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Quality of tumor lysates used for pulsing dendritic cells is influenced by the method used to harvest adherent tumor cells
Gloria Herzog, Ghasem Solgi, Denis S Wiegmann, Christian Nienhaus, Hubert Schrezenmeier, Tatjana Yildiz, Ramin Lotfi
BMC Research Notes , 2011, DOI: 10.1186/1756-0500-4-153
Abstract: Using three individual adherent colorectal tumor cell lines we also faced the difficulty to obtain consistent results regarding maturation inducing effect of tumor lysates on DCs. Therefore, we compared different methods to prepare tumor cell lysate and could demonstrate that trypsinizing as a method to harvest adherent tumor cells has a significant negative impact on biologic activity of tumor lysates. Specifically, we assessed induction of maturation markers CD40, CD80, and CD86 on DCs which were treated with differently prepared lysates.Trypsinizing is a very common way of harvesting adherent cells from culture flasks. Our results shall call investigators' attention to the enzymatic activity of trypsin degrading some possibly important proteins on the surface of cultured cells. Specifically for DC-based vaccination against tumor antigens investigators should avoid trypsin.Lysates from tumor cells are reported to induce maturation of dendritic cells (DCs) and are used as a source for tumor specific antigens [1,2], but as the maturation inducing effect is not strong enough [3], cytokine cocktails are added to DC cultures which are pulsed with tumor lysate [3,4], in the clinical setting of DC-based tumor vaccination. We aimed to enhance the effect of tumor lysate with regard to induction of DC maturation, but as reported by others [3] we had to face the problem that the tumor lysate rather did not induce DC maturation, in terms of upregulation of surface markers CD40, CD80, and CD86. We aimed to clarify the reason for conflicting results concerning lysate-induced DC maturation.Based on its protease activity trypsin cleaves cell surface proteins necessary for adherence. Trypsin at concentrations between 0.25% and 0.5% is generally used to detach and to harvest adherent (tumor) cells at the cost of loss of a portion of surface antigens. Therefore, we hypothesized that trypsin may crucially impact antigenicity of tumor lysate and subsequently its biological activity.By
MORPHOLOGICAL AND CYTOCHEMICAL CHARACTERISTICS OF PURIFIED MURINE SPLENIC DENDRITIC CELLS
A.H. Zarnani,S.M. Moazzeni,R. Shokri,Modzdeh Salehnia
Iranian Journal Of Allergy, Asthma and Immunology , 2003,
Abstract: Dendritic cells function as the main cellular population responsible for professional antigen presentation and hence for induction of primary immune responses. Although they are present in virtually every tissue, nevertheless their number is usually so low that it makes their isolation for studies very difficult.In this study, we purified dendritic cells from mouse spleen by a three-step enrich ment method and evaluated morphological and cytochemical characteristics of isolated cells.We showed that isolated dendritic cells from mouse spleen had all lobulated nuclei with multiple cytoplasmic projections and their morphological features changed after an overnight incubation. It was also shown that typical dendritic cells lacked both Myeloperoxidase (MPO) andNon Specific Esterase (NSE) activity.In conclusion, for reaching a reasonable purity in isolation of dendritic cells from lymphoid tissues, many enrichment steps should be taken, and for determining the pu rity of isolated cells, we recommend that a combination of morphological and cytochemi cal studies be used.
Identification of proteases employed by dendritic cells in the processing of protein purified derivative (PPD)  [cached]
Mohamadzadeh Mansour,Mohamadzadeh Hamid,Brammer Melissa,Sestak Karol
Journal of Immune Based Therapies and Vaccines , 2004, DOI: 10.1186/1476-8518-2-8
Abstract: Dendritic cells (DC) are known to present exogenous protein Ag effectively to T cells. In this study we sought to identify the proteases that DC employ during antigen processing. The murine epidermal-derived DC line Xs52, when pulsed with PPD, optimally activated the PPD-reactive Th1 clone LNC.2F1 as well as the Th2 clone LNC.4k1, and this activation was completely blocked by chloroquine pretreatment. These results validate the capacity of XS52 DC to digest PPD into immunogenic peptides inducing antigen specific T cell immune responses. XS52 DC, as well as splenic DC and DCs derived from bone marrow degraded standard substrates for cathepsins B, C, D/E, H, J, and L, tryptase, and chymases, indicating that DC express a variety of protease activities. Treatment of XS52 DC with pepstatin A, an inhibitor of aspartic acid proteases, completely abrogated their capacity to present native PPD, but not trypsin-digested PPD fragments to Th1 and Th2 cell clones. Pepstatin A also inhibited cathepsin D/E activity selectively among the XS52 DC-associated protease activities. On the other hand, inhibitors of serine proteases (dichloroisocoumarin, DCI) or of cystein proteases (E-64) did not impair XS52 DC presentation of PPD, nor did they inhibit cathepsin D/E activity. Finally, all tested DC populations (XS52 DC, splenic DC, and bone marrow-derived DC) constitutively expressed cathepsin D mRNA. These results suggest that DC primarily employ cathepsin D (and perhaps E) to digest PPD into antigenic peptides.
