The effect of an external magnetic force on cell adhesion and proliferation of magnetically labeled mesenchymal stem cells

<p>Abstract</p> <p>Background</p> <p>As the strategy for tissue regeneration using mesenchymal stem cells (MSCs) for transplantation, it is necessary that MSCs be accumulated and kept in the target area. To accumulate MSCs effectively, we developed a novel technique for a magnetic targeting system with magnetically labeled MSCs and an external magnetic force. In this study, we examined the effect of an external magnetic force on magnetically labeled MSCs in terms of cell adhesion and proliferation.</p> <p>Methods</p> <p>Magnetically labeled MSCs were plated at the bottom of an insert under the influence of an external magnetic force for 1 hour. Then the inserts were turned upside down for between 1 and 24 hours, and the number of MSCs which had fallen from the membrane was counted. The gene expression of MSCs affected magnetic force was analyzed with microarray. In the control group, the same procedure was done without the external magnetic force.</p> <p>Results</p> <p>At 1 hour after the inserts were turned upside down, the average number of fallen MSCs in the magnetic group was significantly smaller than that in the control group, indicating enhanced cell adhesion. At 24 hours, the average number of fallen MSCs in the magnetic group was also significantly smaller than that in control group. In the magnetic group, integrin alpha2, alpha6, beta3 BP, intercellular adhesion molecule-2 (ICAM-2), platelet/endothelial cell adhesion molecule-1 (PECAM-1) were upregulated. At 1, 2 and 3 weeks after incubation, there was no statistical significant difference in the numbers of MSCs in the magnetic group and control group.</p> <p>Conclusions</p> <p>The results indicate that an external magnetic force for 1 hour enhances cell adhesion of MSCs. Moreover, there is no difference in cell proliferation after using an external magnetic force on magnetically labeled MSCs.</p

Peer assessing individual contributions in a group project

Graduates are expected to have good academic knowledge but also the professional skills required in the workplace. One such ‘soft’ skill is the ability to give constructive criticism and provide meaningful but professional feedback. This is particularly relevant when working in a team within industry, where peers need to influence each other to improve their project outcomes and chances of success. The development of student’s skills to generate such feedback should be supported within higher education. Specifically the IPAC Consortium investigates the use of Individual Peer Assessed Contribution to group work. In this context, students create an output directed to their own peers (i.e. a form of external-facing assessment), and prepares students for similar practices in industry. This paper, linked to the roundtable session on external-facing assessments proposed by Grindle and Tong, investigates staff and student perceptions on such practice. Insights gained to this date are presented

The effect of starch-based biomaterials on leukocyte adhesion and activation in vitro

Leukocyte adhesion to biomaterials has long been recognised as a key element to determine their inflammatory potential. Results regarding leukocyte adhesion and activation are contradictory in some aspects of the material’s effect in determining these events. It is clear that together with the wettability or hydrophilicity/hydrophobicity, the roughness of a substrate has a major effect on leukocyte adhesion. Both the chemical and physical properties of a material influence the adsorbed proteins layer which in turn determines the adhesion of cells. In this work polymorphonuclear (PMN) cells and a mixed population of monocytes/macrophages and lymphocytes (mononuclear cells) were cultured separately with a range of starch-based materials and composites with hydroxyapatite (HA). A combination of both reflected light microscopy and scanning electron microscopy (SEM) was used in order to study the leukocyte morphology. The quantification of the enzyme lactate dehydrogenase (LDH) was used to determine the number of viable cells adhered to the polymers. Cell adhesion and activation was characterised by immunocytochemistry based on the expression of several adhesion molecules, crucial in the progress of an inflammatory response. This work supports previous in vitro studies with PMN and monocytes/macrophages, which demonstrated that there are several properties of the materials that can influence and determine their biological response. From our study, monocytes/macrophages and lymphocytes adhere in similar amounts to more hydrophobic (SPCL) and to moderately hydrophilic (SEVA-C) surfaces and do not preferentially adhere to rougher substrates (SCA). Contrarily, more hydrophilic surfaces (SCA) induced higher PMN adhesion and lower activation. In addition, the hydroxyapatite reinforcement induces changes in cell behaviour for some materials but not for others. The observed response to starch-based biodegradable polymers was not significantly different from the control materials. Thus, the results reported herein indicate the low potential of the starch-based biodegradable polymers to induce inflammation especially the HA reinforced composite materials

