62 results on '"Germeraad WT"'
Search Results
2. Efficient retrovirus-mediated gene transduction into murine hematopoietic stem cells and long-lasting expression using a Transwell coculture system
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Germeraad, WT, primary, Asami, N, additional, Fujimoto, S, additional, Mazda, O, additional, and Katsura, Y, additional
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- 1994
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3. Fractionated Radiotherapy with 3 x 8 Gy Induces Systemic Anti-Tumour Responses and Abscopal Tumour Inhibition without Modulating the Humoral Anti-Tumour Response.
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Habets TH, Oth T, Houben AW, Huijskens MJ, Senden-Gijsbers BL, Schnijderberg MC, Brans B, Dubois LJ, Lambin P, De Saint-Hubert M, Germeraad WT, Tilanus MG, Mottaghy FM, Bos GM, and Vanderlocht J
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- Animals, Carcinoma immunology, Carcinoma pathology, Dendritic Cells immunology, Dendritic Cells pathology, Dose Fractionation, Radiation, Female, Immunoglobulin Isotypes blood, Mammary Glands, Animal immunology, Mammary Glands, Animal pathology, Mammary Neoplasms, Experimental immunology, Mammary Neoplasms, Experimental pathology, Membrane Proteins pharmacology, Mice, Mice, Inbred BALB C, T-Lymphocytes immunology, T-Lymphocytes pathology, Antibodies, Monoclonal biosynthesis, Carcinoma radiotherapy, Gamma Rays therapeutic use, Immunity, Humoral, Mammary Glands, Animal radiation effects, Mammary Neoplasms, Experimental radiotherapy
- Abstract
Accumulating evidence indicates that fractionated radiotherapy (RT) can result in distant non-irradiated (abscopal) tumour regression. Although preclinical studies indicate the importance of T cells in this infrequent phenomenon, these studies do not preclude that other immune mechanisms exhibit an addition role in the abscopal effect. We therefore addressed the question whether in addition to T cell mediated responses also humoral anti-tumour responses are modulated after fractionated RT and whether systemic dendritic cell (DC) stimulation can enhance tumour-specific antibody production. We selected the 67NR mammary carcinoma model since this tumour showed spontaneous antibody production in all tumour-bearing mice. Fractionated RT to the primary tumour was associated with a survival benefit and a delayed growth of a non-irradiated (contralateral) secondary tumour. Notably, fractionated RT did not affect anti-tumour antibody titers and the composition of the immunoglobulin (Ig) isotypes. Likewise, we demonstrated that treatment of tumour-bearing Balb/C mice with DC stimulating growth factor Flt3-L did neither modulate the magnitude nor the composition of the humoral immune response. Finally, we evaluated the immune infiltrate and Ig isotype content of the tumour tissue using flow cytometry and found no differences between treatment groups that were indicative for local antibody production. In conclusion, we demonstrate that the 67NR mammary carcinoma in Balb/C mice is associated with a pre-existing antibody response. And, we show that in tumour-bearing Balb/C mice with abscopal tumour regression such pre-existing antibody responses are not altered upon fractionated RT and/or DC stimulation with Flt3-L. Our research indicates that evaluating the humoral immune response in the setting of abscopal tumour regression is not invariably associated with therapeutic effects.
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- 2016
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4. Ascorbic acid serum levels are reduced in patients with hematological malignancies.
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Huijskens MJ, Wodzig WK, Walczak M, Germeraad WT, and Bos GM
- Abstract
In this paper we demonstrate that patients treated with chemotherapy and/or hematopoietic stem cell transplantation (HSCT) have highly significant reduced serum ascorbic acid (AA) levels compared to healthy controls. We recently observed in in vitro experiments that growth of both T and NK cells from hematopoietic stem cells is positively influenced by AA. It might be of clinical relevance to study the function and recovery of immune cells after intensive treatment, its correlation to AA serum levels and the possible effect of AA supplementation.
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- 2016
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5. Pathogen-Associated Molecular Patterns Induced Crosstalk between Dendritic Cells, T Helper Cells, and Natural Killer Helper Cells Can Improve Dendritic Cell Vaccination.
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Oth T, Vanderlocht J, Van Elssen CH, Bos GM, and Germeraad WT
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- Animals, Dendritic Cells immunology, Humans, Killer Cells, Natural immunology, Models, Biological, T-Lymphocytes, Helper-Inducer immunology, Dendritic Cells metabolism, Killer Cells, Natural metabolism, Pathogen-Associated Molecular Pattern Molecules metabolism, T-Lymphocytes, Helper-Inducer metabolism
- Abstract
A coordinated cellular interplay is of crucial importance in both host defense against pathogens and malignantly transformed cells. The various interactions of Dendritic Cells (DC), Natural Killer (NK) cells, and T helper (Th) cells can be influenced by a variety of pathogen-associated molecular patterns (PAMPs) and will lead to enhanced CD8(+) effector T cell responses. Specific Pattern Recognition Receptor (PRR) triggering during maturation enables DC to enhance Th1 as well as NK helper cell responses. This effect is correlated with the amount of IL-12p70 released by DC. Activated NK cells are able to amplify the proinflammatory cytokine profile of DC via the release of IFN-γ. The knowledge on how PAMP recognition can modulate the DC is of importance for the design and definition of appropriate therapeutic cancer vaccines. In this review we will discuss the potential role of specific PAMP-matured DC in optimizing therapeutic DC-based vaccines, as some of these DC are efficiently activating Th1, NK cells, and cytotoxic T cells. Moreover, to optimize these vaccines, also the inhibitory effects of tumor-derived suppressive factors, for example, on the NK-DC crosstalk, should be taken into account. Finally, the suppressive role of the tumor microenvironment in vaccination efficacy and some proposals to overcome this by using combination therapies will be described.
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- 2016
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6. Multipotent adult progenitor cells for hypoxic-ischemic injury in the preterm brain.
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Jellema RK, Ophelders DR, Zwanenburg A, Nikiforou M, Delhaas T, Andriessen P, Mays RW, Deans R, Germeraad WT, Wolfs TG, and Kramer BW
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- Animals, Animals, Newborn, Disease Models, Animal, Fetus, Sheep, Adult Stem Cells transplantation, Hypoxia-Ischemia, Brain therapy, Mesenchymal Stem Cell Transplantation methods, Premature Birth
- Abstract
Background: Preterm infants are at risk for hypoxic-ischemic encephalopathy. No therapy exists to treat this brain injury and subsequent long-term sequelae. We have previously shown in a well-established pre-clinical model of global hypoxia-ischemia (HI) that mesenchymal stem cells are a promising candidate for the treatment of hypoxic-ischemic brain injury. In the current study, we investigated the neuroprotective capacity of multipotent adult progenitor cells (MAPC®), which are adherent bone marrow-derived cells of an earlier developmental stage than mesenchymal stem cells and exhibiting more potent anti-inflammatory and regenerative properties., Methods: Instrumented preterm sheep fetuses were subjected to global hypoxia-ischemia by 25 min of umbilical cord occlusion at a gestational age of 106 (term ~147) days. During a 7-day reperfusion period, vital parameters (e.g., blood pressure and heart rate; baroreceptor reflex) and (amplitude-integrated) electroencephalogram were recorded. At the end of the experiment, the preterm brain was studied by histology., Results: Systemic administration of MAPC therapy reduced the number and duration of seizures and prevented decrease in baroreflex sensitivity after global HI. In addition, MAPC cells prevented HI-induced microglial proliferation in the preterm brain. These anti-inflammatory effects were associated with MAPC-induced prevention of hypomyelination after global HI. Besides attenuation of the cerebral inflammatory response, our findings showed that MAPC cells modulated the peripheral splenic inflammatory response, which has been implicated in the etiology of hypoxic-ischemic injury in the preterm brain., Conclusions: In a pre-clinical animal model MAPC cell therapy improved the functional and structural outcome of the preterm brain after global HI. Future studies should establish the mechanism and long-term therapeutic effects of neuroprotection established by MAPC cells in the developing preterm brain exposed to HI. Our study may form the basis for future clinical trials, which will evaluate whether MAPC therapy is capable of reducing neurological sequelae in preterm infants with hypoxic-ischemic encephalopathy.
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- 2015
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7. Potency of Both Human Th1 and NK Helper Cell Activation is Determined by IL-12p70-Producing PAMP-Matured DCs.
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Oth T, Van Elssen CH, Schnijderberg MC, Senden-Gijsbers BL, Germeraad WT, Bos GM, and Vanderlocht J
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- Cells, Cultured, Coculture Techniques methods, Cytokines immunology, Cytotoxicity, Immunologic immunology, Humans, Interferon-gamma immunology, Klebsiella pneumoniae immunology, Poly I-C immunology, Signal Transduction immunology, Dendritic Cells immunology, Interleukin-12 immunology, Killer Cells, Natural immunology, Lymphocyte Activation immunology, Membranes immunology, T-Lymphocytes, Helper-Inducer immunology, Th1 Cells immunology
- Abstract
Besides T helper (Th) cells, natural killer (NK) cells have also been described to participate in the shaping of dendritic cell (DC)-mediated adaptive immune responses. At present, it remains unclear to what extent the induction of these NK helper cell immune mechanisms is coupled with Th responses and whether both helper immune responses are induced by the same DC upon specific pathogen recognition receptor (PRR) stimulation. In this study, we demonstrate that maturation of DCs with a cocktail containing FMKp (membrane fragments of Klebsiella pneumoniae) mounts both Th cell and NK cell helper responses in a PRR-triggered dose-dependent manner as determined by the capacity of the helper cells to produce IFN-γ. Furthermore, by triggering an additional PRR pathway [FMKp in combination with poly(I:C) lyovec], we reveal that both approaches modulate the amount of DC-derived IL-12p70 and that this cytokine is the key determinant of the DC-induced Th1 and NK cell helper responses. Moreover, all PRR triggers able to induce IL-12-producing mature DCs are sufficient to induce these helper responses. We propose the existence of a single program used by DCs to induce potent cellular immune responses by stimulating both T helper and NK cell helper processes. This knowledge can help to select the proper PRR triggers in preventive and therapeutic vaccine design.
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- 2015
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8. Combination of radiotherapy with the immunocytokine L19-IL2: Additive effect in a NK cell dependent tumour model.
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Rekers NH, Zegers CM, Yaromina A, Lieuwes NG, Biemans R, Senden-Gijsbers BL, Losen M, Van Limbergen EJ, Germeraad WT, Neri D, Dubois L, and Lambin P
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- Animals, Antineoplastic Agents administration & dosage, Combined Modality Therapy, Disease Models, Animal, Drug Administration Schedule, Fibronectins metabolism, Histocompatibility Antigens Class I metabolism, Immunity, Cellular immunology, Mice, Mice, Inbred Strains, Neoplasms immunology, Recombinant Fusion Proteins administration & dosage, Antineoplastic Agents pharmacology, Killer Cells, Natural immunology, Neoplasms radiotherapy, Radioimmunotherapy methods, Recombinant Fusion Proteins pharmacology
- Abstract
Background and Purpose: Recently, we have shown that radiotherapy (RT) combined with the immunocytokine L19-IL2 can induce long-lasting antitumour effects, dependent on ED-B expression and infiltration of cytotoxic T cells. On the other hand, in certain tumours, IL2 treatment can trigger a natural killer cell (NK) immune response. The aim of this study is to investigate the therapeutic effect of our combination therapy in the ED-B positive F9 teratocarcinoma model, lacking MHCI expression and known to be dependent on NK immune responses., Material and Methods: In syngeneic F9 tumour bearing 129/FvHsd mice tumour growth delay was evaluated after local tumour irradiation (10Gy) combined with systemic administration of L19-IL2. Immunological responses were investigated using flow cytometry., Results: Tumour growth delay of L19-IL2 can be further improved by a single dose of RT administered before immunotherapy, but not during immunotherapy. Furthermore, treatment of L19-IL2 favours a NK response and lacks cytotoxic T cell tumour infiltrating immune cells, which may be explained by the absence of MHCI expression., Conclusion: An additive effect can be detected when the NK dependent F9 tumour model is treated with radiotherapy and L19-IL2 and therefore this combination could be useful in the absence of tumoural MHCI expression., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)
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- 2015
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9. Prophylactic Interleukin-2 Treatment Prevents Fetal Gut Inflammation and Injury in an Ovine Model of Chorioamnionitis.
