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Biomedical subjects

Martin Zenke

Publications and source records attributed to Martin Zenke.

At least 19 recordsLinked to original sources
PubMed2006

Fate of Chlamydophila pneumoniae in human monocyte-derived dendritic cells: long lasting infection.

Earlier studies from this group demonstrated that Chlamydophila pneumoniae co-localized with dendritic cells (DC) in temporal artery biopsies from patients with giant cell arteritis (GCA). To investigate the interaction of DC with C. pneumoniae we employed an in vitro cell culture system of human monocyte derived DC. These DC were infected with C. pneumoniae and observed at regular time intervals up to 25 days post infection. Chlamydiae were visualized inside DC by both confocal and electron microscopy. Statistical analysis showed an increase in the number of chlamydial antigen during that period (p < 0.00005, chi2-test). Titration of DC lysates on HEp-2 cells showed that infectious progeny was recovered at various intervals but showed no exponential growth. Additionally, RT-PCR analyses of infected DC identified transcripts from dnaA, ftsK and tal throughout a period of 14 days, indicating viable chlamydiae. Thus, human monocyte-derived DC are susceptible to C. pneumoniae infection. These results indicate that C. pneumoniae-infected DC can play an important role in the transmission of these bacteria in GCA and other chlamydial diseases.

Antigens, Bacterial↗
PubMed2006

Towards an understanding of the transcription factor network of dendritic cell development.

Dendritic cells (DCs) are antigen-presenting cells of the immune system and develop from hematopoietic stem cells through successive steps of lineage commitment and differentiation. The three major DC populations are epidermal Langerhans cells, tissue/interstitial/dermal DCs and plasmacytoid DCs. We review how gene-targeted mutations in mice have contributed to our understanding of how the various DC subpopulations develop. These studies have revealed both overlapping and distinct pathways of DC differentiation and show that there is no obvious correlation between transcription factor knockout phenotypes and a lymphoid or myeloid origin of DCs.

Animals↗
PubMed2005

Immunization with a lentiviral vector stimulates both CD4 and CD8 T cell responses to an ovalbumin transgene.

Lentiviral vectors encoding antigens are promising vaccine candidates because they transduce dendritic cells (DC) in vivo and prime CTL responses. Here we examine their stimulation of antigen-specific CD4(+) T cells, critical for protective immunity against tumors or infectious disease. We constructed lentiviral vectors (lentivectors) expressing ovalbumin, which was secreted (OVA), cytoplasmic (OVAcyt), or fused to either invariant chain (Ii-OVA) or transferrin receptor (TfR-OVA) sequences, targeting the MHC class II presentation pathway. Murine DC infected with the various lentivectors could stimulate OT-I (CD8(+), OVA TCR transgenic) T cells and all except OVAcyt could also stimulate OT-II (CD4(+), OVA TCR transgenic) T cells in vitro. Direct injection of the OVA-, Ii-OVA-, or TfR-OVA-expressing vectors into mice resulted in a CD4(+) T cell response, as shown by expansion of adoptively transferred OT-II T cells and upregulation of CD44 on these cells. The Ii-OVA vector was the most potent inducer of IFN-gamma-secreting CD4(+) and CD8(+) T cells and was the only vector to protect mice completely from challenge with OVA-expressing tumor cells. Therefore directly injected lentivectors can stimulate CD4(+) T cells; both CD4(+) and CD8(+) responses can be enhanced by targeting the antigen to the MHC class II pathway.

Animals↗
PubMed2005

Membrane proteinase 3 expression in patients with Wegener's granulomatosis and in human hematopoietic stem cell-derived neutrophils.