Photopatterned antibodies for selective cell attachment

We present a phototriggerable system that allows for the spatiotemporal controlled attachment of selected cell types to a biomaterial using immobilized antibodies that specifically target individual cell phenotypes.o-Nitrobenzyl caged biotin was used to functionalize chitosan membranes and mediate site-specific coupling of streptavidin and biotinylated antibodies after light activation. The ability of this system to capture and immobilize specific cells on a surface was tested using endothelial-specific biotinylated antibodies and nonspecific ones as controls. Homogeneous patterned monolayers of human umbilical vein endothelial cells were obtained on CD31-functionalized surfaces. This is a simple and generic approach that is applicable to other ligands, materials, and cell types and shows the flexibility of caged ligands to trigger and control the interaction between cells and biomaterials.We thank Martina Knecht (MPIP) for help with the synthesis of caged biotin and Dr. Ron Unger and Prof. C. J. Kirkpatrick (University Clinic Mainz, RepairLab) for providing HUVECs. C.A.C. acknowledges funding support from the Portuguese Foundation for Science and Technology (FCT) (fellowship SFRH/BD/61390/2009) and from the International Max-Planck Research School in Mainz. The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. REGPOT-CT2012-316331-POLARIS

Neoadjuvant in situ gene-mediated cytotoxic immunotherapy improves postoperative outcomes in novel syngeneic esophageal carcinoma models

Esophageal carcinoma is the most rapidly increasing tumor in the United States and has a dismal 15% 5-year survival. Immunotherapy has been proposed to improve patient outcomes; however, no immunocompetent esophageal carcinoma model exists to date to test this approach. We developed two mouse models of esophageal cancer by inoculating immunocompetent mice with syngeneic esophageal cell lines transformed by cyclin-D1 or mutant HRASG12V and loss of p53. Similar to humans, surgery and adjuvant chemotherapy (cisplatin and 5-fluorouracil) demonstrated limited efficacy. Gene-mediated cyototoxic immunotherapy (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir; AdV-tk/GCV) demonstrated high levels of in vitro transduction and efficacy. Using in vivo syngeneic esophageal carcinoma models, combining surgery, chemotherapy and AdV-tk/GCV improved survival (P=0.007) and decreased disease recurrence (P<0.001). Mechanistic studies suggested that AdV-tk/GCV mediated a direct cytotoxic effect and an increased intra-tumoral trafficking of CD8 T cells (8.15% vs 14.89%, P=0.02). These data provide the first preclinical evidence that augmenting standard of care with immunotherapy may improve outcomes in the management of esophageal carcinoma

Real-Time High Resolution 3D Imaging of the Lyme Disease Spirochete Adhering to and Escaping from the Vasculature of a Living Host

Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever, and Lyme borreliosis. They navigate efficiently through dense extracellular matrix and cross the blood–brain barrier by unknown mechanisms. Due to their slender morphology, spirochetes are difficult to visualize by standard light microscopy, impeding studies of their behavior in situ. We engineered a fluorescent infectious strain of Borrelia burgdorferi, the Lyme disease pathogen, which expressed green fluorescent protein (GFP). Real-time 3D and 4D quantitative analysis of fluorescent spirochete dissemination from the microvasculature of living mice at high resolution revealed that dissemination was a multi-stage process that included transient tethering-type associations, short-term dragging interactions, and stationary adhesion. Stationary adhesions and extravasating spirochetes were most commonly observed at endothelial junctions, and translational motility of spirochetes appeared to play an integral role in transendothelial migration. To our knowledge, this is the first report of high resolution 3D and 4D visualization of dissemination of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo

Gene deletion of P-Selectin and ICAM-1 does not inhibit neutrophil infiltration into peritoneal cavity following cecal ligation-puncture