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Nikiforou M, Vanderlocht J, Chougnet CA, Jellema RK, Ophelders DR, Joosten M, Kloosterboer N, Senden-Gijsbers BL, Germeraad WT, Kramer BW, and Wolfs TG
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- Analgesics, Non-Narcotic pharmacology, Animals, CD3 Complex drug effects, Chorioamnionitis blood, Cytokines genetics, Cytokines metabolism, Disease Models, Animal, Female, Ileum immunology, Ileum pathology, Interleukin-2 pharmacology, Intestinal Mucosa injuries, Intestinal Mucosa pathology, Lipopolysaccharides, Lymph Nodes metabolism, Mesentery, Neutrophils drug effects, Pregnancy, Prenatal Injuries etiology, Protective Agents administration & dosage, Protective Agents pharmacology, RNA, Messenger drug effects, Random Allocation, Sheep, T-Lymphocytes, Regulatory metabolism, Analgesics, Non-Narcotic administration & dosage, Chorioamnionitis pathology, Enteritis prevention & control, Interleukin-2 administration & dosage, Prenatal Injuries prevention & control
- Abstract
Background: Chorioamnionitis results from an infection of the fetal membranes and is associated with fetal adverse outcomes notably in the intestine. Using a translational ovine model, we showed that intra-amniotic exposure to inflammatory stimuli decreased the regulatory/effector T (Treg/Teff) cell balance in the gut, which was accompanied by intestinal inflammation and mucosal injury. We thus aimed to augment the Treg/Teff cell ratio in the fetal gut by prophylactic IL-2 treatment and evaluate whether it is sufficient to prevent chorioamnionitis-induced intestinal inflammation and mucosal injury., Methods: Fetal sheep (122 d of gestation) were intra-amniotically exposed to lipopolysaccharide for 2 or 7 days with or without prophylactic IL-2 treatment (4 d). We evaluated the infiltration of inflammatory cells in the ileum and mesenteric lymph nodes. Cytokine gene expression was analyzed in fetal ileum and the inflammatory changes were correlated with gut wall integrity., Results: IL-2 administration preferentially increased intestinal Treg cells and thus the Treg/Teff cell ratio. Prophylactic IL-2 treatment reduced the lipopolysaccharide-induced influx of neutrophils and CD3(+) T cells and decreased the messenger RNA levels of proinflammatory cytokines including IL-6 and IL-17 in the fetal ileum. Importantly, prophylactic IL-2 treatment prevented mucosal damage without inducing fetal adverse treatment outcomes., Conclusions: Our data show that prophylactic IL-2 treatment prevents fetal intestinal inflammation and mucosal injury in the context of experimental chorioamnionitis. Modulation of the Treg/Teff cell balance may contribute to the protective effects of IL-2.
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- 2015
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10. Optimal selection of natural killer cells to kill myeloma: the role of HLA-E and NKG2A.
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Sarkar S, van Gelder M, Noort W, Xu Y, Rouschop KM, Groen R, Schouten HC, Tilanus MG, Germeraad WT, Martens AC, Bos GM, and Wieten L
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- Animals, Cell Degranulation, Cell Line, Tumor, Coculture Techniques, Cytotoxicity, Immunologic, DNA-Binding Proteins genetics, Flow Cytometry, Humans, Interleukin-2 immunology, Mice, Mice, Knockout, Multiple Myeloma immunology, Neoplasm Transplantation, Oxygen metabolism, HLA-E Antigens, Cell Separation methods, Histocompatibility Antigens Class I immunology, Immunotherapy methods, Killer Cells, Natural immunology, Killer Cells, Natural transplantation, Multiple Myeloma therapy, NK Cell Lectin-Like Receptor Subfamily C immunology
- Abstract
Immunotherapy with allogeneic natural killer (NK) cells offers therapeutic perspectives for multiple myeloma patients. Here, we aimed to refine NK cell therapy by evaluation of the relevance of HLA-class I and HLA-E for NK anti-myeloma reactivity. We show that HLA-class I was strongly expressed on the surface of patient-derived myeloma cells and on myeloma cell lines. HLA-E was highly expressed by primary myeloma cells but only marginally by cell lines. HLA-E(low) expression on U266 cells observed in vitro was strongly upregulated after in vivo (bone marrow) growth in RAG-2(-/-) γc(-/-) mice, suggesting that in vitro HLA-E levels poorly predict the in vivo situation. Concurrent analysis of inhibitory receptors (KIR2DL1, KIR2DL2/3, KIR3DL1 and NKG2A) and NK cell degranulation upon co-culture with myeloma cells revealed that KIR-ligand-mismatched NK cells degranulate more than matched subsets and that HLA-E abrogates degranulation of NKG2A+ subsets. Inhibition by HLA-class I and HLA-E was also observed with IL-2-activated NK cells and at low oxygen levels (0.6 %) mimicking hypoxic bone marrow niches where myeloma cells preferentially reside. Our study demonstrates that NKG2A-negative, KIR-ligand-mismatched NK cells are the most potent subset for clinical application. We envision that infusion of high numbers of this subclass will enhance clinical efficacy.
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- 2015
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11. Ascorbic acid promotes proliferation of natural killer cell populations in culture systems applicable for natural killer cell therapy.
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Huijskens MJ, Walczak M, Sarkar S, Atrafi F, Senden-Gijsbers BL, Tilanus MG, Bos GM, Wieten L, and Germeraad WT
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- Cell Differentiation drug effects, Cell Proliferation drug effects, Cells, Cultured, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells drug effects, Hematopoietic Stem Cells metabolism, Humans, K562 Cells, Killer Cells, Natural drug effects, Stem Cells cytology, Stem Cells drug effects, Ascorbic Acid pharmacology, Cell Culture Techniques methods, Killer Cells, Natural cytology, Killer Cells, Natural transplantation
- Abstract
Background Aims: Natural killer (NK) cell-based immunotherapy is a promising treatment for a variety of malignancies. However, generating sufficient cell numbers for therapy remains a challenge. To achieve this, optimization of protocols is required., Methods: Mature NK cells were expanded from peripheral blood mononuclear cells PBMCs in the presence of anti-CD3 monoclonal antibody and interleukin-2. Additionally, NK-cell progenitors were generated from CD34(+) hematopoietic stem cells or different T/NK-cell progenitor populations. Generated NK cells were extensively phenotyped, and functionality was determined by means of cytotoxicity assay., Results: Addition of ascorbic acid (AA) resulted in more proliferation of NK cells without influencing NK-cell functionality. In more detail, PBMC-derived NK cells expanded 2362-fold (median, range: 90-31,351) in the presence of AA and were capable of killing tumor cells under normoxia and hypoxia. Moreover, hematopoietic stem cell-derived progenitors appeared to mature faster in the presence of AA, which was also observed in the NK-cell differentiation from early T/NK-cell progenitors., Conclusions: Mature NK cells proliferate faster in the presence of phospho-L-AA, resulting in higher cell numbers with accurate functional capacity, which is required for adoptive immunotherapy., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)
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- 2015
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12. Long-lasting antitumor effects provided by radiotherapy combined with the immunocytokine L19-IL2.
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Rekers NH, Zegers CM, Germeraad WT, Dubois L, and Lambin P
- Abstract
Recently, we have shown that radiotherapy (RT) combined with L19-IL2 can induce a long-lasting antitumor effect, dependent on ED-B expression and infiltration of cytotoxic T cells. These findings will be translated to a Phase I clinical study (NCT02086721) in patients with oligometastatic solid tumors. See this link for the animation: http://youtu.be/xHbwQuCTkRc.
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- 2015
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13. An acute intake of plant stanol esters alters immune-related pathways in the jejunum of healthy volunteers.
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De Smet E, Mensink RP, Boekschoten MV, de Ridder R, Germeraad WT, Wolfs TG, and Plat J
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- Adult, Anticholesteremic Agents adverse effects, Antigens, Surface blood, Antigens, Surface genetics, Antigens, Surface metabolism, Beverages, Cross-Over Studies, Double-Blind Method, Down-Regulation, Duodenum cytology, Duodenum immunology, Duodenum metabolism, Female, Forkhead Transcription Factors blood, Forkhead Transcription Factors genetics, Forkhead Transcription Factors metabolism, Gene Expression Regulation, Humans, Intestinal Mucosa cytology, Intestinal Mucosa metabolism, Jejunum cytology, Jejunum metabolism, Male, Middle Aged, Sitosterols adverse effects, T-Lymphocytes cytology, T-Lymphocytes metabolism, Young Adult, Anticholesteremic Agents administration & dosage, Immunity, Mucosal, Immunomodulation, Intestinal Mucosa immunology, Jejunum immunology, Sitosterols administration & dosage, T-Lymphocytes immunology
- Abstract
Plant sterols and stanols inhibit intestinal cholesterol absorption and consequently lower serum LDL-cholesterol (LDL-C) concentrations. The underlying mechanisms are not yet known. In vitro and animal studies have suggested that changes in intestinal sterol metabolism are attributed to the LDL-C-lowering effects of plant stanol esters. However, similar studies in human subjects are lacking. Therefore, we examined the effects of an acute intake of plant stanol esters on gene expression profiles of the upper small intestine in healthy volunteers. In a double-blind cross-over design, fourteen healthy subjects (eight female and six male; age 21-55 years), with a BMI ranging from 21 to 29 kg/m², received in random order a shake with or without plant stanol esters (4 g). At 5 h after consumption of the shake, biopsies were taken from the duodenum (around the papilla of Vater) and from the jejunum (20 cm distal from the papilla of Vater). Microarray analysis showed that the expression profiles of genes involved in sterol metabolism were not altered. Surprisingly, the pathways involved in T-cell functions were down-regulated in the jejunum. Furthermore, immunohistochemical analysis showed that the number of CD3 (cluster of differentiation number 3), CD4 (cluster of differentiation number 4) and Foxp3⁺ (forkhead box P3-positive) cells was reduced in the plant stanol ester condition compared with the control condition, which is in line with the microarray data. The physiological and functional consequences of the plant stanol ester-induced reduction of intestinal T-cell-based immune activity in healthy subjects deserve further investigation.
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- 2015
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14. Radiotherapy combined with the immunocytokine L19-IL2 provides long-lasting antitumor effects.
