coculture techniques Research Papers - Academia.edu (original) (raw)

Background: Several synthetic and natural matrices have been described for tissue engineering of bladder but there is little information on the effects of processing on their subsequent mechanical performance or interaction with human cells. Aim: Our aim was to assess the effects of delamination, decellularization and sterilization on the mechanical properties of porcine urinary bladder matrix (UBM) and to then assess the ability of the UBM to act as a scaffold for reconstruction with human bladder cells. Methods: A total of 20 porcine bladders were assessed before and after mechanical delamination and four porcine bladders were followed at every stage through a comparison of several decellularization and terminal sterilization methodologies examining histological and mechanical characteristics. The sterile UBM was seeded with normal human urothelial and bladder stromal cells either as a simultaneous coculture, or with stromal cells followed by urothelial cells. Results: Mechanical delamination, physical rinsing of the resulting bladder stroma in hypotonic buffer, 0.1% sodium dodecyl sulfate solution and 0.1% peracetic acid resulted in an UBM with acceptable mechanical properties capable of supporting urothelial and bladder stromal cells. Terminal sterilization with ethylene oxide resulted in considerable stiffening of the matrix simultaneous coculture and layered seeding of scaffolds with stromal cells followed by epithelial cells gave similar results with good initial urothelial attachment (followed by loss of cells later) and slow stromal cell penetration. Conclusion: We describe a decellularized sterilized porcine UBM with acceptable mechanical properties that shows promise as a scaffold for producing an in vitro tissue-engineered bladder patch material for lower urinary tract reconstruction. Future work now needs to focus on the conditions for achieving secure epithelial attachment.

- by
- •
- Tissue Engineering, Regenerative Medicine, Sterilization, Urinary Tract

In breast cancers, the appearance of metastasis is synonymous with poor prognosis. The metastatic process is usually associated with epithelial-mesenchymal transition (EMT) which is often induced by several soluble factors produced either by the tumour cells themselves or by cells constituting the tumour microenvironment. The aim of the present study was to determine whether the mesenchymal properties given by some molecules such as N-cadherin, for instance, could be acquired by cancer cells via the trogocytosis process with cells of the tumour microenvironment. Hospicells are stromal cells which were first isolated from cancer cell aggregates of patients with ovarian cancer. We recently showed that these cells are immunosuppressive for T lymphocyte functions and confer chemoresistance to cancer cells by the transfer of the MDR protein via trogocytosis. In this study, we showed that a mammary cancer cell line (MDA-MB-231) acquires patches of membrane via oncologic trogocytosis with Hospicells. This unidirectional and active process depends on actin polymerization and can be increased via inhibition of the Src family and decreased via inhibition of PI3K. Trogocytosis between Hospicells and MDA-MB-231 does not lead to the direct acquisition of N-cadherin but rather it leads to the production of soluble factor(s) which induce de novo expression of N-cadherin by the cancer cells. The novelty here is that this factor is produced only if cancer cells interact and undergo trogocytosis with Hospicells. This new expression could confer a more invasive phenotype to the cancer cells and thus can explain the correlation of the presence of Hospicells with the number of invaded lymph nodes in patients with mammary adenocarcinoma.

- by Christophe Klein
- •
- Oncology, Molecular Medicine, Cancer, Cell Cycle

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene (Hoffman et al. 1987), which controls the expression of dystrophin. This protein, normally localized beneath the sarcolemma in normal muscle, is absent in muscle from DMD patients (Bonilla et al. 1988). DMD is also characterized by an abnormally high concentration of intracellular ionized calcium (Bodensteiner & Engel, 1978), which is thought to contribute to the final muscle necrosis. This resting calcium accumulation in DMD cells was also observed in

Her research focuses on human embryonic implantation, uterine receptivity and embryonic stem cells, leading to the publication of 26 peer-reviewed papers. She received awards from the American Fertility Society in 1997 and the Spanish... more

- by Diana Valbuena
- •
- Human Embryonic Stem Cells, Human embryonic stem cell, Cryopreservation, Cell line

Previously we reported that the co-culture of non-brain vascular endothelial cells with glioma cells leads to the induction of a more dierentiated endothelial cell phenotype which exhibits important properties of the blood±brain barrier (BBB). Recognising the potential for improving the model barrier system with agents known to modify the growth and dierentiation of cells in culture we examined the eects of four dierentiating agents (butyric acid, dexamethasone, retinoic acid, and dimethyl sulfoxide) on barrier function. Of these agents only butyric acid and dexamethasone resulted in an enhancement (depending on the dose used) of transendothelial electrical resistance (barrier function). The greatest eect was observed with butyric acid in a dose-dependent manner and was slow in onset and only occurred in the endothelial/glial cell co-cultures. These data indicate that butyric acid may be a bene®cial agent in optimising conditions necessary for induction of BBB properties in in vitro barrier systems.