BACKGROUND: Neutrophil infiltration is one of the critical cellular components of an inflammatory response during peritonitis. The adhesion molecules, P-selectin and intercellular adhesion molecule (ICAM)-1, mediate neutrophil-endothelial cell interactions and the subsequent neutrophil transendothelial migration during the inflammatory response. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy, suggesting that the length of injury might be a critical factor in neutrophil infiltration. Therefore, the objective of this study was to determine the role of P-selectin and ICAM-1 in neutrophil infiltration into the peritoneal cavity during early and late phases of peritonitis. METHODS: Peritonitis was induced in both male wild-type and P-selectin/ICAM-1 double deficient (P/I null) mice by cecal ligation-puncture (CLP). Peripheral blood and peritoneal lavage were collected at 6 and 24 hours after CLP. The total leukocyte and neutrophil contents were determined, and neutrophils were identified with the aid of in situ immunohistochemical staining. Comparisons between groups were made by applying ANOVA and student t-test analysis. RESULTS: CLP induced a severe inflammatory response associated with a significant leukopenia in both wild-type and P/I null mice. Additionally, CLP caused a significant neutrophil infiltration into the peritoneal cavity that was detected in both groups of mice. However, neutrophil infiltration in the P/I null mice at 6 hours of CLP was significantly lower than the corresponding wild-type mice, which reached a similar magnitude at 24 hours of CLP. In contrast, in peritonitis induced by intraperitoneal inoculation of 2% glycogen, no significant difference in neutrophil infiltration was observed between the P/I null and wild-type mice at 6 hours of peritonitis. CONCLUSIONS: The data suggest that alternative adhesion pathway(s) independent of P-selectin and ICAM-1 can participate in neutrophil migration during peritonitis and that the mode of stimuli and duration of the injury modulate the neutrophil infiltration

New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis

A new human hepatocellular carcinoma (HCC) cell line with a highly metastatic potential was established from subcutaneous xenograft of a metastatic model of human HCC in nude mice (LCI-D20) by means of alternating cell culture in vitro and growth in nude mice. The line, designated MHCC97, has been cultivated for 18 months and subcultured for more than 90 passages. The line was showed to be of human origin by karyotype analysis. The cells were either grown as compact colonies (in clusters) or as a monolayered sheet with about 31 h of population-doubling time, exhibited typical malignant epithelial in morphology and were positive for α-fetoprotein (AFP). Flow cytometric analysis of the cell DNA content showed an aneuploid pattern, and its index was 1.5 as compared to that of normal human peripheral blood lymphocytes. Karyotypic analyses of G- and C-banding techniques revealed that all cells presented chromosome abnormalities in number and structure. The number of cell line MHCC97 chromosome ranged from 59 to 65 with a modal number of 60 and 61. At least two common chromosome markers, i(1q) and der(4)t(4;?)(4pter→q35::?), were present in all cells, and deletion of Y chromosome also occurred in all cells. The subcutaneous and intrahepatic xenografts were formed and metastatic lesions in lungs were found after the cells were inoculated into nude mice. The rate of metastasis to lungs was 100% using orthotopic inoculation. Reverse transcription polymerase chain reaction products revealed positive expressions of integrin α5 and β1, urokinase type plasminogen activator receptor (uPAR), vascular endothelial growth factor and nm23-H1 mRNAs of cell line MHCC97. Immunostaining of c-Met, uPAR showed strongly positive in both subcutaneous xenografts and lung metastatic lesions; while positive in xenografts and negative in metastatic lesions for integrin α5, β1. E-cadherin and P53 was not expressed either in xenograft or in the metastatic lesions. PCR products of HBsAg and HBxAg were both positive. The cell line MHCC97 still retained some characteristic features of original tumour. Establishment of cell line MHCC97 should be beneficial to the studies of HCC metastatic mechanisms. © 1999 Cancer Research Campaig

Distinct Effects of IL-18 on the Engraftment and Function of Human Effector CD8+ T Cells and Regulatory T Cells

IL-18 has pleotropic effects on the activation of T cells during antigen presentation. We investigated the effects of human IL-18 on the engraftment and function of human T cell subsets in xenograft mouse models. IL-18 enhanced the engraftment of human CD8+ effector T cells and promoted the development of xenogeneic graft versus host disease (GVHD). In marked contrast, IL-18 had reciprocal effects on the engraftment of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the xenografted mice. Adoptive transfer experiments indicated that IL-18 prevented the suppressive effects of Tregs on the development of xenogeneic GVHD. The IL-18 results were robust as they were observed in two different mouse strains. In addition, the effects of IL-18 were systemic as IL-18 promoted engraftment and persistence of human effector T cells and decreased Tregs in peripheral blood, peritoneal cavity, spleen and liver. In vitro experiments indicated that the expression of the IL-18Rα was induced on both CD4 and CD8 effector T cells and Tregs, and that the duration of expression was less sustained on Tregs. These preclinical data suggest that human IL-18 may have use as an adjuvant for immune reconstitution after cytotoxic therapies, and to augment adoptive immunotherapy, donor leukocyte infusions, and vaccine strategies