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Zegers CM, Rekers NH, Quaden DH, Lieuwes NG, Yaromina A, Germeraad WT, Wieten L, Biessen EA, Boon L, Neri D, Troost EG, Dubois LJ, and Lambin P
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- Animals, Antineoplastic Agents administration & dosage, Cell Line, Tumor, Combined Modality Therapy, Disease Models, Animal, Fibronectins metabolism, Humans, Lymphocyte Depletion, Mice, Neoplasms drug therapy, Neoplasms immunology, Neoplasms metabolism, Neoplasms mortality, Neoplasms radiotherapy, Radiotherapy Dosage, Recombinant Fusion Proteins administration & dosage, T-Lymphocytes, Cytotoxic immunology, T-Lymphocytes, Cytotoxic metabolism, Tumor Burden drug effects, Tumor Burden radiation effects, Xenograft Model Antitumor Assays, Antineoplastic Agents pharmacology, Neoplasms pathology, Recombinant Fusion Proteins pharmacology
- Abstract
Purpose: Radiotherapy modifies the tumor microenvironment and causes the release of tumor antigens, which can enhance the effect of immunotherapy. L19 targets the extra domain B (ED-B) of fibronectin, a marker for tumor neoangiogenesis, and can be used as immunocytokine when coupled to IL2. We hypothesize that radiotherapy in combination with L19-IL2 provides an enhanced antitumor effect, which is dependent on ED-B expression., Experimental Design: Mice were injected with syngeneic C51 colon carcinoma, Lewis lung carcinoma (LLC), or 4T1 mammary carcinoma cells. Tumor growth delay, underlying immunologic parameters, and treatment toxicity were evaluated after single-dose local tumor irradiation and systemic administration of L19-IL2 or equimolar controls., Results: ED-B expression was high, intermediate, and low for C51, LLC, and 4T1, respectively. The combination therapy showed (i) a long-lasting synergistic effect for the C51 model with 75% of tumors being cured, (ii) an additive effect for the LLC model, and (iii) no effect for the 4T1 model. The combination treatment resulted in a significantly increased cytotoxic (CD8(+)) T-cell population for both C51 and LLC. Depletion of CD8(+) T cells abolished the benefit of the combination therapy., Conclusions: These data provide the first evidence for an increased therapeutic potential by combining radiotherapy with L19-IL2 in ED-B-positive tumors. This new opportunity in cancer treatment will be investigated in a phase I clinical study for patients with an oligometastatic solid tumor (NCT02086721). An animation summarizing our results is available at https://www.youtube.com/watch?v=xHbwQuCTkRc., (©2014 American Association for Cancer Research.)
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- 2015
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15. Heat-transfer-method-based cell culture quality assay through cell detection by surface imprinted polymers.
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Eersels K, van Grinsven B, Khorshid M, Somers V, Püttmann C, Stein C, Barth S, Diliën H, Bos GM, Germeraad WT, Cleij TJ, Thoelen R, De Ceuninck W, and Wagner P
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- Cell Culture Techniques, Cell Line, Tumor, Humans, Microsatellite Repeats, Molecular Probes metabolism, Polymers metabolism, Quality Control, Surface Properties, Biomimetics methods, Hot Temperature, Molecular Imprinting, Molecular Probes chemical synthesis, Polymers chemical synthesis
- Abstract
Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time.
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- 2015
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16. Five decades of Dutch immunology.
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Germeraad WT, de Jong EC, and Trouw LA
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- Animals, Anniversaries and Special Events, History, 20th Century, History, 21st Century, Humans, Netherlands, Allergy and Immunology history
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- 2014
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17. Technical advance: ascorbic acid induces development of double-positive T cells from human hematopoietic stem cells in the absence of stromal cells.
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Huijskens MJ, Walczak M, Koller N, Briedé JJ, Senden-Gijsbers BL, Schnijderberg MC, Bos GM, and Germeraad WT
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- Antigens, Differentiation, T-Lymphocyte analysis, Antigens, Differentiation, T-Lymphocyte biosynthesis, Antioxidants pharmacology, Cell Division drug effects, Cells, Cultured, Citrulline metabolism, Coculture Techniques, Filgrastim, Gene Expression Profiling, Gene Rearrangement, T-Lymphocyte, Granulocyte Colony-Stimulating Factor pharmacology, Hematopoietic Stem Cell Mobilization, Hematopoietic Stem Cells cytology, Humans, Immunomagnetic Separation, Integrins analysis, Nitric Oxide metabolism, Primary Cell Culture methods, Receptors, Chemokine analysis, Recombinant Proteins pharmacology, Stromal Cells, omega-N-Methylarginine pharmacology, Ascorbic Acid pharmacology, CD4 Antigens analysis, CD8 Antigens analysis, Hematopoietic Stem Cells drug effects, Lymphopoiesis drug effects, T-Lymphocyte Subsets immunology
- Abstract
The efficacy of donor HSCT is partly reduced as a result of slow post-transplantation immune recovery. In particular, T cell regeneration is generally delayed, resulting in high infection-related mortality in the first years post-transplantation. Adoptive transfer of in vitro-generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled, feeder-free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect, indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore, the addition of AA to feeder cocultures resulted in development of DP and SP T cells, whereas without AA, a preT cell-stage arrest occurred. We conclude that neither DLL4-expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G-CSF-mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy., (© 2014 Society for Leukocyte Biology.)
- Published
- 2014
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18. Stereotactic ablative body radiotherapy combined with immunotherapy: present status and future perspectives.
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Rekers NH, Troost EG, Zegers CM, Germeraad WT, Dubois LJ, and Lambin P
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- Animals, Antibodies, Monoclonal pharmacology, Antibodies, Monoclonal therapeutic use, Bystander Effect, CTLA-4 Antigen antagonists & inhibitors, Cell Death immunology, Cell Death radiation effects, Combined Modality Therapy, Dose Fractionation, Radiation, Forecasting, Humans, Ipilimumab, Mice, Molecular Targeted Therapy, Neoplasm Proteins antagonists & inhibitors, Neoplasms immunology, Neoplasms therapy, Neoplasms, Experimental surgery, Neoplasms, Experimental therapy, Programmed Cell Death 1 Receptor antagonists & inhibitors, Randomized Controlled Trials as Topic, T-Lymphocyte Subsets drug effects, T-Lymphocyte Subsets immunology, Tumor Escape immunology, Tumor Escape radiation effects, Tumor Necrosis Factor Receptor Superfamily, Member 9 antagonists & inhibitors, Immunologic Surveillance radiation effects, Immunotherapy, Neoplasms surgery, Radiosurgery
- Abstract
Radiotherapy is along with surgery and chemotherapy one of the prime treatment modalities in cancer. It is applied in the primary, neoadjuvant as well as the adjuvant setting. Radiation techniques have rapidly evolved during the past decade enabling the delivery of high radiation doses, reducing side-effects in tumour-adjacent normal tissues. While increasing local tumour control, current and future efforts ought to deal with microscopic disease at a distance of the primary tumour, ultimately responsible for disease-progression. This review explores the possibility of bimodal treatment combining radiotherapy with immunotherapy., (Copyright © 2014 Société française de radiothérapie oncologique (SFRO). Published by Elsevier SAS. All rights reserved.)
- Published
- 2014
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19. Monitoring the initiation and kinetics of human dendritic cell-induced polarization of autologous naive CD4+ T cells.
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Oth T, Schnijderberg MC, Senden-Gijsbers BL, Germeraad WT, Bos GM, and Vanderlocht J
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- CD4-Positive T-Lymphocytes metabolism, Cell Differentiation physiology, Cell Polarity physiology, Cells, Cultured, Dendritic Cells metabolism, Flow Cytometry, Humans, Kinetics, Monocytes cytology, Polymerase Chain Reaction, CD4-Positive T-Lymphocytes cytology, Dendritic Cells cytology
- Abstract
A crucial step in generating de novo immune responses is the polarization of naive cognate CD4+ T cells by pathogen-triggered dendritic cells (DC). In the human setting, standardized DC-dependent systems are lacking to study molecular events during the initiation of a naive CD4+ T cell response. We developed a TCR-restricted assay to compare different pathogen-triggered human DC for their capacities to instruct functional differentiation of autologous, naive CD4+ T cells. We demonstrated that this methodology can be applied to compare differently matured DC in terms of kinetics, direction, and magnitude of the naive CD4+ T cell response. Furthermore, we showed the applicability of this assay to study the T cell polarizing capacity of low-frequency blood-derived DC populations directly isolated ex vivo. This methodology for addressing APC-dependent instruction of naive CD4+ T cells in a human autologous setting will provide researchers with a valuable tool to gain more insight into molecular mechanisms occurring in the early phase of T cell polarization. In addition, it may also allow the study of pharmacological agents on DC-dependent T cell polarization in the human system.
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- 2014
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20. Heat-transfer resistance measurement method (HTM)-based cell detection at trace levels using a progressive enrichment approach with highly selective cell-binding surface imprints.
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Bers K, Eersels K, van Grinsven B, Daemen M, Bogie JF, Hendriks JJ, Bouwmans EE, Püttmann C, Stein C, Barth S, Bos GM, Germeraad WT, De Ceuninck W, and Wagner P
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- Animals, CHO Cells, Cells, Cultured, Cricetulus, Glycosylation, Microscopy, Fluorescence, Mucin-1 biosynthesis, Mucin-1 chemistry, Mucin-1 metabolism, Polyurethanes metabolism, Surface Properties, Hot Temperature, Molecular Imprinting, Polyurethanes chemistry
- Abstract
Surface-imprinted polymers allow for specific cell detection based on simultaneous recognition of the cell shape, cell size, and cell membrane functionalities by macromolecular cell imprints. In this study, the specificity of detection and the detection sensitivity for target cells within a pool of non-target cells were analyzed for a cell-specific surface-imprinted polymer combined with a heat-transfer-based read-out technique (HTM). A modified Chinese hamster ovarian cell line (CHO-ldlD) was used as a model system on which the transmembrane protein mucin-1 (MUC1) could be excessively expressed and for which the occurrence of MUC1 glycosylation could be controlled. In specific cancer cells, the overexpressed MUC1 protein typically shows an aberrant apical distribution and glycosylation. We show that surface-imprinted polymers discriminate between cell types that (1) only differ in the expression of a specific membrane protein (MUC1) or (2) only differ in the membrane protein being glycosylated or not. Moreover, surface-imprinted polymers of cells carrying different glycoforms of the same membrane protein do target both types of cells. These findings illustrate the high specificity of cell detection that can be reached by the structural imprinting of cells in polymer layers. Competitiveness between target and non-target cells was proven to negatively affect the detection sensitivity of target cells. Furthermore, we show that the detection sensitivity can be increased significantly by repetitively exposing the surface to the sample and eliminating non-specifically bound cells by flushing between consecutive cell exposures.
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- 2014
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21. Natural killer cells: the secret weapon in dendritic cell vaccination strategies.
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Van Elssen CH, Oth T, Germeraad WT, Bos GM, and Vanderlocht J
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- Animals, Cancer Vaccines immunology, Cell Communication immunology, Cytotoxicity, Immunologic, Dendritic Cells immunology, Dendritic Cells metabolism, Humans, Immunotherapy, Killer Cells, Natural metabolism, Lymphocyte Activation immunology, Neoplasms metabolism, Killer Cells, Natural immunology, Neoplasms immunology, Neoplasms therapy
- Abstract
In cancer therapy, dendritic cell (DC) vaccination is still being explored. Clinical responses, however, are diverse and there is a lack of immunologic readout systems that correspond with clinical outcome. Only in the minority of patients, T-cell responses correlate with clinical outcome, indicating that other immune cells also gain anticancer activity. We still have limited knowledge of the effect of DC vaccination on different immune effector cells. However, it has been shown that bidirectional cross-talk between natural killer (NK) cells and DCs is responsible for enhanced activation of both cell types and increases their antitumor activity. In this review, we postulate the possibility that NK cells are the secret weapons in DC vaccination and studying their behavior together with T-cell activation in vaccinated individuals might predict clinical outcome., (©2014 AACR)
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- 2014
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22. Systemic G-CSF attenuates cerebral inflammation and hypomyelination but does not reduce seizure burden in preterm sheep exposed to global hypoxia-ischemia.