A large membrane proteinase 3 (mPR3)-positive neutrophil subset (mPR3high) is a risk for Wegener's granulomatosis (WG). The relationship between mPR3 expression and clinical manifestations was investigated in 81 WG patients and mPR3 expression was studied in CD34+ stem cell-derived human neutrophils. The mPR3high neutrophil percentage correlated with renal function, anemia, and albumin at the time of presentation. The mPR3high neutrophil percentage and renal failure severity correlated directly after 5 yr. For elucidating mechanisms that govern mPR3 expression, studies were conducted to determine whether the genetic information that governs mPR3 expression resides within the neutrophils, even without stimuli possibly related to disease. CD34+ hematopoietic stem cells were differentiated to neutrophils, and their mPR3 expression was determined. A two-step amplification/differentiation protocol was used to differentiate human CD34+ hematopoietic stem cells into neutrophils with G-CSF. The cells progressively expressed the neutrophil surface markers CD66b, CD35, and CD11b. The ferricytochrome C assay demonstrated a strong respiratory burst at day 14 in response to PMA but none at day 0. Intracellular PR3 was detectable from day 4 by Western blotting. An increasing percentage of a mPR3-positive neutrophil subset became detectable by flow cytometry, whereas a second subset remained negative, consistent with a bimodal expression. Finally, human PR3-anti-neutrophil cytoplasmic autoantibodies induced a stronger respiratory burst, compared with human control IgG in stem cell-derived neutrophils. Taken together, these studies underscore the clinical importance of the WG mPR3 phenotype. The surface mPR3 on resting cells is probably genetically determined rather than being dictated by external factors.

Adult↗
PubMed2005

GandrKB--ontological microarray annotation and visualization.

SUMMARY: The Gandr (gene annotation data representation) knowledgebase is an ontological framework for laboratory-specific gene annotation. Gandr uses Protege 2000 for editing, querying and visualizing microarray data and annotations. Genes can be annotated with provided, newly created or imported ontological concepts. Annotated genes can inherit assigned concept properties and can be related to each other. The resulting knowledgebase can be visualized as interactive network of nodes and edges representing genes and their functional relationships. This allows for immediate and associative gene context exploration. Ontological query techniques allow for powerful data access.

Algorithms↗
PubMed2005

Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro.

Dendritic cells (DC) represent key regulators of the immune system, yet their development from hemopoietic precursors is poorly defined. In this study, we describe an in vitro system for amplification of a Flt3(+)CD11b(+) progenitor from mouse bone marrow with specific cytokines. Such progenitor cells develop into both CD11b(+) and CD11b(-) DC, and CD8alpha(+) and CD8alpha(-) DC in vivo. Furthermore, with GM-CSF, these progenitors synchronously differentiated into fully functional DC in vitro. This two-step culture system yields homogeneous populations of Flt3(+)CD11b(+) progenitor cells in high numbers and allows monitoring the consecutive steps of DC development in vitro under well-defined conditions. We used phenotypic and functional markers and transcriptional profiling by DNA microarrays to study the Flt3(+)CD11b(+) progenitor and differentiated DC. We report here on an extensive analysis of the surface Ag expression of Flt3(+)CD11b(+) progenitor cells and relate that to surface Ag expression of hemopoietic stem cells. Flt3(+)CD11b(+) progenitors studied exhibit a broad overlap of surface Ags with stem cells and express several stem cell Ags such as Flt3, IL-6R, c-kit/SCF receptor, and CD93/AA4.1, CD133/AC133, and CD49f/integrin alpha(6). Thus, Flt3(+)CD11b(+) progenitors express several stem cell surface Ags and develop into both CD11b(+) and CD11b(-) DC, and CD8alpha(+) and CD8alpha(-) DC in vivo, and thus into both of the main conventional DC subtypes.

Adoptive Transfer↗
PubMed2005

In vivo haematopoietic activity is induced in neurosphere cells by chromatin-modifying agents.

Modifications of DNA and chromatin are fundamental for the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. To test whether the developmental potential of fetal brain-derived cells that form floating sphere colonies (neurospheres) can be modified by destabilizing their epigenotype, neurosphere cells were treated with chemical compounds that alter the acetylation and methylation patterns of chromatin and DNA. Intravenous infusion of bulk or clonally derived neurosphere cells treated with a combination of trichostatin A (TSA) plus 5-aza-2'-deoxycytidine (AzaC) (TSA/AzaC neurosphere cells) yielded long-term, multilineage and transplantable neurosphere-derived haematopoietic repopulation. Untreated neurosphere cells exhibited no haematopoietic repopulation activity. The neurosphere-derived haematopoietic cells showed a diploid karyotype, indicating that they are unlikely to be products of cell fusion events, a conclusion strengthened by multicolour fluorescence in situ hybridization. Our results indicate that altering the epigenotype of neurosphere cells followed by transplantation enables the generation of neurosphere-derived haematopoietic cells.