- by Roger Hurst
- •
- Neurochemistry, Dexamethasone, Cell Differentiation, Glioma
- by Sufang Yang
- •
- Stem Cells, Biology, Stem Cell, Medicine
- by Andras Kapus
- •
- Biology, Inflammation, Apoptosis, Medicine

The present pilot study aims to evaluate the frequency and the function of regulatory T (Treg) cells in patients with diffuse cutaneous SSc (dcSSc) before and after autologous hematopoietic SCT (aHSCT). Peripheral blood lymphocytes from seven dcSSc patients were analyzed before and 24 months after aHSCT and were compared with those from seven healthy donors (controls). Immunophenotyping of CD4 þ CD25 high FoxP3 þ natural Treg (nTreg), CD4 þ CD25 þ TGF-b þ and CD4 þ CD25 þ IL-10 þ adaptive Treg (aTreg) cell subsets was performed using four-color flow cytometry. Treg-suppressive capability was measured after coculture with autologous T effector cells by evaluation of T-cell proliferation using 3 H-thymidine incorporation. Peripheral CD4 þ CD25 high FoxP3 þ (2 ± 0.5 vs 4.2 ± 1.1, Po0.01), CD4 þ CD25 þ TGF-b þ (6.9 ± 1.8 vs 14.6 ± 5.0, Po0.05) and CD4 þ CD25 þ IL-10 þ (10.7 ± 0.5 vs 16.1±3.2, Po0.01) Tregs as well as CD4 þ CD25 high CD127 low Tregs suppressive capacity (Po0.05) were decreased in dcSSc patients vs controls. After aHSCT (n ¼ 7), the percentages of CD4 þ CD25 high FoxP3 þ (4.1 ± 1.8) and CD4 þ CD25 þ IL-10 þ (15.7 ± 2.2) Treg cells and the suppressive activity of CD4 þ CD25 high CD127 low were restored to the levels in controls. The decreased frequency and the functional defect of peripheral Treg cells from patients with dcSSc are reversed following aHSCT to reach those observed in controls. This pilot study brings evidence of an effective restoration of nTreg and aTreg subsets, and recovery of nTreg suppressive function following aHSCT.

Preface VII in Volume 2 collect 61 chapters covering protocols for 59 plant species. The plants are grouped according to their practical utilization rather than their botanical classification. The significant expansion of this section reflects the remarkable advancements in plant transformation technology during the past decade. Part VIII in Volume 2 (Non-plants) contains six chapters with protocols for introducing DNA into non-plant species such as bacteria, fungi, algae, and mammalian cells. The description of this unique capacity of Agrobacterium is a new addition to this edition.

- by Esteban Hopp
- •
- Soil, Regeneration, Genetic Engineering, Gene transfer techniques
- by Abhaya Dandekar
- •
- Gene transfer techniques, Juglans, Glucuronidase, Germination

Agrobacterium tumefaciens is a soil bacterium that for more than a century has been known as a pathogen causing the plant crown gall disease. Unlike many other pathogens, Agrobacterium has the ability to deliver DNA to plant cells and permanently alter the plant genome. The discovery of this unique feature 30 years ago has provided plant scientists with a powerful tool to genetically transform plants for both basic research purposes and for agricultural development.

- by William Powell
- •
- Methods, Rhizobium, Plant Roots, Fagaceae

All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology TM is a trademark of The Preface Agrobacterium tumefaciens is a soil bacterium that for more than a century has been known as a pathogen causing the plant crown gall disease. Unlike many other pathogens, Agrobacterium has the ability to deliver DNA to plant cells and permanently alter the plant genome. The discovery of this unique feature 30 years ago has provided plant scientists with a powerful tool to genetically transform plants for both basic research purposes and for agricultural development. Compared to physical transformation methods such as particle bombardment or electroporation, Agrobacterium-mediated DNA delivery has a number of advantages. One of the features is its propensity to generate single or a low copy number of integrated transgenes with defined ends. Integration of a single transgene copy into the plant genome is less likely to trigger "gene silencing" often associated with multiple gene insertions. When the first edition of Agrobacterium Protocols was published in 1995, only a handful of plants could be routinely transformed using Agrobacterium. Agrobacterium-mediated transformation is now commonly used to introduce DNA into many plant species, including monocotyledon crop species that were previously considered non-hosts for Agrobacterium. Most remarkable are recent developments indicating that Agrobacterium can also be used to deliver DNA to non-plant species including bacteria, fungi, and even mammalian cells. While the list of organisms that can be infected by Agrobacterium has increased significantly over the past decade, the success in transformation also relies on culture responsiveness of the target cells/tissues subsequent to the cocultivation with Agrobacterium. Essentially, the dynamic interactions between the two living organisms are critical for development of transformation methods. The second edition of Agrobacterium Protocols contains 80 chapters (two volumes) divided into 14 parts. In addition to basic Agrobacterium handing techniques and model plant transformation methods (Parts I and II in Volume 1), this edition collects 61 chapters covering protocols for 59 plant species. Volume 2 contains 35 of 59 plant protocols (Parts I to VII). The plants are grouped according to their practical utilization rather than their botanical classification. The significant expansion of this section reflects the remarkable advancements in plant transformation technology during the past decade. Part vii viii Preface VIII (Non-Plants) contains six chapters with protocols for introducing DNA into non-plant species such as bacteria, fungi, algae, and mammalian cells. The description of this unique capacity of Agrobacterium is a new addition to this edition. Agrobacterium Protocols provides a bench-top manual for tested protocols involving Agrobacterium-mediated transformation. All chapters are written in the same format as that used in the Methods in Molecular Biology series. Each chapter is contributed by authors who are leaders or veterans in the respective areas. The Abstract and Introduction sections provide outlines of protocols, the rationale for selection of particular target tissues, and overall transformation efficiency. The Materials section lists the host materials, Agrobacterium strains and vectors, stock solutions, media, and other supplies necessary for carrying out these transformation experiments. The Methods section is the core of each chapter. It provides a detailed step-by-step description of the entire transformation procedure from the preparation of starting materials to the harvest of transgenic plants. To ensure the reproducibility of each protocol, the Notes section supplies additional information on possible pitfalls in the protocol and alternative materials or methods for generating transgenic plants. Typically, most laboratories only work on one or a few plant species. Of course, each laboratory or individual researcher has his/her own favorite variation or modification of any given plant transformation protocol. The protocols presented in this edition represent the most efficient methods used in the laboratories of these contributors. They are by no means the only methods for successful transformation of your plant of interest. The broad range of target tissue selection and in vitro culture procedures indicate the complexity in plant transformation. It is the intention of this book to facilitate the transfer of this rapidly developing technology to all researchers for use in both fundamental and applied biology. I take this opportunity to thank all my colleagues whose time and effort made this edition possible. Special thanks go to my family for their unconditional love and support during the process of editing this book.