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Jellema RK, Lima Passos V, Ophelders DR, Wolfs TG, Zwanenburg A, De Munter S, Nikiforou M, Collins JJ, Kuypers E, Bos GM, Steinbusch HW, Vanderlocht J, Andriessen P, Germeraad WT, and Kramer BW
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- Animals, Disease Models, Animal, Electrocardiography, Electroencephalography, Encephalitis etiology, Fetal Hypoxia pathology, Fetus, Flow Cytometry, Hematopoietic Stem Cell Mobilization, Hypoxia-Ischemia, Brain pathology, Immunohistochemistry, Nerve Fibers, Myelinated drug effects, Seizures etiology, Sheep, Encephalitis pathology, Fetal Hypoxia complications, Granulocyte Colony-Stimulating Factor pharmacology, Hypoxia-Ischemia, Brain complications, Neuroprotective Agents pharmacology
- Abstract
Hypoxic-ischemic encephalopathy (HIE) is common in preterm infants, but currently no curative therapy is available. Cell-based therapy has a great potential in the treatment of hypoxic-ischemic preterm brain injury. Granulocyte-colony stimulating factor (G-CSF) is known to mobilize endogenous hematopoietic stem cells (HSC) and promotes proliferation of endogenous neural stem cells. On these grounds, we hypothesized that systemic G-CSF would be neuroprotective in a large translational animal model of hypoxic-ischemic injury in the preterm brain. Global hypoxia-ischemia (HI) was induced by transient umbilical cord occlusion in instrumented preterm sheep. G-CSF treatment (100μg/kg intravenously, during five consecutive days) was started one day before the global HI insult to ascertain mobilization of endogenous stem cells within the acute phase after global HI. Mobilization of HSC and neutrophils was studied by flow cytometry. Brain sections were stained for microglia (IBA-1), myelin basic protein (MBP) and myeloperoxidase (MPO) to study microglial proliferation, white matter injury and neutrophil invasion respectively. Electrographic seizure activity was analyzed using amplitude-integrated electroencephalogram (aEEG). G-CSF effectively mobilized CD34-positive HSC in the preterm sheep. In addition, G-CSF caused marked mobilization of neutrophils, but did not influence enhanced invasion of neutrophils into the preterm brain after global HI. Microglial proliferation and hypomyelination following global HI were reduced as a result of G-CSF treatment. G-CSF did not cause a reduction of the electrographic seizure activity after global HI. In conclusion, G-CSF induced mobilization of endogenous stem cells which was associated with modulation of the cerebral inflammatory response and reduced white matter injury in an ovine model of preterm brain injury after global HI. G-CSF treatment did not improve neuronal function as shown by seizure analysis. Our study shows that G-CSF treatment has neuroprotective potential following hypoxic-ischemic injury in the preterm brain., (© 2013.)
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- 2013
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23. Mesenchymal stem cells induce T-cell tolerance and protect the preterm brain after global hypoxia-ischemia.
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Jellema RK, Wolfs TG, Lima Passos V, Zwanenburg A, Ophelders DR, Kuypers E, Hopman AH, Dudink J, Steinbusch HW, Andriessen P, Germeraad WT, Vanderlocht J, and Kramer BW
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- Animals, Base Sequence, DNA Primers, Disease Models, Animal, Magnetic Resonance Imaging, Polymerase Chain Reaction, Seizures prevention & control, Sheep, Brain embryology, Hypoxia-Ischemia, Brain immunology, Immune Tolerance, Mesenchymal Stem Cells immunology, T-Lymphocytes immunology
- Abstract
Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 10(6) MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE.
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- 2013
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24. Hypoxia induced impairment of NK cell cytotoxicity against multiple myeloma can be overcome by IL-2 activation of the NK cells.
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Sarkar S, Germeraad WT, Rouschop KM, Steeghs EM, van Gelder M, Bos GM, and Wieten L
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- Cell Degranulation drug effects, Cell Hypoxia drug effects, Cell Membrane drug effects, Cell Membrane metabolism, Granzymes metabolism, Histocompatibility Antigens Class I metabolism, Humans, K562 Cells, Killer Cells, Natural drug effects, Killer Cells, Natural physiology, Ligands, NK Cell Lectin-Like Receptor Subfamily K metabolism, Oxygen metabolism, Perforin metabolism, Receptors, IgG metabolism, Cytotoxicity, Immunologic drug effects, Interleukin-2 pharmacology, Killer Cells, Natural immunology, Killer Cells, Natural pathology, Lymphocyte Activation drug effects, Multiple Myeloma immunology, Multiple Myeloma pathology
- Abstract
Background: Multiple Myeloma (MM) is an incurable plasma cell malignancy residing within the bone marrow (BM). We aim to develop allogeneic Natural Killer (NK) cell immunotherapy for MM. As the BM contains hypoxic regions and the tumor environment can be immunosuppressive, we hypothesized that hypoxia inhibits NK cell anti-MM responses., Methods: NK cells were isolated from healthy donors by negative selection and NK cell function and phenotype were examined at oxygen levels representative of hypoxic BM using flowcytometry. Additionally, NK cells were activated with IL-2 to enhance NK cell cytotoxicity under hypoxia., Results: Hypoxia reduced NK cell killing of MM cell lines in an oxygen dependent manner. Under hypoxia, NK cells maintained their ability to degranulate in response to target cells, though, the percentage of degranulating NK cells was slightly reduced. Adaptation of NK- or MM cells to hypoxia was not required, hence, the oxygen level during the killing process was critical. Hypoxia did not alter surface expression of NK cell ligands (HLA-ABC, -E, MICA/B and ULBP1-2) and receptors (KIR, NKG2A/C, DNAM-1, NCRs and 2B4). It did, however, decrease expression of the activating NKG2D receptor and of intracellular perforin and granzyme B. Pre-activation of NK cells by IL-2 abrogated the detrimental effects of hypoxia and increased NKG2D expression. This emphasized that activated NK cells can mediate anti-MM effects, even under hypoxic conditions., Conclusions: Hypoxia abolishes the killing potential of NK cells against multiple myeloma, which can be restored by IL-2 activation. Our study shows that for the design of NK cell-based immunotherapy it is necessary to study biological interactions between NK- and tumor cells also under hypoxic conditions.
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- 2013
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25. Cerebral inflammation and mobilization of the peripheral immune system following global hypoxia-ischemia in preterm sheep.
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Jellema RK, Lima Passos V, Zwanenburg A, Ophelders DR, De Munter S, Vanderlocht J, Germeraad WT, Kuypers E, Collins JJ, Cleutjens JP, Jennekens W, Gavilanes AW, Seehase M, Vles HJ, Steinbusch H, Andriessen P, Wolfs TG, and Kramer BW
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- Animals, Animals, Newborn, Female, Fetus immunology, Fetus pathology, Immunity, Innate, Microglia immunology, Microglia pathology, Pregnancy, Sheep, Brain immunology, Brain pathology, Cell Movement immunology, Hypoxia-Ischemia, Brain immunology, Hypoxia-Ischemia, Brain pathology
- Abstract
Background: Hypoxic-ischemic encephalopathy (HIE) is one of the most important causes of brain injury in preterm infants. Preterm HIE is predominantly caused by global hypoxia-ischemia (HI). In contrast, focal ischemia is most common in the adult brain and known to result in cerebral inflammation and activation of the peripheral immune system. These inflammatory responses are considered to play an important role in the adverse outcomes following brain ischemia. In this study, we hypothesize that cerebral and peripheral immune activation is also involved in preterm brain injury after global HI., Methods: Preterm instrumented fetal sheep were exposed to 25 minutes of umbilical cord occlusion (UCO) (n = 8) at 0.7 gestation. Sham-treated animals (n = 8) were used as a control group. Brain sections were stained for ionized calcium binding adaptor molecule 1 (IBA-1) to investigate microglial proliferation and activation. The peripheral immune system was studied by assessment of circulating white blood cell counts, cellular changes of the spleen and influx of peripheral immune cells (MPO-positive neutrophils) into the brain. Pre-oligodendrocytes (preOLs) and myelin basic protein (MBP) were detected to determine white matter injury. Electro-encephalography (EEG) was recorded to assess functional impairment by interburst interval (IBI) length analysis., Results: Global HI resulted in profound activation and proliferation of microglia in the hippocampus, periventricular and subcortical white matter. In addition, non-preferential mobilization of white blood cells into the circulation was observed within 1 day after global HI and a significant influx of neutrophils into the brain was detected 7 days after the global HI insult. Furthermore, global HI resulted in marked involution of the spleen, which could not be explained by increased splenic apoptosis. In concordance with cerebral inflammation, global HI induced severe brain atrophy, region-specific preOL vulnerability, hypomyelination and persistent suppressed brain function., Conclusions: Our data provided evidence that global HI in preterm ovine fetuses resulted in profound cerebral inflammation and mobilization of the peripheral innate immune system. These inflammatory responses were paralleled by marked injury and functional loss of the preterm brain. Further understanding of the interplay between preterm brain inflammation and activation of the peripheral immune system following global HI will contribute to the development of future therapeutic interventions in preterm HIE.
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- 2013
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26. Phage display generation of a novel human anti-CD1A monoclonal antibody with potent cytolytic activity.
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Bechan GI, Lee DW, Zajonc DM, Heckel D, Xian R, Throsby M, van Meijer M, Germeraad WT, Kruisbeek AM, Egeler RM, and Arceci RJ
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- Animals, Antibodies, Monoclonal isolation & purification, Antibodies, Monoclonal, Humanized immunology, Antibodies, Monoclonal, Humanized isolation & purification, Antibody Affinity immunology, Antibody Specificity immunology, Antigens, CD1 metabolism, Cell Line, Tumor, Humans, Immunoglobulin G immunology, Kinetics, Melanoma, Experimental immunology, Melanoma, Experimental metabolism, Mice, Mice, Inbred NOD, Mice, SCID, Peptide Library, Precursor Cell Lymphoblastic Leukemia-Lymphoma immunology, Precursor Cell Lymphoblastic Leukemia-Lymphoma metabolism, Single-Chain Antibodies immunology, Single-Chain Antibodies isolation & purification, Antibodies, Monoclonal immunology, Antibody-Dependent Cell Cytotoxicity immunology, Antigens, CD1 immunology, Cell Surface Display Techniques
- Abstract
CD1A is a cell surface protein expressed on Langerhans cells and cortical thymocytes that could potentially be used as an immunotherapeutic target in Langerhans Cell Histiocytosis (LCH), the cortical subtype of T-cell acute lymphocytic leukaemia (T-ALL) and other CD1A-positive tumours. The monoclonal antibody (mAb) CR2113 was selected from a panel of six fully human mAbs isolated from a semi-synthetic phage display library, based on specificity and avidity against cells expressing CD1 antigen variants. CR2113 recognized CD1A in T-ALL cell lines and patient samples. Confocal microscopy revealed that the CR2113-CD1A complex was internalized at 37°C. Furthermore, while CR2113 induced moderate complement-dependent cytotoxicity (CDC), potent antibody-dependent cell cytotoxicity (ADCC) activity was observed against CD1A expressing cell lines as well as T-ALL cell lines and T-ALL patient samples. In vivo experiments showed that CR2113 as a naked antibody has modest but specific anti-tumour activity against CD1A-expressing tumours. CR2113 is a high-affinity human anti-CD1A mAb with significant ADCC activity. These properties make CR2113 a candidate for clinical diagnostic imaging and therapeutic targeting of LCH as well as potential use in other clinical applications., (© 2012 Blackwell Publishing Ltd.)
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- 2012
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27. Ovine fetal thymus response to lipopolysaccharide-induced chorioamnionitis and antenatal corticosteroids.