Animals↗
PubMed2005

Infection of mature dendritic cells with herpes simplex virus type 1 dramatically reduces lymphoid chemokine-mediated migration.

Herpes simplex virus type 1 (HSV-1) is able to establish latency in infected individuals. In order to characterize potential new immune-escape mechanisms, mature dendritic cells (DCs) were infected with HSV-1 and total cellular RNA was isolated from infected and mock-infected populations at different time points. RNA profiling on Affymetrix Human Genome U133A arrays demonstrated a dramatic downregulation of the migration-mediating surface molecules CCR7 and CXCR4, an observation that was further confirmed by RT-PCR and fluorescence-activated cell sorting analyses. Furthermore, migration assays revealed that, upon infection of mature DCs, CCR7- and CXCR4-mediated migration towards the corresponding CCL19 and CXCL12 chemokine gradients was strongly reduced. It is noteworthy that the infection of immature DCs with HSV-1 prior to maturation led to a failure of CCR7 and CXCR4 upregulation during DC maturation and, as a consequence, also induced a block in their migratory capacity. Additional migration assays with a Deltavhs mutant virus lacking the virion host shutoff (vhs) gene, which is known to degrade cellular mRNAs, suggested a vhs-independent mechanism. These results indicate that HSV-1-infected mature DCs are limited in their capacity to migrate to secondary lymphoid organs, the areas of antigen presentation and T-cell stimulation, thus inhibiting an antiviral immune response. This represents a novel, previously unrecognized mechanism for HSV-1 to escape the human immune system.

Cell Differentiation↗
PubMed2005

RNA transfer and its use in dendritic cell-based immunotherapy.

RNA is a key macromolecule for the mobilisation and interpretation of genetic information. Research has sought to exploit the inherent properties of RNA, such as the direct production of proteins in the cytoplasm without the need for nuclear translocation. This property makes the delivery of genes into postmitotic cells especially attractive. Recently, RNA transfer into postmitotic dendritic cells (DCs) has emerged as a potential new therapeutic agent in the area of immunotherapy. DCs are the most important regulators of the immune system. Thus, transfecting DCs with RNA allows the specific manipulation of immune responses and, thereby, the treatment of a variety of diseases, such as cancer. Preclinical studies have demonstrated that RNA-transduced DCs efficiently stimulate antigen-specific T cell responses in vitro and in animal tumour models. In addition, the clinical data from Phase I and II trials of tumour patients indicate that RNA-transduced DCs represent a promising approach for the development of future vaccination strategies. The use of RNA molecules as therapeutic agents is a relatively new approach in the treatment of diseases, such as cancer, but has received increasing attention during the past decade. Especially in the field of immunotherapy, the inherent properties of RNA molecules in combination with immunostimulating dendritic cells (DCs) are being investigated at present for their beneficial therapeutic effect. Immunotherapy is based on the stimulation of the patient's immune system to recognise and eliminate infected cells or tumour cells in an antigen-specific manner. Current approaches focus on the stimulation of CD8(+) cytotoxic T lymphocyte responses, as well as on the induction of CD4(+) T helper cell responses, in order to obtain optimal and sustained immune responses capable of eliminating altered cells. This review mainly focuses on the potential use of RNA-transduced DCs as a therapeutic strategy in the treatment of cancer, as current studies on the treatment of infectious diseases are just beginning.

Animals↗
PubMed2004

Immunomodulatory derivative of thalidomide (IMiD CC-4047) induces a shift in lineage commitment by suppressing erythropoiesis and promoting myelopoiesis.