- by Guoqing Song
- •
- Regeneration, Biology, Medicine, Genetic Engineering
- by Suel-Kee Kim
- •
- Gastroenterology, Immunohistochemistry, Cell separation, Cell Differentiation
- by Rachel Waddington
- •
- Dentistry, Biology, Medicine, Cell Death

Background: Mycobacterium tuberculosis-specific CD8+ and CD4+ T lymphocyte responses restrict the spread of extracellular pathogens by limiting M.tuberculosis replication. Alterations in cytolytic function, inappropriate maturation/differentiation, and limited proliferation could reduce their ability to control M.tuberculosis replication. Methods: In an attempt to further characterize the immune responses during M.tuberculosis infection, we enumerated γδ and αβ receptor-bearing T cells expressing CD8 or CD4 phenotype and analyzed the differentiation phenotypes of CD8+ and CD4+ T lymphocyte subpopulations in 47 cases (23 new cases and 24 multidrug resistant patients) and 20 control subjects, using flowcytometry. Results: We found that the CD4/CD8 ratio was significantly lower in newly-diagnosed M.tuberculosis patients compared to multidrug resistant and control subjects (P < 0.003). Also, we found that a large proportion of CD8+ T lymphocytes in newly-diagnosed patients was defined by increased surface expression of CD57 as compared to the two other settings (P < 0.002). This increase was more profound in patients with an inverted CD4/CD8 ratio. Analysis of the late activation antigen revealed that this was predominantly HLA-DR+ (P < 0.003). No significant changes were observed in the percentages of CD8+CD57+ T cells between the different settings. Moreover, the co-stimulatory molecule CD28+ tended to be underexpressed by CD8+ T cells in multidrug resistant patients when compared to newly-diagnosed subjects (P < 0.002), but not to the control subjects. In contrast, the frequency of CD28+ marker on CD4+ T cells was higher in the setting of multidrug resistant compared with those of new cases (P < 0.0001). No significant changes were observed in percentages of γδ receptor-bearing T cells between different groups. Conclusion: We suggest that the increase in the proportion of CD57+ within CD8+ T cells in newly-diagnosed patients results from M.tuberculosis antigenic stimulation, which is a hallmark of many infections and that the protracted accumulation of CD57+ T lymphocytes might reflect an end-stage differentiation phenotype.

- by zinat entezami
- •
- Immune response, Flow Cytometry, Cell Differentiation, Tuberculosis

This focus article introduces the concept of NutriChip, an integrated microfluidic platform for investigating the potential of the immuno-modulatory function of dairy food. The core component of the NutriChip is a miniaturized artificial human gastrointestinal tract (GIT), which consists of a confluent layer of epithelial cells separated from a co-culture of immune cells by a permeable membrane. This setting creates conditions mimicking the human GIT and allows studying processes that characterize the passage of nutrients though the human GIT, including the activation of immune cells in response to the transfer of nutrients across the epithelial layer. The NutriChip project started by developing a biologically active in vitro cellular system in a commercial Transwell co-culture system. This Transwell system serves as a reference for the micro-scale device which is being developed. The microfluidic setup of NutriChip allows monitoring of the response of immune cells to pro-inflammatory stimuli, such as lipid polysaccharide (LPS), and to the application of potentially anti-inflammatory dairy food. This differential response will be quantified by measuring the variation in expression of pro-inflammatory cytokines, including interleukin 1 (IL-1) and interleukin 6 (IL-6), secreted by the immune cells, and this is achieved by using a dedicated optical imager. A series of dairy products will be screened for their anti-inflammatory properties using the NutriChip system and, finally, the outcome of the NutriChip will be validated by a human nutrition trial. Therefore, the NutriChip platform offers a new option to evaluate the influence of food quality on health, by monitoring the expression of relevant immune cell biomarkers.

- by Qasem Ramadan
- •
- Engineering, Nutrition, Fluorescence Microscopy, Calcium

The blood-brain barrier consists of the cerebral microvascular endothelium, pericytes, astrocytes, and neurons. In this study, we analyzed the influence of primary porcine brain capillary pericytes on the barrier integrity of primary porcine brain capillary endothelial cells in a species-consistent in vitro coculture model. We were able to show a barrier integrity-decreasing impact of pericytes by transendothelial electrical resistance (TEER) and 14 C-sucrose permeability measurements. The morphology analysis revealed serrated cell borders and a shift of the endothelial morphology towards a cobblestone shape under the influence of pericytes. The analysis of the two major barrier integrity modulators vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) displayed higher MMP activity and higher levels VEGF, MMP-2, and MMP-9 in the coculture, whereas VEGF levels were decreased by the MMP inhibitor GM6001, indicating a complex interplay of both. Inhibition experiments with neutralizing VEGF antibody and GM6001 increased the TEER, which proves the involvement of VEGF and MMPs in the barrier-decreasing process. Analysis of occludin yielded decreased protein content and discontinuous expression at the endothelial cell borders under the influence of pericytes. These results together reveal the potential of pericytes to regulate the endothelial barrier integrity via MMPs and VEGF.