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Kuypers E, Collins JJ, Jellema RK, Wolfs TG, Kemp MW, Nitsos I, Pillow JJ, Polglase GR, Newnham JP, Germeraad WT, Kallapur SG, Jobe AH, and Kramer BW
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- Adrenal Cortex Hormones therapeutic use, Animals, Apoptosis drug effects, Bone Morphogenetic Protein 4 genetics, Bone Morphogenetic Protein 4 metabolism, CD3 Complex metabolism, Cell Proliferation drug effects, Chorioamnionitis immunology, Chorioamnionitis metabolism, Female, Fetus metabolism, Forkhead Transcription Factors genetics, Forkhead Transcription Factors metabolism, Gene Expression Regulation drug effects, Hedgehog Proteins genetics, Hedgehog Proteins metabolism, Pregnancy, T-Lymphocytes cytology, T-Lymphocytes drug effects, T-Lymphocytes metabolism, Thymus Gland drug effects, Thymus Gland metabolism, Time Factors, Toll-Like Receptors genetics, Toll-Like Receptors metabolism, Adrenal Cortex Hormones pharmacology, Chorioamnionitis chemically induced, Chorioamnionitis drug therapy, Fetus drug effects, Lipopolysaccharides pharmacology, Sheep embryology, Thymus Gland embryology
- Abstract
Rationale: Chorioamnionitis is associated with preterm delivery and involution of the fetal thymus. Women at risk of preterm delivery receive antenatal corticosteroids which accelerate fetal lung maturation and improve neonatal outcome. However, the effects of antenatal corticosteroids on the fetal thymus in the settings of chorioamnionitis are largely unknown. We hypothesized that intra-amniotic exposure to lipopolysaccharide (LPS) causes involution of the fetal thymus resulting in persistent effects on thymic structure and cell populations. We also hypothesized that antenatal corticosteroids may modulate the effects of LPS on thymic development., Methods: Time-mated ewes with singleton fetuses received an intra-amniotic injection of LPS 7 or 14 days before preterm delivery at 120 days gestational age (term = 150 days). LPS and corticosteroid treatment groups received intra-amniotic LPS either preceding or following maternal intra-muscular betamethasone. Gestation matched controls received intra-amniotic and maternal intra-muscular saline. The fetal intra-thoracic thymus was evaluated., Results: Intra-amniotic LPS decreased the cortico-medullary (C/M) ratio of the thymus and increased Toll-like receptor (TLR) 4 mRNA and CD3 expression indicating involution and activation of the fetal thymus. Increased TLR4 and CD3 expression persisted for 14 days but Foxp3 expression decreased suggesting a change in regulatory T-cells. Sonic hedgehog and bone morphogenetic protein 4 mRNA, which are negative regulators of T-cell development, decreased in response to intra-amniotic LPS. Betamethasone treatment before LPS exposure attenuated some of the LPS-induced thymic responses but increased cleaved caspase-3 expression and decreased the C/M ratio. Betamethasone treatment after LPS exposure did not prevent the LPS-induced thymic changes., Conclusion: Intra-amniotic exposure to LPS activated the fetal thymus which was accompanied by structural changes. Treatment with antenatal corticosteroids before LPS partially attenuated the LPS-induced effects but increased apoptosis in the fetal thymus. Corticosteroid administration after the inflammatory stimulus did not inhibit the LPS effects on the fetal thymus.
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- 2012
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28. Inflammation-restraining effects of prostaglandin E2 on natural killer-dendritic cell (NK-DC) interaction are imprinted during DC maturation.
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Van Elssen CH, Vanderlocht J, Oth T, Senden-Gijsbers BL, Germeraad WT, and Bos GM
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- Alprostadil analogs & derivatives, Alprostadil pharmacology, Bucladesine pharmacology, Cell Differentiation, Cell Movement drug effects, Cells, Cultured drug effects, Cells, Cultured immunology, Chemokines biosynthesis, Chemokines genetics, Coculture Techniques, Cytokines biosynthesis, Cytokines genetics, Cytotoxicity, Immunologic drug effects, Dendritic Cells cytology, Dendritic Cells immunology, Dendritic Cells metabolism, Down-Regulation drug effects, Gene Expression Regulation drug effects, Humans, Immunosuppression Therapy, Interferon-gamma biosynthesis, Interferon-gamma genetics, Killer Cells, Natural immunology, Klebsiella pneumoniae immunology, Misoprostol pharmacology, Palatine Tonsil cytology, T-Lymphocytes, Helper-Inducer immunology, Dendritic Cells drug effects, Dinoprostone pharmacology, Inflammation immunology, Killer Cells, Natural drug effects
- Abstract
Among prostaglandins (PGs), PGE2 is abundantly expressed in various malignancies and is probably one of many factors promoting tumor growth by inhibiting tumor immune surveillance. In the current study, we report on a novel mechanism by which PGE2 inhibits in vitro natural killer-dendritic cell (NK-DC) crosstalk and thereby innate and adaptive immune responses via its effect on NK-DC crosstalk. The presence of PGE2 during IFN-γ/membrane fraction of Klebsiella pneumoniae DC maturation inhibits the production of chemokines (CCL5, CCL19, and CXCL10) and cytokines (IL-12 and IL-18), which is cAMP-dependent and imprinted during DC maturation. As a consequence, these DCs fail to attract NK cells and show a decreased capacity to trigger NK cell IFN-γ production, which in turn leads to reduced T-helper 1 polarization. In addition, the presence of PGE2 during DC maturation impairs DC-mediated augmentation of NK-cell cytotoxicity. Opposed to their inhibitory effects on peripheral blood-derived NK cells, PGE2 matured DCs induce IL-22 secretion of inflammation constraining NKp44(+) NK cells present in mucosa-associated lymphoid tissue. The inhibition of NK-DC interaction is a novel regulatory property of PGE2 that is of possible relevance in dampening immune responses in vivo.
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- 2011
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29. Flow cytometry-based assay to evaluate human serum MUC1-Tn antibodies.
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Van Elssen CH, Clausen H, Germeraad WT, Bennet EP, Menheere PP, Bos GM, and Vanderlocht J
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- Amino Acid Sequence, Animals, Antigens, Neoplasm genetics, Breast Neoplasms immunology, Breast Neoplasms therapy, CHO Cells, Cancer Vaccines administration & dosage, Cancer Vaccines genetics, Cancer Vaccines immunology, Case-Control Studies, Cricetinae, Cricetulus, Female, Glycosylation, Humans, Molecular Sequence Data, Mucin-1 genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins immunology, Transfection, Vaccines, Subunit administration & dosage, Vaccines, Subunit genetics, Vaccines, Subunit immunology, Antibodies, Neoplasm blood, Antigens, Neoplasm chemistry, Flow Cytometry methods, Mucin-1 chemistry, Mucin-1 immunology
- Abstract
Mucin-1 (MUC1) is a heavily O-glycosylated, transmembrane protein that is expressed on the apical surface of most secretory epithelia. In malignantly transformed epithelia, MUC1 has lost its apical distribution, is underglycosylated and is secreted into the circulation. Due to the underglycosylation of MUC1, cancer-specific MUC1-Tn/STn antigens, which are highly immunogenic, become exposed. We aimed at developing a system that allows detection of antibodies directed to the native form of MUC1 and the underglycosylated MUC1-Tn epitopes. To this end, we made use of the Chinese Hamster Ovary (CHO) ldlD cell line stably transfected with MUC1. This cell line has a glycosylation defect, which can be reversed by addition of different monosaccharides to the cell culture and enables the production of cells expressing the MUC1-Tn glycoforms. After validation with glycospecific antibodies, the CHO-ldlD MUC1 system was used to detect serum MUC1 and MUC1-Tn antibodies. Using this system, we could confirm the presence of MUC1-Tn antibodies in the serum of a patient vaccinated with a truncated MUC1 peptide. This indicates that the CHO-ldlD MUC1 system represents a flow cytometry-based technique to detect antibodies binding to the underglycosylated MUC1 protein. This cellular system is complementary to the previously published methods to detect MUC1 serum antibodies, since the antibodies to the native protein are evaluated and therefore it can be effectively used for MUC1 antibody monitoring in vaccination studies as well as for functional assays., (Copyright © 2010 Elsevier B.V. All rights reserved.)
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- 2011
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30. T cells fail to develop in the human skin-cell explants system; an inconvenient truth.
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Meek B, Van Elssen CH, Huijskens MJ, van der Stegen SJ, Tonnaer S, Lumeij SB, Vanderlocht J, Kirkland MA, Hesselink R, Germeraad WT, and Bos GM
- Subjects
- Animals, Cell Culture Techniques, Cell Line, Cells, Cultured, Coculture Techniques, Fibroblasts cytology, Fibroblasts metabolism, Flow Cytometry, Fluorescent Antibody Technique, Hematopoietic Stem Cells metabolism, Humans, Intercellular Signaling Peptides and Proteins genetics, Intercellular Signaling Peptides and Proteins metabolism, Intracellular Signaling Peptides and Proteins, Keratinocytes cytology, Keratinocytes metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, T-Lymphocytes metabolism, Cell Differentiation, Hematopoietic Stem Cells cytology, Skin cytology, T-Lymphocytes cytology
- Abstract
Background: Haplo-identical hematopoietic stem cell (HSC) transplantation is very successful in eradicating haematological tumours, but the long post-transplant T-lymphopenic phase is responsible for high morbidity and mortality rates. Clark et al. have described a skin-explant system capable of producing host-tolerant donor-HSC derived T-cells. Because this T-cell production platform has the potential to replenish the T-cell levels following transplantation, we set out to validate the skin-explant system., Results: Following the published procedures, while using the same commercial components, it was impossible to reproduce the skin-explant conditions required for HSC differentiation towards mature T-cells. The keratinocyte maturation procedure resulted in fragile cells with minimum expression of delta-like ligand (DLL). In most experiments the generated cells failed to adhere to carriers or were quickly outcompeted by fibroblasts. Consequently it was not possible to reproduce cell-culture conditions required for HSC differentiation into functional T-cells. Using cell-lines over-expressing DLL, we showed that the antibodies used by Clark et al. were unable to detect native DLL, but instead stained 7AAD+ cells. Therefore, it is unlikely that the observed T-lineage commitment from HSC is mediated by DLL expressed on keratinocytes. In addition, we did confirm expression of the Notch-ligand Jagged-1 by keratinocytes., Conclusions: Currently, and unfortunately, it remains difficult to explain the development or growth of T-cells described by Clark et al., but for the fate of patients suffering from lymphopenia it is essential to both reproduce and understand how these co-cultures really "work". Fortunately, alternative procedures to speed-up T-cell reconstitution are being established and validated and may become available for patients in the near future.
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- 2011
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31. Klebsiella pneumoniae-triggered DC recruit human NK cells in a CCR5-dependent manner leading to increased CCL19-responsiveness and activation of NK cells.
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Van Elssen CH, Vanderlocht J, Frings PW, Senden-Gijsbers BL, Schnijderberg MC, van Gelder M, Meek B, Libon C, Ferlazzo G, Germeraad WT, and Bos GM
- Subjects
- Cells, Cultured, Gene Expression Regulation immunology, Humans, Interferon-gamma immunology, Receptors, CCR7 immunology, Th1 Cells immunology, Toll-Like Receptor 2 immunology, Toll-Like Receptor 4 immunology, Cell Movement immunology, Chemokine CCL19 immunology, Dendritic Cells immunology, Killer Cells, Natural immunology, Klebsiella Infections immunology, Klebsiella pneumoniae immunology, Lymphocyte Activation immunology, Receptors, CCR5 immunology
- Abstract
Besides their role in destruction of altered self-cells, NK cells have been shown to potentiate T-cell responses by interacting with DC. To take advantage of NK-DC crosstalk in therapeutic DC-based vaccination for infectious diseases and cancer, it is essential to understand the biology of this crosstalk. We aimed to elucidate the in vitro mechanisms responsible for NK-cell recruitment and activation by DC during infection. To mimic bacterial infection, DC were exposed to a membrane fraction of Klebsiella pneumoniae, which triggers TLR2/4. DC matured with these bacterial fragments can actively recruit NK cells in a CCR5-dependent manner. An additional mechanism of DC-induced NK-cell recruitment is characterized by the induction of CCR7 expression on CD56(dim) CD16(+) NK cells after physical contact with membrane fraction of K. pneumoniae-matured DC, resulting in an enhanced migratory responsiveness to the lymph node-associated chemokine CCL19. Bacterial fragment-matured DC do not only mediate NK-cell migration but also meet the prerequisites needed for augmentation of NK-cell cytotoxicity and IFN-γ production, the latter of which contributes to Th1 polarization., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
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- 2010
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32. Expression of aberrantly glycosylated Mucin-1 in ovarian cancer.