Immunomodulatory derivative (IMiD) CC-4047, a new analog of thalidomide, directly inhibits growth of B-cell malignancies in vivo and in vitro and exhibits stronger antiangiogenic activity than thalidomide. However, there is little information on whether CC-4047 affects normal hematopoiesis. Here we investigated the effect of CC-4047 on lineage commitment and differentiation of hematopoietic stem cells. We found that CC-4047 effectively inhibits erythroid cell colony formation from CD34+ cells and increases the frequency of myeloid colonies. We also demonstrate that development of both erythropoietin-independent and erythropoietin-dependent red cell progenitors was strongly inhibited by CC-4047, while terminal red cell differentiation was unaffected. DNA microarray analysis revealed that red cell transcription factors, including GATA-1, GATA-2, erythroid Kruppel-like factor (EKLF), and growth factor independence-1B (Gfi-1b), were down-regulated in CC-4047-treated CD34+ cells, while myeloid transcription factors such as CCAAT/enhancer binding protein-alpha (C/EBPalpha), C/EBPdelta, and C/EBPepsilon were induced. Analysis of cytokine secretion indicated that CC-4047 induced secretion of cytokines that enhance myelopoiesis and inhibit erythropoiesis. In conclusion, these data indicate that CC-4047 might directly influence lineage commitment of hematopoietic cells by increasing the propensity of stem and/or progenitor cells to undergo myeloid cell development and concomitantly inhibiting red cell development. Therefore, CC-4047 provides a valuable tool to study the mechanisms underlying lineage commitment.

Cell Differentiation↗
PubMed2004

Immunoglobulin-like transcripts ILT2, ILT3 and ILT7 are expressed by human dendritic cells and down-regulated following activation.

Immunoglobulin-like transcripts (ILT) represent novel immunoglobulin superfamily receptors that are expressed in myeloid, lymphoid and dendritic cells (DC). Here, we studied by gene expression profiling with DNA microarrays ILT expression in different DC subsets, including plasmacytoid DC (PDC), monocyte-derived DC (Mo-DC) and DC obtained by in vitro differentiation from CD34(+) progenitor cells, and DC activated in the presence of different activating agents. ILT2 and ILT3 were expressed in PDC, Mo-DC and DC obtained from CD34(+) cells. ILT7 mRNA was present in PDC, but absent in Mo-DC and DC obtained from CD34(+) cells, indicating that ILT7 mRNA expression seems to be a marker for PDC. CpG-DNA and inflammatory stimuli, such as TNF alpha, prostaglandin E2 (PGE2) and soluble CD40 ligand (sCD40L), and different combinations thereof are frequently employed for DC activation. Here, we demonstrate that ILT2 and ILT3 expression is down-regulated following DC activation by CpG-DNA and inflammatory stimuli at both mRNA and protein levels. Thus, activation of human DC with such stimuli involves down-regulation of inhibitory ILT2 and ILT3 receptors, and this could represent a novel mechanism contributing to DC activation.

Antigens, CD↗
PubMed2004

RNA-containing adenovirus/polyethylenimine transfer complexes effectively transduce dendritic cells and induce antigen-specific T cell responses.

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cells in initiating primary immune responses. Given the unique properties of DCs, gene-modified DCs represent a particularly attractive approach for immunotherapy of diseases such as cancer. METHODS: Gene-modified DCs were obtained by a receptor-mediated gene delivery system using adenovirus (Ad) particles as ligand and RNA or DNA condensed by polyethylenimine (PEI). In vitro transcribed polyadenylated or non-polyadenylated RNA was used. RNA-transduced DCs were generated expressing chicken ovalbumin (OVA) or chimeric constructs thereof, and compared with DNA-transduced DCs. RESULTS: Ad/PEI transfection complexes efficiently delivered RNA into DCs. Such RNA-transduced DCs induced OVA-specific T cell responses more effectively than DNA-transduced DCs. Furthermore, DCs transduced with polyadenylated RNA were more potent in stimulating CD4(+) and CD8(+) T cell responses than DCs transduced with non-polyadenylated RNA and this was particularly important for CD4(+) T cell responses. CONCLUSIONS: Ad/PEI/RNA transfection is an efficient means for generating RNA-transduced DCs and for stimulating antigen-specific T cell responses. Polyadenylation of RNA enhances CD8(+) T cell responses and is essential for CD4(+) T cell responses.

Adenoviridae↗
PubMed2004

Gene expression profiling of dendritic cells by DNA microarrays.

Dendritic cells (DC) are professional antigen presenting cells that play a pivotal role in initiating primary immune responses and have been implicated in determining the balance between immunity and tolerance. DC originate from hematopoietic stem cells in bone marrow and different DC subsets have been identified according to their phenotype, function, activation state and location. Gene expression analysis of DC by DNA microarrays represents a systemic approach to the underlying complexity of DC biology and is expected to provide important clues to the application of DC in the clinic. Here we review the recent findings from the use of DNA microarrays in DC biology and discuss the challenges emerging from these studies.