- by Hans-Joachim Galla
- •
- Cognitive Science, Membrane Proteins, Enzyme Inhibitors, In Vitro

The anti-α4 monoclonal antibody natalizumab inhibits lymphocyte extravasation into the central nervous system and increases peripheral T and B lymphocytes in multiple sclerosis patients. To investigate whether the lymphocyte accumulation was due to a higher lymphocyte production, an altered homeostasis, or a differential transmigration of lymphocyte subsets through endothelia, T-cell receptor excision circles and kappa-deleting recombination excision circles were quantified before and after treatment, T-cell receptor repertoire was analyzed by spectratyping, and T-and B-lymphocyte subset migration was studied using transwell coated with vascular and lymphatic endothelial cells. We found that the number of newly produced T and B lymphocytes is increased because of a high release and of a low propensity of naïve subsets to migrate across endothelial cells. In some patients this resulted in an enlargement of T-cell heterogeneity. Because new lymphocyte production ensures the integrity of immune surveillance, its quantification could be used to monitor natalizumab therapy safety.

- by Emirena Michela Garrafa
- •
- Immunology, Multiple sclerosis, Clinical immunology, Child

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (<10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth betwe...

- by Yayu Chiang
- •
- Neuroscience, Cell line, High throughput screening, Neurons

BACKGROUND AND OBJECTIVES: The role of bone marrow-mesenchymal stem cells (BM-MSC) in leukaemic cell control is controversial. The purpose of this work was to evaluate BM-MSC role regarding the viability, proliferation and immunophenotype of normal B-cell precursors from control (Ct) patients and leukaemic cells from B-acute lymphoblastic leukaemia (B-ALL) patients. PATIENTS AND METHODS: BM-MSC were isolated and characterised from voluntary donors. Mononuclear cells isolated from Ct and BALL bone marrow samples were cultured in the presence or absence of BM-MSC for 7 days. Cell viability was determined with LIVE/DEAD and proliferation index evaluated by CFSE labelling. Cell population immunophenotypes were characterised by estimating CD19, CD10, CD20 and CD45 antigens by flow cytometry. RESULTS: After co-culture, BALL cells exhibited higher viability (20-40%) as compared to just cells (3-10%). Ct and BALL absolute cell counts were higher in the presence of BM-MSC (Ct: 25/mm 3 cf 8/mm 3 , BALL: 15/ mm 3 cf 3/mm 3). Normal B-cell subpopulations in co-culture had increased expression of CD19 and CD10 (Prepre B) and CD45 and CD20 antigens (Pre-B). BALL cells co-cultured with BM-MSC showed an increase in CD19 and CD20, although the greatest increase was observed in the CD10 antigen. CONCLUSIONS: Lymphoid cell maintenance, at early stages of differentiation, was significantly promoted by BM-MSC in normal and leukaemic cells. Co-cultures also modulated the expression of antigens associated with the BALL asynchronous phenotype as CD10 co-expressed with CD19 and CD20. To our knowledge, this is the first time that CD10, CD19 and CD20 leukaemic antigens have been reported as being regulated by BM-MSC.

- by jose augusto ocampo aristizabal
- •
- Cluster Analysis, Phenotype, Cell Shape, Immunophenotyping
- by Amy Hu
- •
- Gap Junctions, Fluorescence Microscopy, Multidisciplinary, Biocatalysis

BACKGROUND. Tissue factor (TF) is a cell surface glycoprotein intricately related to blood coagulation and inflammation. This study was performed to investigate the role of monocytelineage cells in prostate cancer cell TF expression and cell invasion. METHODS. Prostate cancer cell invasion was tested with and without added peripheral blood monocytes or human monocyte-lineage cell lines. TF neutralizing antibodies were used to determine the TF requirement for prostate cancer cell invasion activity. Immunohistochemistry was performed to identify prostate tissue CD68 positive monocyte-derived cells and prostate epithelial TF expression. RESULTS. Co-culture of PC-3, DU145, and LNCaP cells with isolated human monocytes significantly stimulated prostate cancer cell invasion activity. TF expression was greater in highly invasive prostate cancer cells and was induced in PC-3, DU145, and LNCaP cells by co-culture with U-937 cells, but not with THP-1 cells. TF neutralizing antibodies inhibited PC-3 cell invasion in cocultures with monocyte-lineage U-937 or THP-1 cells. Prostate cancer tissues contained more CD68 positive cells in the stroma and epithelium (145 AE 53/mm 2) than benign prostate (108 AE 31/mm 2). Samples from advanced stage prostate cancer tended to contain more CD68 positive cells when compared with lower stage lesions. Prostatic adenocarcinoma demonstrated significantly increased TF expression compared with benign prostatic epithelium. CONCLUSIONS. This study shows that co-culture with monocyte-lineage cells induced prostate cancer cell invasion activity. PC-3 invasion and TF expression was induced in co-culture with U-937 cells and partially inhibited with TF neutralizing antibodies.