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Van Elssen CH, Frings PW, Bot FJ, Van de Vijver KK, Huls MB, Meek B, Hupperets P, Germeraad WT, and Bos GM
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- Adenocarcinoma pathology, Biomarkers, Tumor analysis, Biomarkers, Tumor metabolism, Epitopes metabolism, Female, Glycosylation, Humans, Immunohistochemistry, Ovarian Neoplasms pathology, Adenocarcinoma metabolism, Mucin-1 metabolism, Ovarian Neoplasms metabolism
- Abstract
Aims: Mucin 1 (MUC1) is an important tumour-associated antigen (TAA), both overexpressed and aberrantly glycosylated in adenocarcinomas. The aim of this study was to examine the MUC1-glycosylation status of primary ovarian adenocarcinomas and metastatic lesions., Methods and Results: Paraffin-embedded tissue sections of 37 primary ovarian adenocarcinomas representing all histotypes (22 serous, five mucinous, two clear-cell, eight endometrioid), four serous borderline tumours with intraepithelial carcinoma, seven sections of ovarian endometriosis and 13 metastatic lesions were analysed by immunohistochemistry. Non-neoplastic ovarian surface epithelium and serous cystadenomas were used as controls. All epithelia expressed MUC1 protein. Of primary tumours, 76% expressed the differentiation-dependent glycoform and 84% the cancer-associated glycoform (Tn/Sialyl-Tn-epitopes). In metastatic lesions this was 77% and 85%, respectively. Notably, in 57% of ovarian endometriosis and 75% of intraepithelial lesions, the cancer-associated MUC1 epitopes were expressed, whereas normal ovarian surface epithelium and serous cystadenomas did not express these epitopes., Conclusions: The underglycosylated MUC1 epitopes are expressed by all histotypes of primary ovarian adenocarcinomas, by the vast majority of metastatic lesions and by possible ovarian cancer precursor lesions, but not by normal ovarian tissue. These results indicate that MUC1-associated Tn/STn-epitopes are important targets for immunotherapy and diagnostic imaging in ovarian cancer patients., (© 2010 Blackwell Publishing Limited.)
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- 2010
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33. Thymic cysts originate from Foxn1 positive thymic medullary epithelium.
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Vroegindeweij E, Crobach S, Itoi M, Satoh R, Zuklys S, Happe C, Germeraad WT, Cornelissen JJ, Cupedo T, Holländer GA, Kawamoto H, and van Ewijk W
- Subjects
- Animals, Cysts embryology, Cysts pathology, Embryo, Mammalian embryology, Embryo, Mammalian pathology, Epithelium embryology, Epithelium pathology, Forkhead Transcription Factors genetics, Mice, Mice, Transgenic, Thymus Gland embryology, Thymus Gland pathology, Cysts immunology, Embryo, Mammalian immunology, Epithelium immunology, Forkhead Transcription Factors immunology, Thymus Gland immunology
- Abstract
Thymic epithelial cells (TECs), derived from polarized two-dimensional (2D) oriented endodermal cells, are distinguished from other epithelial cells by their unique three-dimensional (3D) phenotype. However, some polarized epithelial cells remain present in the normal thymus, forming thymic cysts at the cortico-medullary junction. Here, we analyse the dynamics, origin and phenotype of such thymic cysts. In time-course experiments, we show a reverse correlation between thymic cyst expansion and the presence of thymocytes, suggesting a default pathway for the development of TECs in the absence of thymocytes. By transplanting isolated TEC populations into E15 fetal thymic lobes, we provide evidence that medullary thymic epithelial cells (mTECs), rather than cortical thymic epithelial cells (cTECs) contribute to the formation of thymic cysts. Finally, thymi of reporter mice reveal that the cysts originate from epithelia committed to a thymic fate, as indicated by the expression of Foxn1. The 2D-phenotype of cyst-lining TECs is not caused by a downregulation of Foxn1 expression, since a significant proportion of these cells in the embryonic and adult thymus continues to express Foxn1 at the protein level., ((c) 2009 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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34. In vitro-differentiated T/natural killer-cell progenitors derived from human CD34+ cells mature in the thymus.
- Author
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Meek B, Cloosen S, Borsotti C, Van Elssen CH, Vanderlocht J, Schnijderberg MC, van der Poel MW, Leewis B, Hesselink R, Manz MG, Katsura Y, Kawamoto H, Germeraad WT, and Bos GM
- Subjects
- Animals, Cell Differentiation, DNA-Binding Proteins genetics, DNA-Binding Proteins immunology, DNA-Binding Proteins metabolism, Hematopoietic Stem Cell Transplantation, Humans, Killer Cells, Natural cytology, Killer Cells, Natural immunology, Lymphoid Progenitor Cells cytology, Lymphoid Progenitor Cells immunology, Mice, Mice, Knockout, T-Lymphocytes cytology, T-Lymphocytes immunology, Thymus Gland cytology, Thymus Gland immunology, Transplantation, Heterologous, Transplantation, Homologous, Antigens, CD34, Killer Cells, Natural metabolism, Lymphoid Progenitor Cells metabolism, T-Lymphocytes metabolism, Thymus Gland metabolism
- Abstract
Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a treatment option for patients with hematopoietic malignancies that is hampered by treatment-related morbidity and mortality, in part the result of opportunistic infections, a direct consequence of delayed T-cell recovery. Thymic output can be improved by facilitation of thymic immigration, known to require precommitment of CD34(+) cells. We demonstrate that Delta-like ligand-mediated predifferentiation of mobilized CD34(+) cells in vitro results in a population of thymocyte-like cells arrested at a T/natural killer (NK)-cell progenitor stage. On intrahepatic transfer to Rag2(-/-)gamma(c)(-/-) mice, these cells selectively home to the thymus and differentiate toward surface T-cell receptor-alphabeta(+) mature T cells considerably faster than animals transplanted with noncultured CD34(+) cells. This finding creates the opportunity to develop an early T-cell reconstitution therapy to combine with HSCT.
- Published
- 2010
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35. Increased tumor-specific CD8+ T cell induction by dendritic cells matured with a clinical grade TLR-agonist in combination with IFN-gamma.
- Author
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Vanderlocht J, Van Elssen CH, Senden-Gijsbers BL, Meek B, Cloosen S, Libon C, Bos GM, and Germeraad WT
- Subjects
- Cell Movement, Cytokines biosynthesis, Dendritic Cells drug effects, Dendritic Cells immunology, Humans, Immunologic Memory, Mucin-1 immunology, T-Lymphocytes, Helper-Inducer immunology, CD8-Positive T-Lymphocytes immunology, Dendritic Cells physiology, Interferon-gamma pharmacology, Neoplasms immunology, Toll-Like Receptors agonists
- Abstract
The limited response rate of cancer patients treated with dendritic cell (DC)-based vaccines indicates that vast improvements remain necessary. In many murine tumour models it has been demonstrated that the use of innate triggers (e.g. TLR triggers) in the maturation of DC results in higher efficacy. However, as few of these innate triggers are generated clinical grade, there remains a great necessity to fill the gap between fundamental mouse studies and a clinical trial in humans. In the present study we used a TLR2/4-agonist (FMKp which is available clinical grade) in combination with IFN-gamma (FIcocktail) in the maturation of elutriated monocyte-derived DC and compared it with the most used DC in current clinical trials (TNF-alpha/PGE-2, i.e. TP-cocktail). In addition to the assessment of CD4+ T cell polarizing capacity, we compared the quantity and intrinsic quality of induced CD8+ T cells of 2 different DC maturation protocols with all cells from the same donor. Besides differences in the cytokine profile, which could be coupled to increased Th1 and Th17 polarization, we demonstrate in this study that FMKp/IFN-gamma matured DC are twice as effective in inducing cytotoxic T cells against known tumor antigens. Both DCs induced phenotypically equivalent effector memory CD8+ T cells that did not show a significant difference in their intrinsic capacity to kill tumor cells. These findings point to the therapeutic applicability of FI-DC as superior inducers of functional antigen-specific T cells. Their increased chemokine secretion is suggestive of a mechanism by which these DC may compensate for the limited migration observed for all ex vivo cultured DC when applied in patients.
- Published
- 2010
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36. Notch activation in thymic epithelial cells induces development of thymic microenvironments.
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Masuda K, Germeraad WT, Satoh R, Itoi M, Ikawa T, Minato N, Katsura Y, van Ewijk W, and Kawamoto H
- Subjects
- Animals, B-Lymphocytes metabolism, B-Lymphocytes physiology, Cell Lineage genetics, Cell Lineage physiology, Cells, Cultured, Embryo, Mammalian, Female, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins physiology, Membrane Proteins genetics, Membrane Proteins physiology, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Transgenic, Models, Biological, Organogenesis genetics, Organogenesis physiology, Pregnancy, Receptors, Notch genetics, Receptors, Notch physiology, T-Lymphocytes metabolism, T-Lymphocytes physiology, Thymus Gland physiology, Transfection, Epithelial Cells metabolism, Receptors, Notch metabolism, Thymus Gland embryology, Thymus Gland metabolism
- Abstract
The development and maintenance of thymic microenvironments depends on sustained crosstalk signals derived from developing thymocytes. However, the molecular basis for the initial phase in the lymphoid dependent development of thymic epithelial cells (TECs) remains unclear. Here we show that similarly to regular thymocytes, developing B cells enforced to express the Notch ligand Delta-like-1 (DLL1) efficiently induce the non-polarized, three-dimensional (3D) meshwork architecture of cortical TECs in fetal thymic organ culture. Moreover, the DLL1-overexpressing B cells induce well-developed distinct medullae. Such medullae also arose in lobes reconstituted with Rag2(-/-) thymocytes overexpressing DLL1. Our present findings thus strongly suggest that Notch signaling from thymocytes to TECs induces TEC development at an early phase of thymic organogenesis. The present approach using non-T lineage cells for the in vitro construction of thymic environments may also provide a novel tool for thymus regeneration and T cell production in immunocompromised individuals.
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- 2009
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37. Surface Mucin-1 does not play a role in dendritic cell migration.
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Cloosen S, Caberg JH, Huls MB, Vanderlocht J, Senden-Gijsbers BL, Roncarati P, Hubert P, Delvenne P, Germeraad WT, and Bos GM
- Subjects
- Cell Movement drug effects, Cells, Cultured, Cytokines pharmacology, Dendritic Cells drug effects, Humans, Intercellular Adhesion Molecule-1 immunology, Intercellular Adhesion Molecule-1 metabolism, Mucin-1 drug effects, Cell Movement immunology, Dendritic Cells immunology, Mucin-1 immunology
- Abstract
Mucin-1 (MUC1) is a transmembrane glycoprotein that is upregulated upon maturation of dendritic cells (DC) in vitro or in vivo. One of the proposed functions of surface expressed MUC1 is its involvement in migration of cells. We hypothesized that MUC1 is involved in DC migration since mature DC (mDC) are highly migratory cells and MUC1 is upregulated on the surface of DC upon maturation. In this study we cultured DC using two maturation cocktails, one cocktail containing IL-4, GM-CSF, TNFalpha, PGE2, IL-1 beta and IL-6 (TP1,6-DC) and the other IL-13, GM-CSF, Ribomunyl and IFN-gamma (RI-DC). Both maturation cocktails render DC with a similar surface phenotype including CCR7 expression, but only the former induces a migratory capacity of DC to a CCL19 gradient. To analyze the role of surface-expression of MUC1 on TP1,6-DC, that are capable of migration, expression of MUC1 was prevented by adding an anti-MUC1 antibody (Ab) during the maturation process. Compared with matured DC in the absence of the Ab, no difference was observed in chemokine-induced migratory behaviour between the MUC1+ and MUC1- DC populations in a standard Transwell chemotaxis assay, nor in organotypic cultures. Our data clearly demonstrate that surface MUC1 on DC does not influence intrinsic cell-motility, nor is it involved in cell-cell and cell-matrix dependent migration.
