mechanical loading. On the other hand, osteocytes are also considered as the unloading sensors in vivo. Previous studies showed that the mechanosensation and mechanotransduction of osteocytes may play an essential role in mediating bone response to microgravity, and one of the most important molecular signaling pathway involved in the mechanotransduction is the Wnt/ s-catenin signaling pathway. In order to investigate the effect of simulated microgravity on the Wnt/ s-catenin signaling pathway in osteocytes, MLO-Y4 cells (an osteocyte-like cell line) were cultured under controlled rotation to simulate microgravity for 5 days. The cytoskeleton and s-catenin nuclear translocation of MLO-Y4 cells were detected by laser scanning confocal microscope and the fluorescence intensity was quantified; the mRNA expressions of upstream and downstream key components in Wnt canonical signaling were detected with RT-PCR. Two regulators of the Wnt/ s-catenin pathway, NMP4/CIZ and Smads, were also investigated by RT-PCR; finally the expression of Wnt target genes and Sost protein level were detected with the absence or presence of the Sclerostin antibody (Scl-AbI) under simulated microgravity. The results showed that under simulated microgravity, (1) F-actin filaments were disassembled and some short dendritic processes appeared at the cell periphery; (2) the gene expression of Wnt3a, Wnt5a, DKK1, CyclinD1, LEF-1 and CX43 in the simulated microgravity group were significantly lower whereas Wnt1 and Sost in the simulated microgravity group were significantly higher than the control group; (3) the gene and protein level of s-catenin were reduced, and no s-catenin nuclear translocation observed; (4) the gene expression of Smad1, Smad4 and Smad7 were significantly lower whereas NMP4/CIZ and Smad3 in the simulated microgravity were significantly higher than the control group; (5) Scl-AbI partially inhibited the down-regulation of simulated microgravity to Wnt target gene expression and Sclerostin protein expression. The results suggested that firstly the cytoskeleton was disturbed in MLO-Y4 by simulated microgravity; secondly the activity of Wnt/ s-catenin signaling pathway was depressed, with the nuclear translocation of s-catenin suppressed by simulated microgravity; thirdly the Wnt/ s-catenin signaling pathway positive regulators (Smads) were decreased, while the negative regulator (NMP4/CIZ) was increased under simulated microgravity; finally Scl-AbI could partially restore the adverse effect of simulated microgravity to Wnt signaling. This study may help us to understand the mechanotransduction alteration of Wnt/ s-catenin signaling pathway in osteocytes under simulated microgravity, and further may partly clarify the mechanism of microgravity-induced osteoporosis.

Cellular basis of neuroepithelial bending during mouse spinal neural tube closure

SummaryBending of the neural plate at paired dorsolateral hinge points (DLHPs) is required for neural tube closure in the spinal region of the mouse embryo. As a step towards understanding the morphogenetic mechanism of DLHP development, we examined variations in neural plate cellular architecture and proliferation during closure. Neuroepithelial cells within the median hinge point (MHP) contain nuclei that are mainly basally located and undergo relatively slow proliferation, with a 7 h cell cycle length. In contrast, cells in the dorsolateral neuroepithelium, including the DLHP, exhibit nuclei distributed throughout the apico-basal axis and undergo rapid proliferation, with a 4 h cell cycle length. As the neural folds elevate, cell numbers increase to a greater extent in the dorsolateral neural plate that contacts the surface ectoderm, compared with the more ventromedial neural plate where cells contact paraxial mesoderm and notochord. This marked increase in dorsolateral cell number cannot be accounted for solely on the basis of enhanced cell proliferation in this region. We hypothesised that neuroepithelial cells may translocate in a ventral-to-dorsal direction as DLHP formation occurs, and this was confirmed by vital cell labelling in cultured embryos. The translocation of cells into the neural fold, together with its more rapid cell proliferation, leads to an increase in cell density dorsolaterally compared with the more ventromedial neural plate. These findings suggest a model in which DLHP formation may proceed through ‘buckling’ of the neuroepithelium at a dorso-ventral boundary marked by a change in cell-packing density.