- by André Kajdacsy-Balla
- •
- Nephrology, Andrology, Nonparametric Statistics, Cancer

DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the "Taverne" license above, please follow below link for the End User Agreement:

- by Sourabh Ghosh
- •
- Materials Science, Tissue Engineering, Hydrogels, Regenerative Medicine
- by mwiya mwiya
- •
- Immunology, Immunology of the Gut, Medicine, Dendritic Cells

Background Mast cells contribute to the pathogenesis of asthma and allergy through the release of a plethora of pro-inflammatory mediators and cytokines. Their study is hampered by the difficult access to human tissue samples. Human mast cells have been cultured from CD34 1 progenitors in the bone marrow of normal volunteers following iliac crest bone marrow biopsy but this is invasive. Hip bone marrow could provide a more convenient less invasive source of mast cell progenitors. Objective To characterize mast cells cultured from human bone marrow obtained at routine hip surgery. Methods Mononuclear cells were isolated from the bone marrow reamings of patients undergoing routine hip replacement surgery and were cultured with recombinant stem cell factor (SCF), IL-6 and IL-10. Cell surface markers were examined using flow cytometry, protease expression monitored using immunohistochemistry, histamine measured by radioenzymic assay, Ca 21 responses analysed using ratiometric Ca 21 imaging, and ion currents recorded via the patch-clamp technique. Results Mast cells were absent at baseline, but accounted for 65 AE 7% of cells after 8-12 weeks of culture, equating to a mean 0.6 AE 0.14 Â 10 6 mast cells per culture. Fifty-three percent of tryptase 1 cells also contained chymase. The remaining cells comprised a population of large CD1a 1 HLA-DR 1 and FceRIa 1 cells, most likely dendritic cells. All mast cells expressed CD117 and the highaffinity IgE receptor a-chain (FceRIa) constitutively, and developed a Ca 21 response following IgEdependent activation. These cells exhibited 7.8 AE 2.9% net IgE-dependent histamine release, and demonstrated a similar ion channel profile to human lung mast cells. In particular, the intermediate conductance Ca 21-activated K 1 channel opened following IgE-dependent activation. Conclusions Mast cells grown from human hip marrow provide a rich non-invasive source of functionally mature mast cells. In addition, this culture system may be useful for the generation of FceRIa 1 dendritic cells.

- by Patrick Berger
- •
- Immunology, Electrophysiology, Flow Cytometry, Biology

Development of new, anti-metastatic drugs from natural products has been substantially constrained by the lack of a reliable in vitro screening system. Such a system should ideally mimic the native, three-dimensional (3D) tumor microenvironment involving different cell types and allow quantitative analysis of cell behavior critical for metastasis. These requirements are largely unmet in the current model systems, leading to poor predictability of the in vitro collected data for in vivo trials, as well as prevailing inconsistency among different in vitro tests. In the present study, we report application of a 3D, microfluidic device for validation of the anti-metastatic effects of twelve natural compounds. This system supports co-culture of endothelial and cancer cells in their native 3D morphology as in the tumor microenvironment, and provides real-time monitoring of the cells treated with each compound. We found that three compounds, namely sanguinarine, nitidine, and resveratrol, exhibited significant anti-metastatic or anti-angiogenic effects. Each compound was further examined for its respective activity with separate conventional biological assays, and the outcomes were in agreement with the findings collected from the microfluidic system. In summary, we recommend use of this biomimetic model system as a new engineering tool for high-throughput evaluation of more diverse natural compounds with varying anti-cancer potentials.

- by Roger D. Kamm
- •
- Chemistry, Microfluidics, Biomimetics, Biology

Objective—To characterize the role of a vascular-expressed class 3 semaphorin (semaphorin 3G [Sema3G]). Methods and Results—Semaphorins have been identified as axon guidance molecules. Yet, they have more recently also been characterized as attractive and repulsive regulators of angiogenesis. Through a transcriptomic screen, we identified Sema3G as a molecule of angiogenic endothelial cells. Sema3G-deficient mice are viable and exhibit no overt vascular phenotype. Yet, LacZ expression in the Sema3G locus revealed intense arterial vascular staining in the angiogenic vasculature, starting at E9.5, which was detectable throughout adolescence and downregulated in adult vasculature. Sema3G is expressed as a full-length 100-kDa secreted molecule that is processed by furin proteases to yield 95- and a 65-kDa Sema domain–containing subunits. Full-length Sema3G binds to NP2, whereas processed Sema3G binds to NP1 and NP2. Expression profiling and cellular experiments identified autocrine effe...

- by Constantinos Mikelis
- •
- RNA interference, Endothelial Cells, Mice, Phenotype

Invariant natural killer T (iNKT) cells regulate the T helper (Th) 1/2 balance and elicit either enhancement or suppression of the immune responses. However, the exact mechanism by which iNKT cells exert these contrasting functions has remained elusive. We demonstrate herein that two major distinct subsets of human iNKT cells, CD4 + CD8b-(CD4 +) and CD4-CD8b-(double negative; DN) cells, express functional CD40 ligand (CD40L), but they differentially regulate the dendritic cell (DC) function by reciprocal NKT-DC interactions, thereby influencing the subsequent Th response. The CD4 subset stimulated by a-galactosylceramide (a-GalCer)-loaded DC immediately produced massive amounts of IL-4 and IL-13, which together with IFN-c enhanced CD40L-induced IL-12 production by DC. In contrast, the DN subset eliminated the DC by cytolysis and changed the living DC into a default subtype, in turn markedly down-regulating the levels of IL-12. Therefore, the DC stimulated by the CD4 subset preferentially induced Th1 responses, whereas the DC reacted with the DN subset induced a shift toward Th2 responses. These findings may provide an important insight into better understanding the contribution of iNKT-DC crosstalk governing the Th1/2 balance and the diverse influences of iNKT cells in various diseases.