- Published
- 2009
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38. Immunohistological analysis of peptide-induced delayed-type hypersensitivity in advanced melanoma patients treated with melanoma antigen-pulsed mature monocyte-derived dendritic cell vaccination.
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Nakai N, Katoh N, Germeraad WT, Kishida T, Ueda E, Takenaka H, Mazda O, and Kishimoto S
- Subjects
- Aged, Antigen Presentation immunology, Antigens, Neoplasm immunology, Biopsy, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes pathology, Cancer Vaccines immunology, Female, Humans, Hypersensitivity, Delayed immunology, Langerhans Cells immunology, Male, Melanoma immunology, Melanoma pathology, Middle Aged, Skin pathology, Skin Neoplasms immunology, Skin Neoplasms pathology, Th1 Cells immunology, Th1 Cells pathology, Th2 Cells immunology, Th2 Cells pathology, Time Factors, Antigens, Neoplasm therapeutic use, Cancer Vaccines therapeutic use, Hypersensitivity, Delayed pathology, Langerhans Cells pathology, Melanoma drug therapy, Skin Neoplasms drug therapy
- Abstract
Background: In melanoma patients vaccinated with monocyte-derived melanoma peptide-pulsed dendritic cells (DC), the delayed-type hypersensitivity (DTH) reactions have been examined as a surrogate marker to determine if acquired immunity is induced by DC vaccination. To date, however, only limited information has been reported as for histopathological analyses of DTH., Objective: To evaluate tumor-specific immunomonitoring histopathologically after DC vaccination in melanoma patients., Methods: Seven patients previously vaccinated with monocyte-derived melanoma peptide-pulsed DCs were challenged with recall antigenic peptide injection in the skin of the forearm. Using immunohistochemical techniques, the presence of immune cells and the expression of CD4, CD8, interleukin (IL)-2, IL-4, IL-10, Foxp3, CD1a, CD1d, and interferon (IFN)-gamma was investigated at the site of injection where a DTH reaction developed., Results: Strong DTH reactions from infiltrated erythema to bullae formation were detected in all 7 cases. Biopsies taken from the DTH site revealed heavy infiltration of mononuclear cells and eosinophils in the dermis and subcutaneous tissue. Cells staining positively for CD4, CD8, IL-2, IL-4, Foxp3, CD1d, and IFN-gamma were increased at the site 48h after antigen injection in all cases. Cells positive for IL-10 were never found in any patient. Regulatory T cells appeared 6h after injection and reached their maximum at day 7., Conclusions: The significant induction of CD8(+)T cells as well as both Th1 and Th2-type cells at the site of DTH suggests that effective antigen presentation leading to anti-tumor immune responses has taken place. Inhibitory mechanisms may also develop as the disappearance of the DTH response could be related to an increase in Foxp3+ cells.
- Published
- 2009
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39. Increased release of sMD-2 during human endotoxemia and sepsis: a role for endothelial cells.
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Wolfs TG, Dunn-Siegrist I, van't Veer C, Hodin CM, Germeraad WT, van Zoelen MA, van Suylen RJ, Peutz-Kootstra CJ, Elson G, Pugin J, and Buurman WA
- Subjects
- Adult, Case-Control Studies, Endothelial Cells drug effects, Endothelial Cells immunology, Endothelial Cells pathology, Endotoxemia immunology, Female, Hematopoietic System cytology, Hematopoietic System drug effects, Humans, Kinetics, Lipopolysaccharides pharmacology, Liver drug effects, Liver immunology, Liver pathology, Lung drug effects, Lung immunology, Lung pathology, Lymphocyte Antigen 96 blood, Male, Middle Aged, Sepsis blood, Sepsis immunology, Solubility drug effects, Tumor Necrosis Factor-alpha pharmacology, Endothelial Cells metabolism, Endotoxemia metabolism, Lymphocyte Antigen 96 metabolism, Sepsis metabolism
- Abstract
MD-2 is the crucial cofactor of TLR4 in the detection of LPS. Here, we show that soluble MD-2 (sMD-2) circulates in plasma of healthy individuals as a polymeric protein. The total amount of sMD-2 in septic plasma was strongly elevated and contained both sMD-2 polymers and monomers, the latter representing the putative biologically active form of MD-2. Moreover, during experimental human endotoxemia, the monomeric and total sMD-2 content in plasma increased with the kinetics of an acute phase protein. The increase in sMD-2 monomers was paralleled by enhanced TLR4 costimulatory activity. The presence of functional sMD-2 during endotoxemia and sepsis was confirmed by immunodepletion. Immunohistochemistry revealed that MD-2 expression in septic patients was strongly enhanced on endothelium and multiple inflammatory cells in lung and liver. In vitro studies showed that sMD-2 release appears to be restricted to endothelial cells and dendritic cells. Release of sMD-2 by endothelial cells was strongly enhanced by LPS and TNF-alpha stimulation. Taken together, this study demonstrates the increase of both circulating polymeric and functional monomeric sMD-2 during endotoxemia and sepsis, and evidence is provided that the endothelium is involved in this process.
- Published
- 2008
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40. Expression of tumor-associated differentiation antigens, MUC1 glycoforms and CEA, in human thymic epithelial cells: implications for self-tolerance and tumor therapy.
- Author
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Cloosen S, Arnold J, Thio M, Bos GM, Kyewski B, and Germeraad WT
- Subjects
- Antigens, Neoplasm immunology, Antigens, Neoplasm metabolism, Carcinoembryonic Antigen immunology, Carcinoembryonic Antigen metabolism, Child, Preschool, Epithelial Cells immunology, Epithelial Cells metabolism, Glycosylation, Humans, Infant, Infant, Newborn, Mucin-1, Mucins immunology, Mucins metabolism, Protein Isoforms, Self Tolerance immunology, Thymus Gland cytology, Thymus Gland metabolism, Antigens, Neoplasm biosynthesis, Carcinoembryonic Antigen biosynthesis, Mucins biosynthesis, Thymus Gland immunology
- Abstract
Expression of tissue-restricted self-antigens in the thymus, termed promiscuous gene expression, imposes T cell tolerance and protects from autoimmune diseases. This antigen pool also includes various types of tumor-associated antigens (TAA) previously thought to be secluded from the immune system. The scope of promiscuous gene expression has been defined by mRNA analysis at the global level of isolated medullary thymic epithelial cells (mTECs). Information at the protein level on the frequency of mTECs expressing a given antigen, on coexpression patterns, and post-translational modifications is largely missing. We report here promiscuous expression at the protein level of two TAA, MUC1 and CEA, in situ and in purified human mTECs. Both antigens are expressed in 1% to 3% of mTECs, either individually or coexpressed in the same cell. Using a panel of anti-MUC1 monoclonal antibodies recognizing different post-translational modifications, i.e., glycoforms of MUC1, we show that only fully glycosylated forms of MUC1 and the differentiation-dependent glycoforms were detected on mTECs, but not the cancer-associated glycoforms. Our findings imply that MUC1 and CEA are amenable to central tolerance induction, which might, however, be incomplete in case of tumor cell-restricted MUC1 glycoforms. Knowledge of these subtleties in promiscuous gene expression may, in the future, assist the selection of T cell tumor vaccines for clinical trials.
- Published
- 2007
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41. Cancer specific Mucin-1 glycoforms are expressed on multiple myeloma.
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Cloosen S, Gratama J, van Leeuwen EB, Senden-Gijsbers BL, Oving EB, von Mensdorff-Pouilly S, Tarp MA, Mandel U, Clausen H, Germeraad WT, and Bos GM
- Subjects
- Acute Disease, Antibodies, Neoplasm metabolism, Antigens, Neoplasm immunology, Bone Marrow immunology, Flow Cytometry, Glycosylation, Humans, Leukemia, Myeloid metabolism, Mucin-1, Mucins immunology, Multiple Myeloma immunology, Antigens, Neoplasm metabolism, Mucins metabolism, Multiple Myeloma metabolism, Neoplasm Proteins metabolism
- Abstract
Present therapies cannot cure the large majority of patients with multiple myeloma (MM) and therefore new treatment strategies are imperative. This study analysed the different glycosylation profiles of Mucin-1 (MUC1) on MM and acute myeloid leukaemia (AML) cells using a series of anti-MUC1 antibodies. Seventy-three per cent of the MM patients had plasma cells that expressed the fully glycosylated forms of MUC1. In contrast to controls, normal bone marrow cells and AML cells, the differentiation-dependent and cancer-associated glycoforms of MUC1 were present on 59% and 36% MM tumour cells respectively. This indicated that aberrantly glycosylated MUC1 is a potential immunotherapeutic target in MM patients.
- Published
- 2006
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42. Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector.
- Author
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van Leeuwen EB, Cloosen S, Senden-Gijsbers BL, Agervig Tarp M, Mandel U, Clausen H, Havenga MJ, Duffour MT, García-Vallejo JJ, Germeraad WT, and Bos GM
- Subjects
- Antibodies, Monoclonal immunology, Antigen Presentation immunology, Antigens, Neoplasm genetics, Antigens, Neoplasm metabolism, Cells, Cultured, Dendritic Cells cytology, Flow Cytometry, Genetic Vectors, Glycosylation, Humans, Mucin-1, Mucins genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Sialyltransferases genetics, Sialyltransferases metabolism, Adenoviridae genetics, Adenoviridae physiology, Dendritic Cells immunology, Dendritic Cells metabolism, Mucins metabolism, Transduction, Genetic
- Abstract
Background: DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides., Methods: In this study we used a fiber-modified adenoviral vector (rAd5F35) containing full-length tumor Ag cDNA to transduce human monocyte (Mo)-derived DC in vitro. Cells were efficiently transduced and survived for at least 3 days after adenoviral transduction. Phenotype and function after maturation of Mo-DC were not impaired by infection with adenovirus particles. Expression of the tumor-associated Ag mucin-1 (MUC1) was detected using MAb defining different MUC1 glycoforms., Results: Non-transduced mature Mo-DC express endogenous MUC1 with normal glycosylation. After transduction with the rAd5F35-MUC1 adenoviral vector, Mo-DC also expressed MUC1 with tumor-associated glycosylation (Tn and T glycoforms), although no changes in mRNA levels of relevant glycosyltransferases could be demonstrated., Discussion: The presence of aberrantly glycosylated MUC1 may influence Ag presentation of the tumor glycoforms of MUC1 to immune cells, affecting tumor cell killing. These findings could be highly relevant to developing strategies for cancer immunotherapy based on DC vaccines using MUC1 as tumor Ag.
- Published
- 2006
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43. Transduction with a fiber-modified adenoviral vector is superior to non-viral nucleofection for expressing tumor-associated Ag mucin-1 in human DC.