Improved anti-melanoma effect of a transdermal mitoxantrone ethosome gel

Melanomas are malignant tumors characterized by early metastasis, rapid development, poor prognosis and high mortality. A highly effective and convenient method is necessary for long-term treatment of melanomas. Mitoxantrone (MTO) was topically applied for melanoma therapy using an MTO ethosome gel. Firstly, an ethosome was prepared from MTO, phospholipids, ethanol and water followed by addition of hydroxypropyl methylcellulose to obtain an ethosome gel. The ethosome was characterized. The cytotoxicity on B16 melanoma cells was evaluated on an electrical cell-substrate impedance sensing system with a novel modified chip. In vivo anti-melanoma effect of the ethosome gel was explored. Immunohistochemical and flow cytometric investigations were done. The MTO ethosomes had the size of 78nm and the zeta potential of -55mV. The ethosomes were flexible vesicles and showed much higher in vitro permeability across the rat skin than MTO aqueous solutions. The ethosomes had significant cytotoxicity and higher in vivo anti-melanoma effect than MTO solutions. The calreticulin membrane translocation of B16 cells was improved by the MTO ethosomes and the cell uptake of MTO was confirmed. The MTO ethosome gel is a promising transdermal delivery system for melanoma therapy with the advantages of non-invasion and no significant side effects.

The dormant blood microbiome in chronic, inflammatory diseases.

Blood in healthy organisms is seen as a ‘sterile’ environment: it lacks proliferating microbes. Dormant or not-immediately-culturable forms are not absent, however, as intracellular dormancy is well established. We highlight here that a great many pathogens can survive in blood and inside erythrocytes. ‘Non-culturability’, reflected by discrepancies between plate counts and total counts, is commonplace in environmental microbiology. It is overcome by improved culturing methods, and we asked how common this would be in blood. A number of recent, sequence-based and ultramicroscopic studies have uncovered an authentic blood microbiome in a number of non-communicable diseases. The chief origin of these microbes is the gut microbiome (especially when it shifts composition to a pathogenic state, known as ‘dysbiosis’). Another source is microbes translocated from the oral cavity. ‘Dysbiosis’ is also used to describe translocation of cells into blood or other tissues. To avoid ambiguity, we here use the term ‘atopobiosis’ for microbes that appear in places other than their normal location. Atopobiosis may contribute to the dynamics of a variety of inflammatory diseases. Overall, it seems that many more chronic, non-communicable, inflammatory diseases may have a microbial component than are presently considered, and may be treatable using bactericidal antibiotics or vaccines.

Saikosaponin a, an active compound of Radix Bupleuri, attenuates inflammation in hypertrophied 3T3-L1 adipocytes via ERK/NF-κB signaling pathways.

Bupleurum falcatum L. is employed in oriental medicine in Korea. This root has been used for anti-inflammatory, anti-pyretic, and anti-hepatotoxic effects in the treatments of common cold, fever, and hepatitis. One of major bioactive compounds of Radix Bupleuri is the saikosaponin a (SSNa). However, little is known concerning the effects of SSNa on obesity associated with a state of low-grade inflammation. Consequently, this study was conducted to determine the inhibition of the inflammation pathway of SSNa in obesity. MTT assay was conducted for cytotoxicity and viability; nuclear and cytoplasmic fractions were extracted from adipocytes for translocation of nuclear factor-κB cells (NF-κB); nitric oxide (NO) production and secretion using Griess reagent; reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting for mRNA and protein levels associated with inflammation in the hypertrophied adipocytes. The results revealed that SSNa significantly decreased the expression of tumor necrosis factor-α (TNFα), interleukin (IL)-1β and IL-6 as proinflammatory cytokines, compared to that of non-treated control cells. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as inflammatory factors were reduced by treatment of these cells with SSNa and also suppressed NO production. Phosphorylation of IκBα was inhibited and translocation of NF-κB was suppressed via the ERK pathway in response to SSNa treatment. In conclusion, the results demonstrated that SSNa can inhibit the expression of inflammatory-associated genes in hypertrophied 3T3-L1 adipocytes and is a potent inhibitor of NF-κB activation. Thus these results suggest that SSNa is a novel therapeutic agent against that can be used against obesity-associated inflammation.