- by Osamu Ishihara
- •
- Immunology, Cytokines, Dendritic Cells, Cell Differentiation

Microvesicles have been shown to mediate varieties of intercellular communication. Work in murine species has shown that lung-derived microvesicles can deliver mRNA, transcription factors, and microRNA to marrow cells and alter their phenotype. The present studies evaluated the capacity of excised human lung cancer cells to change the genetic phenotype of human marrow cells. We present the first studies on microvesicle production by excised cancers from human lung and the capacity of these microvesicles to alter the genetic phenotype of normal human marrow cells. We studied 12 cancers involving the lung and assessed nine lung-specific mRNA species (aquaporin, surfactant families, and clara cell-specific protein) in marrow cells exposed to tissue in co-culture, cultured in conditioned media, or exposed to isolated lung cancerderived microvesicles. We assessed two or seven days of co-culture and marrow which was unseparated, separated by ficoll density gradient centrifugation or ammonium chloride lysis. Under these varying conditions, each cancer derived from lung-mediated marrow expression of between one and seven lung-specific genes. Microvesicles were identified in the pellet of ultracentrifuged conditioned media and shown to enter marrow cells and induce lung-specific mRNA expression in marrow. A lung melanoma and a sarcoma also induced lung-specific mRNA in marrow cells. These data indicate that lung cancer cells may alter the genetic phenotype of normal cells and suggest that such perturbations might play a role in tumor progression, tumor

- by Gerald Colvin
- •
- Genetics, Biology, Medicine, Lung Cancer

Most of the commercial probiotic products are dairy-based, and the development of non-dairy probiotic products could be an alternative for new functional products. The peanut-soy milk (PSM(1)) was inoculated with six different lactic acid bacteria (LAB), including probiotic strains and yeasts and fermentation was accomplished for 24h at 37 °C and afterwards, another 24h at ±4 °C. The Pediococcus acidilactici (UFLA BFFCX 27.1), Lactococcus lactis (CCT 0360), Lactobacillus rhamnosus (LR 32) probiotic LAB, and the Lactobacillus delbrueckii subsp. bulgaricus (LB 340) yogurt starter culture reached cell concentrations of about 8.3log CFU/mL during fermentation. However, these strains were not able to acidify the substrate when inoculated as pure culture. The Lactobacillus acidophilus (LACA 4) probiotic produced significant amounts of lactic acid (3.35 g/L) and rapidly lowered the pH (4.6). Saccharomyces cerevisiae (UFLA YFFBM 18.03) did not completely consume the available sugars in PSM ...

- by Rosane Schwan
- •
- Microbiology, Probiotics, Biology, Industrial Biotechnology

Background: Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood-brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. Method: To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. Results: A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values 200 Ω⋅cm 2. After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. Conclusion: These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes.

- by Masami Niwa
- •
- Nanomedicine, Transmission Electron Microscopy, Nanotechnology, Medicine

Human astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools to recapitulate this complex interaction. To address this barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC)-derived astrocytes (hAstros) combined with neurons generated from hPSC-derived neural stem cells (hNeurons) or directly induced via Neurogenin 2 overexpression (iNeurons). Our systematic methods rapidly produce structurally complex hAstros and synapses in high-density coculture with iNeurons in precise numbers, allowing for improved studies of neural circuit function, disease modeling, and drug screening. We conclude that these bioengineered neural circuit model systems are reliable and scalable tools to accurately study aspects of human astrocyte-neuron functional properties whi...

- by Erik Ullian
- •
- Cell Differentiation, Neurons, Astrocytes, SYNAPSES
- by Bonnie Sloane
- •
- Cancer, Mice, Bone Metastasis, Bone marrow
- by David Hannaway
- •
- Regeneration, Genetic Engineering, Sterilization, Seeds

In Parkinson's disease (PD), neurogenesis is impaired in the subventricular zone (SVZ) of postmortem human PD brains, in primate nonhuman and rodent models of PD. The vital role of Wingless-type MMTV integration site (Wnt)/β-catenin signaling in the modulation of neurogenesis, neuroprotection, and synaptic plasticity coupled to our recent findings uncovering an active role for inflammation and Wnt/β-catenin signaling in MPTP-induced loss and repair of nigrostriatal dopaminergic (DAergic) neurons prompted us to study the impact of neuroinflammation and the Wnt/β-catenin pathway in the response of SVZ neuroprogenitors (NPCs) in MPTP-treated mice. In vivo experiments, using bromodeoxyuridine and cell-specific markers, and ex vivo time course analyses documented an inverse correlation between the reduced proliferation of NPCs and the generation of new neuroblasts with the phase of maximal exacerbation of microglia reaction, whereas a shift in the microglia proinflammatory phenotype...

- by B. Marchetti
- •
- Neuroscience, Stem Cells, Neurogenesis, Biology

- by André Kajdacsy-Balla
- •
- Nephrology, Andrology, Nonparametric Statistics, Cancer

Background: Mesenchymal stem cells have prominent immune modulatory properties, which may have clinical applications; however their major source, bone marrow, is of limited availability. On the other hand, mesenchymal stem cells derived from human exfoliated deciduous teeth (SHEDs) are readily accessible, but their immune regulatory properties have not been completely investigated. This study was designed, therefore, to evaluate the SHEDs influence on DCs differentiation, maturation, ability to activate T cells and to expand CD4 + Foxp3 + T cells. Methodology/Principal Findings: The experiments were based in cellular co-culture during differentiation and maturation of monocyte derived-DCs (moDCs), with, or not, presence of SHEDs. After co-culture with SHEDs, (moDCs) presented lower expression of BDCA-1 and CD11c, in comparison to DC cultivated without SHEDs. CD40, CD80, CD83 and CD86 levels were also decreased in mature DCs (mDCs) after co-cultivation with SHEDs. To assess the ability of SHEDs-exposed moDCs to modulate T cell responses, the former were separated from SHEDs, and co-cultured with peripheral blood lymphocytes. After 5 days, the proliferation of CD4 + and CD8 + T cells was evaluated and found to be lower than that induced by moDCs cultivated without SHEDs. In addition, an increase in the proportion of CD4 + Foxp3 + IL-10 + T cells was observed among cells stimulated by mature moDCs that were previously cultivated with SHEDs. Soluble factors released during co-cultures also showed a reduction in the pro-inflammatory cytokines (IL-2, TNF-a and IFN-c), and an increase in the anti-inflammatory molecule IL-10. Conclusion/Significance: This study shows that SHEDs induce an immune regulatory phenotype in moDCs cells, evidenced by changes in maturation and differentiation rates, inhibition of lymphocyte stimulation and ability to expand CD4 + Foxp3 + T cells. Further characterization and validation of this phenomenon could support the use of SHEDs, directly or indirectly for immune modulation in the clinical practice.