A new approach for the large-scale generation of mature dendritic cells from adherent PBMC using roller bottle technology  [cached]
Campbell-Anson Ryan E,Kentor Diane,Wang Yi J,Bushnell Kathryn M
Journal of Immune Based Therapies and Vaccines , 2008, DOI: 10.1186/1476-8518-6-1
Abstract: Background Human monocyte-derived DC (mDC) loaded with peptides, protein, tumor cell lysates, or tumor cell RNA, are being tested as vaccines against multiple human malignancies and viral infection with great promise. One of the factors that has limited more widespread use of these vaccines is the need to generate mDC in large scale. Current methods for the large-scale cultivation of mDC in static culture vessels are labor- and time- intensive, and also require many culture vessels. Here, we describe a new method for the large-scale generation of human mDC from human PBMC from leukopheresis or buffy coat products using roller bottles, never attempted before for mDC generation. We have tested this technology using 850 cm2 roller bottles compared to conventional T-175 flat-bottom static culture flasks. Methods DC were generated from adherent human PBMC from buffy coats or leukopherisis products using GM-CSF and IL-4 in T-175 static flasks or 850 cm2 roller bottles. The cells were matured over two days, harvested and analyzed for cell yield and mature DC phenotype by flow cytometry, and then functionally analyzed for their ability to activate allogeneic T-cell or recall antigen peptide-specific T-cell responses. Results Monocytes were found to adhere inside roller bottles to the same extent as in static culture flasks. The phenotype and function of the mDC harvested after maturation from both type of culture systems were similar. The yield of mDC from input PBMC in the roller bottle system was similar as in the static flask system. However, each 850 cm2 roller bottle could be seeded with 4–5 times more input PBMC and could yield 4–5 times as many mDC per culture vessel than the static flasks as a result. Conclusion Our results indicate that the roller bottle technology can generate similar numbers of mDC from adherent PBMC as traditional static flask methods, but with having to use fewer culture vessels. Thus, this may be a more practical method to generate mDC in large-scale cutting down on the amount of laboratory manipulations, and can save both time and labor costs.
Immunotherapy Using Dendritic Cells against Multiple Myeloma: How to Improve?
Thanh-Nhan Nguyen-Pham,Yoon-Kyung Lee,Hyeoung-Joon Kim,Je-Jung Lee
Clinical and Developmental Immunology , 2012, DOI: 10.1155/2012/397648
Abstract: Multiple myeloma (MM) is a good target disease in which one can apply cellular immunotherapy, which is based on the graft-versus-myeloma effect. This role of immune effector cells provides the framework for the development of immune-based therapeutic options that use antigen-presenting cells (APCs) with increased potency, such as dendritic cells (DCs), in MM. Current isolated idiotype (Id), myeloma cell lysates, myeloma dying cells, DC-myeloma hybrids, or DC transfected with tumor-derived RNA has been used for immunotherapy with DCs. Immunological inhibitory cytokines, such as TGF-β, IL-10, IL-6 and VEGF, which are produced from myeloma cells, can modulate antitumor host immune response, including the abrogation of DC function, by constitutive activation of STAT3. Therefore, even the immune responses have been observed in clinical trials, the clinical response was rarely improved following DC vaccinations in MM patients. We are going to discuss how to improve the efficacy of DC vaccination in MM.