Regulation of Glucocorticoid Receptor (GR) Translocation of Airway Smooth Muscle Cells (ASM) By Pparγ Agonist Rosiglitazone and Insulin

Regulation of Glucocorticoid Receptor (GR) Translocation of Airway Smooth Muscle Cells (ASM) By Pparγ Agonist Rosiglitazone and Insulin

Volumetric measurement of human red blood cells by MOSFET-based microfluidic gate.

In this paper, we present a MOSFET-based (metal oxide semiconductor field-effect transistor) microfluidic gate to characterize the translocation of red blood cells (RBCs) through a gate. In the microfluidic system, the bias voltage modulated by the particles or biological cells is connected to the gate of MOSFET. The particles or cells can be detected by monitoring the MOSFET drain current instead of DC/AC-gating method across the electronic gate. Polystyrene particles with various standard sizes are utilized to calibrate the proposed device. Furthermore, RBCs from both adults and newborn blood sample are used to characterize the performance of the device in distinguishing the two types of RBCs. As compared to conventional DC/AC current modulation method, the proposed device demonstrates a higher sensitivity and is capable of being a promising platform for bioassay analysis.

The configuration of events and cell growth activity in the storeyed cambium of the linden (Tilia cordata Mill.)

A sample of wood from the trunk of the studied linden showed a periodic change in the orientation of the grain, occurring in an approximately two-year cycle. These changes in the grain were accompanied by cyclic changes in the configuration of events in the rays, that is, in the splitting and uniting of rays in the cambium. Intrusive growth on the lateral, radial edges near the ends of fusiform cells caused the translocation of cells on the storey borders, resulting in the splitting and uniting of rays. The growth activity of fusiform cells was determined on the basis of analysis of events in the rays through comparison of successive layers of the terminal parenchyma of the wood. Grouping of the ends of intrusively growing cells occurred here. The distribution of growth activity on the surface of the cambium was not uniform. It was possible to identify areas of cambium significantly differing from each other in respect to the duration of their growth activity. This period did not coincide with the duration of the cycle of changes in the configuration of events.

Use of frequency swept ultrasound to manipulate Nb2-11 live cells in a microfluidic channel

Recently microfluidic manipulation of live cell has been attracting attention because it is necessary for fast and accurate bioassays of cells within miniaturized devices like a lab-on-a-chip. This work concerns the alignment, translocation and concentration of live cells in a microfluidic channel by utilizing an ultrasonic standing wave. Among many ultrasonic manipulation methods, the technique using frequency swept ultrasound which was proposed in a previous study offers excellent particle manipulation performance, but that has been demonstrated only for biocompatible polystyrene microspheres with several different diameters, not actual biological cells. However, the technique needs to be verified for real cells because sizes and properties are little different with each other. Thus, in this work, the feasibility of using the ultrasonic frequency sweep method to manipulate live Nb2-11 cells was investigated experimentally The cells were aligned in a plane, moved to one side of a channel, and finally filtered out by sending them to one outlet with ultrasound. The frequency region of the applied ultrasound was between 1.75 MHz and 3.05 MHz. From experimental results, the cell behavior showing perfect alignment, almost perfect translocation and hence considerable aggregation proved the feasibility of using the frequency sweep method though the force acting on the Nb2-11 cells are much smaller than the force acting on polystyrene beads due to the differences in their sizes and mechanical properties.

Abstract A100: Helicobacter hepaticus contributes to mammary gland carcinogenesis through bacterial translocation and subsequent expansion of cancer-promoting myeloid-derived suppressor cells