- by Fernando silva
- •
- Engineering, Physics, Chemistry, Biology

Bone marrow mesenchymal cells (MSCs) can protect leukemic cells from chemotherapy, thus increasing their survival rate. We studied the potential molecular mechanisms underlying this effect in acute lymphoblastic leukemia (ALL) cells. Coculture of ALL cells with MSCs induced on the lymphoblast plasma membrane the expression of a signaling complex formed by hERG1 (human ether-à-go-go-related gene 1) channels, the β1-integrin subunit, and the chemokine receptor CXC chemokine receptor-4. The assembly of such a protein complex activated both the extracellular signal-related kinase 1/2 (ERK1/2) and the phosphoinositide 3-kinase (PI3K)/Akt prosurvival signaling pathways. At the same time, ALL cells became markedly resistant to chemotherapy-induced apoptosis. hERG1 channel function appeared to be important for both the initiation of prosurvival signals and the development of drug resistance, because specific channel blockers decreased the protective effect of MSCs. NOD/SCID mice engrafted w...

- by Amedeo Amedei
- •
- Western blotting, Signal Transduction, RNA interference, Mice

Suspended and immobilized cocultures of the thermotolerant yeast, Kluyveromyces marxianus DMKU 3-1042 and the mesophilic flocculent yeast, Saccharomyces cerevisiae M30 were studied for their abilities to improve production and stability of ethanol fermentation. Sugarcane juice and blackstrap molasses, at initial sugar concentrations of 220 g/L, were used as carbon sources. The results indicated that the coculture system could improve ethanol production from both sugarcane juice and blackstrap molasses when the operating temperature ranged between 33 C and 45 C. High temperature tolerances were achieved when the coculture was immobilized. The immobilized coculture was more effective in high-temperature ethanol fermentation than the suspended cultures. The coculture immobilized on thin-shell silk cocoon and fermented at 37 C and 40 C generated maximal ethanol concentrations of 81.4 and 77.3 g/L, respectively, which were 5.9e8.7% and 16.8e39.0% higher than those of the suspended cultures, respectively.

- by Savitree Limtong
- •
- Engineering, Chemistry, Technology, Medicine

Highlights d Splice isoform signatures distinguish normal and malignant progenitor cell aging d Pro-survival splice isoform switching is a feature of secondary AML LSC d Splice isoform biomarkers provide diagnostic and therapeutic targets... more

- by Anil Sadarangani
- •
- Biology, Medicine, Hematopoiesis, Biological Sciences

We show here that PRL-3 protein is expressed in fetal heart, developing blood vessels, and pre-erythrocytes but not in their mature counterparts. These observations imply that PRL-3 may be involved in the early development of the circulatory system. Because PRL-3 mRNA had been reported to be consistently elevated in metastatic samples derived from colorectal cancers, we attempted to investigate if PRL-3 might be involved in tumor angiogenesis and if PRL-3-expressing cells could cross-talk to human umbilical vascular endothelial cells (HUVEC) by using an in vitro coculture system. HUVECs were grown with fibroblasts, which were later overlaid with PRL-3-expressing cells. We observed that both PRL-3expressing Chinese hamster ovary (CHO) cells and PRL-3-expressing DLD-1 human colon cancer cells could redirect the migration of HUVECs toward them; in addition, PRL-3-expressing DLD-1 cells could enhance HUVEC vascular formation. In vivo injection of PRL-3-expressing CHO cells into nude mice to form local tumors resulted in the recruitment of host endothelial cells into the tumors and initiation of angiogenesis. We further showed that PRL-3-expressing cells reduced interleukin-4 (IL-4) expression levels and thus attenuated IL-4 inhibitory effects on the HUVEC vasculature. Our findings provide direct evidence that PRL-3 may be involved in triggering angiogenesis and establishing microvasculature and it may serve as an attractive therapeutic target with respect to both angiogenesis and cancer metastasis.

- by Samantha Quah
- •
- Cancer, Colorectal cancer, Hematopoiesis, Hematopoietic Stem Cells

Eighteen fungi isolated from soil by hair bating method were tested against soil inhabiting Microsporum equinum, Microsporum fulvum, Microsporum gypseum and Microsporum racemosum for their antagonistic interactions. Colony inhibition during dual cultures showed inhibition of all the four Microsporum species. The maximum inhibition of M. equinum, M. fulvum, M. gypseum and M. racemosum was caused by Chrysosporium keratinophilum, Chrysosporium tropicum, Curvularia lunata and Chrysosporium lucknowense in dual cultures. On the other hand, M. fulvum showed maximum inhibition of Macrophomina phaseolina (70.1%) while M. equinum, M. gypseum and M. racemosum showed maximum inhibition of Colletotrichum gloeosporoides. Staling products of C. lucknowense accelerated growth of all Microsporum species, C. keratinophilum 3 and Chrysosporium evolceaunui and M. phaseolina accelerated growth of two species of Microsporum. Staling product of Alternaria alternata was most inhibitory. Culture filtrates o...