Role of plasminogen activators and inhibitors in reproduction
Yixun Liu
Chinese Science Bulletin , 1999, DOI: 10.1007/BF02909700
Abstract: The recent progress in the studies on the role of local and directed fibrinolysis controlled by plasminogen activators (PAs) and regulated by their inhibitors (PAIs) in reproduction is summarized. Hormone-induced coordinated expression of tissue type PA (tPA) and PAI type-1 (PAI-1) in the ovary is involved in the processes of ovulation and luteal regression; increases in urokinase type PA (uPA) and PAI-1 activity in the early stage of luteinized follicles may be responsible for ovarian tissue remodeling and angiogenesis; the PA system has been found to play an important role in spermatogenesis in testis and modulation of sperm maturation in epididymis. PA and matrix matalloproteanase (MMP) and their respective inhibitors have also been identified in trophoblast and uterus. The targeted proteolytic activity generated by the two systems may play an essential role in the processes of the cyclic uterine angiogenesis, implantation and placentation as well as parturition.
Dendritic Cell  [PDF]
Sevda S?ker
Dicle Medical Journal , 2005,
Abstract: Dendritic cells, a member of family of antigen presenting cells, are most effective cells in the primary immune response. Dendritic cells originated from dendron, in mean of tree in the Greek, because of their long and elaborate cytoplasmic branching processes. Dendritic cells constitute approximately 0.1 to 1 percent of the blood’s mononuclear cell. Dendritic cells are widely distributed, and specialized for antigen capture and T cell stimulation. In this article, structures and functions of dendritic cells are reviewed.
Regulation of Tumor Immunity by Tumor/Dendritic Cell Fusions  [PDF]
Shigeo Koido,Sadamu Homma,Eiichi Hara,Yoshihisa Namiki,Akitaka Takahara,Hideo Komita,Eijiro Nagasaki,Masaki Ito,Toshifumi Ohkusa,Jianlin Gong,Hisao Tajiri
Clinical and Developmental Immunology , 2010, DOI: 10.1155/2010/516768
Abstract: The goal of cancer vaccines is to induce antitumor immunity that ultimately will reduce tumor burden in tumor environment. Several strategies involving dendritic cells- (DCs)- based vaccine incorporating different tumor-associated antigens to induce antitumor immune responses against tumors have been tested in clinical trials worldwide. Although DCs-based vaccine such as fusions of whole tumor cells and DCs has been proven to be clinically safe and is efficient to enhance antitumor immune responses for inducing effective immune response and for breaking T-cell tolerance to tumor-associated antigens (TAAs), only a limited success has occurred in clinical trials. This paper reviews tumor immune escape and current strategies employed in the field of tumor/DC fusions vaccine aimed at enhancing activation of TAAs-specific cytotoxic T cells in tumor microenvironment.
Potential Therapeutic Use of PPAR-Programed Human Monocyte-Derived Dendritic Cells in Cancer Vaccination Therapy  [PDF]
Adrienn Gy ngy si,László Nagy
PPAR Research , 2008, DOI: 10.1155/2008/473804
Abstract: Dendritic cells (DCs) can regulate all elements of the immune system, and therefore are an ideal target for vaccination. During the last two decades, as a result of extensive research, DCs became the primary target of antitumor vaccination as well. A critical issue of antitumor vaccination is the phenotype of the dendritic cell used. It has been recently shown that several nuclear hormone receptors, and amongst them the lipid-activated nuclear receptor and peroxisome proliferator-activated receptor gamma (PPAR), have important roles in effecting the immunophenotype of human dendritic cells. It regulates primarily lipid metabolism and via this it influences the immunophenotype of DCs by altering lipid antigen uptake, presentation, and also other immune functions. In this review, we summarize the principles of antitumor vaccination strategies and present our hypothesis on how PPAR-regulated processes might be involved and could be exploited in the design of vaccination strategies.
Discovery of Novel Small Molecule Activators of β-Catenin Signaling  [PDF]
Folkert Verkaar,Mario van der Stelt,W. Matthijs Blankesteijn,Antoon A. van der Doelen,Guido J. R. Zaman
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0019185
Abstract: Wnt/β-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/β-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed bona fide activators of β-catenin signaling. The compounds stabilized cytoplasmic β-catenin and activated β–catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate β-catenin signaling has yet to be determined, several key regulators of Wnt/β-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced β-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/β-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/β-catenin signaling.
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