Abstract Introduction: Gastrointestinal (GI) tract dysbiosis and resulting inflammation are implicated in breast carcinogenesis. CD11b+GR1+ myeloid- derived suppressor cells (MDSCs) are important drivers of breast cancer. Here we report that in APCMin/+ mice treated with enteric Helicobacter hepaticus (H. hepaticus) MDSCs cells are dramatically expanded and, along with the pathogen H. hepaticus, are directly recruited to mammary gland lymph node, where they suppress anti-carcinogenic cytotoxic T-cells and promote procarcinogenic inflammation, respectively. Methods: In our H. hepaticus and specific pathogen free colony we inoculated female APCMin/+ mice with H. hepaticus and compared neoplastic and inflammatory outcomes to the non-infected controls. Results: Successful infection was confirmed by H. hepaticus specific qPCR and fluorescence in situ hybridization (FISH). H. hepaticus was identified not only within the small intestine but also within the mammary gland lymph node and mammary tumor tissue of the treated group. In APCMin/+ mice treated with chow diet, H. hepaticus-induced procarcinogenic CD11b+GR1+ cells within the lamina propria of the small intestine, the surrounding mesenteric lymph nodes, the spleen, peripheral blood and the mammary gland lymph nodes. In H. hepaticus infected mice, this was accompanied by increased splenic expression of IL-1β, IL-6 and GM-CSF and the increased expression of IL-1β, IL-6 and GM-CSF, IL-23a and IL-17a in the small intestine. Interestingly, adding high fat diet to H. hepaticus infected mice appeared to further promote the trafficking of MDSCs from the spleen into the peripheral blood and regional lymph nodes with increased serum inflammatory cytokines production cytokines, including IL-6. This appears to have been mediated, at least in part, by adipocyte signaling to myeloid precursors. Conclusions: We show the presence of H. hepaticus in mammary gland and intestine, which correlated with mammary gland tumor incidence and increased intestinal tumor multiplicity. The co-existence of enteric H. hepaticus infection appears to promote mammary tumorigenesis through upregulation of IL-1β, IL-6 and GM-CSF and the resulting trafficking of tumor promoting CD11b+GR1+ MDSCs. We are currently examining the route and broader relevance of H. hepaticus extra-intestinal translocation and whether this has direct effects on tumorigenesis, consistent with findings in other Helicobacter spp. Citation Format: Xiaowei Chen, Daniel L. Worthley, Zhongming Ge, Yagnesh Tailor, Christian Kaufman, Lenzie Cheaney, Lesley Kline, Gladys Y. Valeriano, Christoph B. Westphalen, Susan E. Erdman, James G. Fox, Timothy C. Wang. Helicobacter hepaticus contributes to mammary gland carcinogenesis through bacterial translocation and subsequent expansion of cancer-promoting myeloid-derived suppressor cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A100.

Cysts About the Knee: Evaluation and Management

Popliteal (Baker) cysts, meniscal cysts, proximal tibiofibular joint cysts, and cruciate ligament ganglion cysts are cystic masses commonly found about the knee. Popliteal cysts form when a bursa swells with synovial fluid, with or without a clear inciting etiology. Presentation ranges from asymptomatic to painful, limited knee motion. Management varies based on symptomatology and etiology. Meniscal cysts form within or adjacent to the menisci. These collections of synovial fluid are thought to develop from translocation of synovial cells or extravasation of synovial fluid into the meniscus through a tear. Joint-line pain and swelling are common symptoms. Management entails partial meniscectomy with cyst decompression or excision. Proximal tibiofibular joint cysts are rare, and their etiology remains unclear. Pain and swelling secondary to local tissue invasion is common, and management consists of surgical excision. Cruciate ligament ganglion cysts have no clear etiology but are associated with mucoid degeneration of the anterior and posterior cruciate ligaments, knee trauma, and synovial translocation into these ligaments. Knee pain and limited range of motion, especially with exercise, are common presenting symptoms. In symptomatic cases, arthroscopic excision is commonly performed.

Anoikis: A Potential Target to Prevent Lung Cancer Metastasis?

169 ISSN 1758-1966 10.2217/LMT.13.13 © 2013 Future Medicine Ltd Lung Cancer Manage. (2013) 2(3), 169–171 The translocation of cancer cells to other parts of the body is the process of cancer metastasis, and this term has frightened people with lung cancer for years. Recently, any method that prevents cancer metas­ tasis is of great interest in cancer research prospects. Based on the fact that without metastasis, the tumor can be eliminated entirely at its primary site, a better under­ standing of the body’s key defense mecha­ nism against metastasis, named anoikis, may result in new strategies that prevent the progression of cancers, aid conven­ tional cancer treatment and reduce cancer relapse.

Abstract 4129: Efficient cancer cell separation using size and deformability based filtration.