- Author
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van Leeuwen EB, Cloosen S, Senden-Gijsbers BL, Germeraad WT, and Bos GM
- Subjects
- Adenoviridae physiology, Antibodies, Monoclonal immunology, Antigens, Neoplasm genetics, Antigens, Neoplasm metabolism, Cell Proliferation, Cell Survival, Cells, Cultured, Dendritic Cells cytology, Electroporation, Genetic Vectors genetics, Green Fluorescent Proteins metabolism, Humans, Interleukin-10 biosynthesis, Interleukin-12, Lymphocyte Culture Test, Mixed, Mucin-1, Mucins genetics, Phenotype, Viral Load, Adenoviridae genetics, Dendritic Cells immunology, Dendritic Cells metabolism, Mucins metabolism, Transduction, Genetic
- Abstract
Background: DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase the chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. Methodologically, several recombinant DNA delivery techniques have been used., Methods: In this study we compared nucleofection, an optimized form of electroporation, and adenoviral transduction regarding their efficiency to transduce human monocyte-derived (Mo-) DC in vitro. Expression of the tumor-associated Ag mucin-1 (MUC1) after adenoviral transduction (rAd5Fib35-MUC1) was determined using two MAb., Results: We showed that the viability of cells and percentage of green fluorescent protein (GFP)-positive cells after transduction with a fiber-modified adenoviral vector (rAd5F35-GFP) was much higher than after nucleofection. Furthermore, phenotype and function of DC were not impaired by infection with adenovirus particles. Cells matured normally; up-regulation of CD40, CD80, CD83, CD86 and HLA-DR was not affected by adenoviral transduction. The capacity to stimulate naive T-cell proliferation was preserved and no change in IL-10 production was observed. Production of IL-12 increased up to 500-fold upon adenoviral transduction, considered to contribute positively to an anti-tumor immune response. Non-transduced mature DC expressed low levels of endogenous MUC1. After transduction with the rAd5F35-MUC1 adenoviral vector, a 100-fold increase in MUC1 expression by DC was observed., Discussion: The use of the fiber-modified adenoviral vector presented here may therefore be favorable compared with non-viral gene delivery systems for DC that will be used in cancer immunotherapy.
- Published
- 2006
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44. Mucin-1 is expressed on dendritic cells, both in vitro and in vivo.
- Author
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Cloosen S, Thio M, Vanclée A, van Leeuwen EB, Senden-Gijsbers BL, Oving EB, Germeraad WT, and Bos GM
- Subjects
- Animals, Antigen Presentation immunology, Cells, Cultured, Dendritic Cells transplantation, Humans, Immunotherapy, Adoptive, Mice, Mice, Transgenic, Mucin-1 genetics, Neoplasms pathology, Neoplasms therapy, Dendritic Cells immunology, Gene Expression Regulation immunology, Mucin-1 immunology, T-Lymphocytes, Cytotoxic immunology, T-Lymphocytes, Helper-Inducer immunology
- Abstract
Dendritic cells (DCs) are the best professional antigen-presenting cells to stimulate cytotoxic as well as T helper cells and are therefore appropriate candidates for establishing immunotherapy. The concept of our vaccination program is to introduce the tumor-associated antigen mucin-1 (MUC1) into DCs. Analysis of immature and mature DCs--before transducing the antigen MUC1--already demonstrated expression of MUC1 on in vitro monocyte-derived DCs upon maturation. Different culture methods as well as maturation cocktails showed similar results concerning the upregulation of MUC1 expression. Furthermore, we studied the expression of MUC1 on DCs in vivo. No MUC1 expression was found on blood DCs, or on thymic or tonsil DCs. On the other hand, synovial fluid from patients with arthritis contained DCs that were found to express MUC1. This study shows for the first time that the tumor-associated antigen MUC1 is expressed on in vivo DCs. We further show that MUC1 is also expressed on in vitro cultured bone marrow-derived DCs of human MUC1 transgenic mice, supporting the relevance of this mouse model to the human situation. The observation that MUC1 is present on in vivo DCs suggests a functional role, but this physiological function remains to be elucidated.
- Published
- 2004
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45. Development of thymic microenvironments in vitro is oxygen-dependent and requires permanent presence of T-cell progenitors.
- Author
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Germeraad WT, Kawamoto H, Itoi M, Jiang Y, Amagai T, Katsura Y, and van Ewijk W
- Subjects
- Animals, Cell Communication, Cell Differentiation, Epithelial Cells cytology, Hyaluronan Receptors metabolism, Immunohistochemistry, Mice, Mice, Inbred C57BL, Partial Pressure, Receptors, Interleukin-2 metabolism, Signal Transduction, Stromal Cells cytology, T-Lymphocytes metabolism, Thymus Gland embryology, Oxygen physiology, T-Lymphocytes cytology, Thymus Gland cytology
- Abstract
Development of a mature T-cell repertoire in the thymus depends on lympho-stromal interaction between thymocytes and stromal cells. To facilitate intercellular contact, the epithelium in the thymus has differentiated into a unique three-dimensionally (3D)-oriented network. Here we analyze factors influencing induction and maintenance of the 3D configuration of the epithelial network in fetal thymic lobes in vitro. We show that the 3D configuration of the thymic stroma depends on (a) the oxygen pressure in vitro and (b) permanent physical contact between stromal cells and developing thymocytes. This latter feature is demonstrated by incubation of fetal thymic lobes with deoxyguanosine (d-Guo), inducing a 2D-organized thymic stroma, with thymic cysts appearing. Reconstitution of d-Guo-treated lobes with a limited number of flow-sorted T-cell progenitors restores the 3D configuration of the thymic epithelium, but only at high oxygen pressure. This study underlines the plasticity of thymic epithelium and shows that the unique organization of the thymic epithelium is dependent on both oxygen and crosstalk signals derived from developing thymocytes.
- Published
- 2003
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46. Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease.
- Author
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Havenga MJ, Lemckert AA, Ophorst OJ, van Meijer M, Germeraad WT, Grimbergen J, van Den Doel MA, Vogels R, van Deutekom J, Janson AA, de Bruijn JD, Uytdehaag F, Quax PH, Logtenberg T, Mehtali M, and Bout A
- Subjects
- Animals, Bone and Bones, Cell Line, Gene Transfer Techniques, Genetic Vectors, Humans, Mice, Organ Culture Techniques, Prenatal Diagnosis, Rats, Recombinant Fusion Proteins, Serotyping, Tissue Engineering, Viral Vaccines, Adenoviruses, Human classification, Adenoviruses, Human genetics, Capsid genetics, Capsid Proteins, Cardiovascular Diseases prevention & control, Genetic Therapy methods
- Abstract
Since targeting of recombinant adenovirus vectors to defined cell types in vivo is a major challenge in gene therapy and vaccinology, we explored the natural diversity in human adenovirus tissue tropism. Hereto, we constructed a library of Ad5 vectors carrying fibers from other human serotypes. From this library, we identified vectors that efficiently infect human cells that are important for diverse gene therapy approaches and for induction of immunity. For several medical applications (prenatal diagnosis, artificial bone, vaccination, and cardiovascular disease), we demonstrate the applicability of these novel vectors. In addition, screening cell types derived from different species revealed that cellular receptors for human subgroup B adenoviruses are not conserved between rodents and primates. These results provide a rationale for utilizing elements of human adenovirus serotypes to generate chimeric vectors that improve our knowledge concerning adenovirus biology and widen the therapeutic window for vaccination and many different gene transfer applications.
- Published
- 2002
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47. Developing thymocytes organize thymic microenvironments.
- Author
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van Ewijk W, Kawamoto H, Germeraad WT, and Katsura Y
- Subjects
- Cell Communication, Epithelial Cells, Stromal Cells, T-Lymphocytes, Thymus Gland embryology
- Published
- 2000
- Full Text
- View/download PDF
48. Thymic microenvironments, 3-D versus 2-D?
- Author
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van Ewijk W, Wang B, Hollander G, Kawamoto H, Spanopoulou E, Itoi M, Amagai T, Jiang YF, Germeraad WT, Chen WF, and Katsura Y
- Subjects
- Animals, Cell Differentiation immunology, Humans, Stromal Cells immunology, T-Lymphocytes immunology, Thymus Gland embryology, Thymus Gland immunology, Cell Communication immunology, Stromal Cells cytology, T-Lymphocytes cytology, Thymus Gland cytology
- Abstract
Lympho-stromal interactions in the thymus crucially de- termine the fate of developing T cells. Epithelial cells, inter- digitating reticular cells, macrophages and fibroblasts all play a role in the shaping of the T cell repertoire. Recently published evidence shows that lympho-stromal interaction acts bi-directional. Developing T cell themselves, at different stages of differentiation, control the microarchitecture of thymic microenvironments, a phenomenon designated as 'crosstalk'. This paper reviews experiments showing that developing T cells crosstalk to different thymic epithelial cells in a stepwise fashion. In this way, correctly organized thymic microenvironments guarantee normal thymopoiesis., (Copyright 1999 Academic Press.)
- Published
- 1999
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49. Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments.
- Author
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Van Ewijk W, de Kruif J, Germeraad WT, Berendes P, Röpke C, Platenburg PP, and Logtenberg T
- Subjects
- Animals, Antibodies isolation & purification, Antibody Specificity, Bacteriophages, Humans, Immunodominant Epitopes, Immunohistochemistry, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Peptide Library, Antibodies immunology, Stromal Cells immunology, Thymus Gland immunology
- Abstract
In the murine thymus, the stroma forms microenvironments that control different steps in T cell development. To study the architecture of such microenvironments and more particularly the nature of communicative signals in lympho-stromal interaction during T cell development, we have employed the phage antibody display technology, with the specific aim of isolating thymic stromal cell-specific single-chain antibodies from a semisynthetic phage library. A subtractive approach using intact, mildly fixed thymic fragments as target tissue and lymphocytes as absorber cells generated monoclonal phages (MoPhabs) detecting subsets of murine thymic stromal cells. In the present paper we report on the reactivity of single-chain antibodies derived from three MoPhabs, TB4-4, TB4-20, and TB4-28. While TB4-4 and TB4-20 are both epithelium specific, TB4-28 detects an epitope expressed on both epithelial- and mesenchymal-derived stromal cells. TB4-4 reacts with all cortical epithelial cells and with other endoderm-derived epithelia, but this reagent leaves the majority of medullary epithelial cells unstained. In contrast, MoPhab TB4-20 detects both cortical and medullary thymic epithelial cells, as well as other endoderm- and ectoderm-derived epithelial cells. Cross-reaction of single-chain antibodies to human thymic stromal cells shows that our semisynthetic phage antibody display library, in combination with the present subtractive approach, permits detection of evolutionary conserved epitopes expressed on subsets of thymic stromal cells.
- Published
- 1997
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50. Gene transduction into murine primitive hematopoietic cells with 2-gene retroviral vectors using a Transwell coculture system.
- Author
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Asami N, Germeraad WT, Fujimoto S, Nagai S, Izumi T, and Katsura Y
- Subjects
- Animals, Bone Marrow Cells, Cells, Cultured, Escherichia coli genetics, Gene Expression, Genes, Bacterial, Genes, Reporter, Genetic Vectors, Mice, Mice, Inbred C57BL, Recombinant Proteins, Retroviridae genetics, Time Factors, Tissue Distribution, beta-Galactosidase genetics, Gene Transfer Techniques, Hematopoietic Stem Cells, Transduction, Genetic
- Abstract
The present study aims at expressing a reporter gene in hematopoietic cells in vivo by introducing it into primitive hematopoietic cells with a 2-gene retroviral vector. Various constructs of retroviral vectors containing the human IL-2 receptor alpha chain gene (TAC) as the reporter and the neomycin phosphotransferase gene (neo) as a selectable marker were engineered, and the effectiveness of these vectors for expression of the reporter gene was evaluated after transfection into the packaging cell line GP + E86. It was found that the highest levels of reporter gene expression were attained with constructs ordered 5' long terminal repeat (LTR)-TAC-internal promoter-neo-3' LTR. In experiments investigating the expression of a reporter gene in hematopoietic cells, we used the Escherichia coli beta-galactosidase gene (lacZ) instead of TAC, because a very sensitive detection method was available for lacZ. For transduction of hematopoietic progenitors, packaging cell lines producing recombinant viruses were cultured in a Transwell hung into a Dexter-type bone marrow (BM) culture. The BM cells were selected with G418, and transferred into irradiated recipient mice. LacZ enzyme activity was detectable in the peripheral blood lymphocytes (PBL) of recipients taken 8 wk after reconstitution.
- Published
- 1996
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