- by R.K.S. Kushwaha
- •
- Soil, Fungi, Soil Microbiology, Clinical Sciences

Human graft endothelial cells (ECs) can act as antigen-presenting cells to initiate allograft rejection by host memory T cells. Rapamycin, an mTOR inhibitor used clinically to suppress T cell responses, also acts on DCs, rendering them tolerogenic. Here, we report the effects of rapamycin on EC alloimmunogenicity. Compared with mock-treated cells, rapamycin-pretreated human ECs (rapa-ECs) stimulated less proliferation and cytokine secretion from allogeneic CD4 + memory cells, an effect mimicked by shRNA knockdown of mTOR or raptor in ECs. The effects of rapamycin persisted for several days and were linked to upregulation of the inhibitory molecules PD-L1 and PD-L2 on rapa-ECs. Additionally, rapa-ECs produced lower levels of the inflammatory cytokine IL-6. CD4 + memory cells activated by allogeneic rapa-ECs became hyporesponsive to restimulation in an alloantigen-specific manner and contained higher percentages of suppressive CD4 + CD25 hi CD127 lo FoxP3 + cells that did not produce effector cytokines. In a human-mouse chimeric model of allograft rejection, rapamycin pretreatment of human arterial allografts increased graft EC expression of PD-L1 and PD-L2 and reduced subsequent infiltration of allogeneic effector T cells into the artery intima and intimal expansion. Preoperative conditioning of allograft ECs with rapamycin could potentially reduce immune-mediated rejection. Conflict of interest: The authors have declared that no conflict of interest exists.

- by Tai Yi
- •
- Gene expression, Mice, Immunosuppression, Cell Proliferation

Preservation of smoked salmon from bacterial spoilage, and especially from Listeria monocytogenes, by bacteriocin producers is a promising challenge. Over a hundred lactic acid bacteria, isolated from commercial vacuum packaged cold smoked salmon, were screened for their antagonistic activity against L. innocua. Twenty-two strains were able to produce bacteriocin-like proteinaceous substances. These strains were characterized physiologically and biochemically as Carnobacterium strains. Three different groups were determined by pulsed-field gel electrophoresis after Sma I and Apa I DNA digestion. Peptidoglycan hydrolases patterns completed the characterization of these strains. All were confirmed as being Carnobacterium piscicola. Growth and bacteriocin production of three strains of each group and two well known bacteriocin producers (C. divergens V41 and C. piscicola V1) were tested in a simulated cold smoked fish system at 48C. These strains 8 21 were able to reach 10 cfu ml in 21 days and to produce as much bacteriocin activities in the cold smoked fish system as in the rich media. Carnobacterium divergens V41 and C. piscicola V1 were the most effective strains in co-culture experiments, inhibiting L. monocytogenes as early as day 4, whereas C. piscicola SF668 inhibiting effect was observed at day 13. The potential for using such biopreservation treatments on whole smoked salmon is discussed.

- by patrick boyaval
- •
- Microbiology, Food, Refrigeration, Industrial Biotechnology
- by thuc le
- •
- Cancer, Macrophages, Adipose tissue, Tumor microenvironment

To determine changes in the spatial and temporal distribution of cell-cell adhesion molecules during transendothelial migration of monocytes, we examined an in vitro model system of diapedesis using high resolution laser scanning confocal microscopy. Human arterial endothelial cells were cultured to confluence on coverslips coated with Matrigel and activated with IL-1beta before the addition of monocytic THP-1 cells. Seventy per cent of monocytes transmigrated through the endothelium within one hour. Diapedesis, but not adhesion and spreading, was inhibited 8-fold in co-cultures that contained endothelial cell conditioned medium, suggesting the release of an endothelial derived inhibitor. Double immunofluorescence labeling with antibodies to LFA-1, alpha- and beta-catenin, VE-cadherin and with Texas Red phalloidin, identified a circular transmigration passage in endothelial cell-cell contact regions. This passage was formed by an LFA-1-containing pseudopodium that penetrated between...

- by Martin Sandig
- •
- Confocal Microscopy, Biological Sciences, Vascular endothelium, Beta-Catenin

In early chick embryo, the precardiac cells reside within distinct groups of mesodermal cells known as presumptive heart forming regions (HFRs). HFRs are located on the lateral sides of the Hensen's node. In an effort to study fate of HFRs in isolation, HFRs were excised from early gastrulating chick embryos and cultured in vitro. A very small proportion of HFRs from 18 h incubated embryos differentiated into beating cardiomyocytes whereas about 43% of HFRs from embryos incubated for longer durations (20, 23 and 28 h) showed beating activity. The potential of HFRs, from 18 h incubated embryos, to differentiate into cardiomyocytes increased significantly in presence of Hensen's node. About one third of the HFR cells underwent spontaneous differentiation into adipocytes in culture. Simultaneously, some of the cells derived from HFRs exhibited alkaline phosphatase (AP) activity indicating presence of stem cells in the culture. HFR cells were positive for vimentin indicating their mesenchymal origin. FGF supplement increased the proportion of AP-positive cells in a dose dependent manner. The present study demonstrates that HFRs can serve as a source of mesenchymal stem cells which can be gainfully employed for various purposes. The results also suggest that even though the in vitro cultured HFRs from 18 h incubated HH stage 4 chick embryo retain the potential to undergo cardiac differentiation, certain instructive signals from Hensen's node may reinforce the fate.