Abstract Break-free cancer cells in the blood stream are statistically larger (mainly >15 microns) than the majority of normal cells (5-11 microns). Therefore, filters with engineered pores, for example, 8 μm circular pores, could be used to isolate rare cancer cells from billions of hematologic cells in the blood sample. Size exclusion based filtration, however, is not immune to methodological constraint. Loss of cells smaller than the size cutoff and failure of separating cancer cells from normal cells of similar sizes have been the problems. Incorporating a second separation parameter by controlling cell deformability during the enrichment process could minimize the cell loss and increase the purity of the product. Recent advances using column-wall or crescent-post microfluidic chips have demonstrated the potential of utilizing cell size and deformability based separation techniques for rare cell enrichment. In this paper, we propose an innovative filter that aims to provide similar enrichment effect at quicker time (∼minutes/mL blood) and lower manufacturing cost. The key to realize efficient filtration process is the pattern design of highly dense pores. The effect of pore size on cell separation was first studied using rectangular shaped Polydimethylsiloxane (PDMS) microchannels, which were microfabricated by the technique of soft lithography. In the experiments, intact breast cancer cells (MDA-MB-231 and MDA-MB-453) or human leukocytes were guided through the PDMS channels of different sizes. The flow rates and pressure drop were monitored. The deformation of the cells was visualized using a high-speed camera. The optimal pore size was selected to be the microchannel size that most leukocytes passed through, but not for the majority of the cancer cells, at high flow rate and low pressure-drop. As a second step, the effect of pore shape on cell separation was studied. We used the method of focused ion beam (FIB) to micromill pores into a thin polymer membrane, which have the size equal to or smaller than the optimal size obtained and a variety of shapes. FIB is an electron/ ion dual beam system, in which high-speed ion flow carries high momentum to engrave micro-sized pattern into substrate materials with high precision. The micromilled membrane was then integrated into a microfluidic flow chamber for cell separation. Pressure drop and cell deformation were recorded during the translocation of cells on the membrane. We conclude that both pore size and shape affect the size cutoff of cells to be separated. Under the pressure conditions that preserve the cell integrity and viability, cancer cells can be separated from normal cells at high flow rate, purity (>70%) and recovery (>80%). Our results could provide a detailed guidance for developing an efficient cell filter that can be used for rapid enrichment of rare cancer cells in blood for cancer detection and characterization. Citation Format: Yuhao Xu, Ali Hashmi, Wael A. Harb, Bin Hong, Jie Xu. Efficient cancer cell separation using size and deformability based filtration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4129. doi:10.1158/1538-7445.AM2013-4129

Impact of Host Membrane Pore Formation by the Yersinia pseudotuberculosis Type III Secretion System on the Macrophage Innate Immune Response

Type III secretion systems (T3SSs) are used by Gram-negative pathogens to form pores in host membranes and deliver virulence-associated effector proteins inside host cells. In pathogenic Yersinia, the T3SS pore-forming proteins are YopB and YopD. Mammalian cells recognize the Yersinia T3SS, leading to a host response that includes secretion of the inflammatory cytokine interleukin-1β (IL-1β), Toll-like receptor (TLR)-independent expression of the stress-associated transcription factor Egr1 and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), and host cell death. The known Yersinia T3SS effector proteins are dispensable for eliciting these responses, but YopB is essential. Three models describe how the Yersinia T3SS might trigger inflammation: (i) mammalian cells sense YopBD-mediated pore formation, (ii) innate immune stimuli gain access to the host cytoplasm through the YopBD pore, and/or (iii) the YopB-YopD translocon itself or its membrane insertion is proinflammatory. To test these models, we constructed a Yersinia pseudotuberculosis mutant expressing YopD devoid of its predicted transmembrane domain (YopD(ΔTM)) and lacking the T3SS cargo proteins YopHEMOJTN. This mutant formed pores in macrophages, but it could not mediate translocation of effector proteins inside host cells. Importantly, this mutant did not elicit rapid host cell death, IL-1β secretion, or TLR-independent Egr1 and TNF-α expression. These data suggest that YopBD-mediated translocation of unknown T3SS cargo leads to activation of host pathways influencing inflammation, cell death, and response to stress. As the YopD(ΔTM) Y. pseudotuberculosis mutant formed somewhat smaller pores with delayed kinetics, an alternative model is that the wild-type YopB-YopD translocon is specifically sensed by host cells.