Animals↗
PubMed2003

GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter.

GATA-1 and c-Myb transcription factors represent key regulators of red blood cell development. GATA-1 is upregulated and c-myb proto-oncogene expression is downregulated when red cell progenitors differentiate into erythrocytes. Here we have employed a culture system, that faithfully recapitulates red blood cell differentiation in vitro, to follow the kinetics of GATA-1 and c-myb expression. We show that c-myb proto-oncogene expression is high in progenitors and effectively downregulated at the time when nuclear GATA-1 accumulates and cells differentiate into erythrocytes. Additionally, we identified two GATA-1 binding sites within the c-myb promoter and demonstrate that GATA-1 protein binds to these sites in vitro. Furthermore, GATA-1 represses c-myb expression through one of the GATA-1 binding sites in transient transfection experiments and this requires FOG-1. Thus, our study provides evidence for a direct molecular link between GATA-1 activity and c-myb proto-oncogene expression during terminal red cell differentiation.

Animals↗
PubMed2003

Transcriptional profiling identifies Id2 function in dendritic cell development.

Dendritic cells (DCs) are potent antigen-presenting cells with a pivotal role in antigen-specific immune responses. Here, we found that the helix-loop-helix transcription factor Id2 is up-regulated during DC development in vitro and crucial for the development of distinct DC subsets in vivo. Id2-/- mice lack Langerhans cells (LCs), the cutaneous contingent of DCs, and the splenic CD8alpha+ DC subset is markedly reduced. Mice deficient for transforming growth factor (TGF)-beta also lack LCs, and we demonstrate here that, in DCs, TGF-beta induces Id2 expression. We also show that Id2 represses B cell genes in DCs. These findings reveal a TGF-beta-Id2 signaling pathway in DCs and suggest a mechanism by which Id2 affects the lineage choice of B cell and DC progenitors.

B-Lymphocytes↗
PubMed2003

Cryopreservation of mature monocyte-derived human dendritic cells for vaccination: influence on phenotype and functional properties.

In the past decade there has been increasing evidence that tumor antigen-loaded dendritic cells (DC) are able to elicit anti-tumor T-cell responses. Initial clinical data for different tumor entities are encouraging, with objective tumor regressions being observed in some patients. Since GMP production of DC for clinical vaccination protocols is a time- and cost-intensive procedure, cryopreservation of DC in aliquots ready for clinical use would significantly facilitate DC-based vaccination in the clinic. We asked whether freezing and thawing alters the phenotype or functional properties of DC. DC from healthy volunteers and from patients with chronic myeloid leukemia (CML) were analyzed after freezing and thawing for their viability, morphology, immunophenotype (FACS profile), T-cell stimulatory capacity (mixed lymphocyte reaction) and mobility (time-lapse cinemicroscopy). Our results demonstrate that cryopreservation does not cause significant changes in the phenotype or function of DC, neither in DC from healthy volunteers nor in those from CML patients. Our data indicate that cryopreserved aliquots of DC are suitable for clinical application in DC-based immunotherapy protocols.

Cancer Vaccines↗
PubMed2002

Nuclear factor kappaB-dependent gene expression profiling of Hodgkin's disease tumor cells, pathogenetic significance, and link to constitutive signal transducer and activator of transcription 5a activity.

Constitutive nuclear nuclear factor (NF)-kappaB activity is observed in a variety of hematopoietic and solid tumors. Given the distinctive role of constitutive NF-kappaB for Hodgkin and Reed-Sternberg (HRS) cell viability, we performed molecular profiling in two Hodgkin's disease (HD) cell lines to identify NF-kappaB target genes. We recognized 45 genes whose expression in both cell lines was regulated by NF-kappaB. The NF-kappaB-dependent gene profile comprises chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors, the majority of which maintain a marker-like expression in HRS cells. Remarkably, we found 17 novel NF-kappaB target genes. Using chromatin immunoprecipitation we demonstrate that NF-kappaB is recruited directly to the promoters of several target genes, including signal transducer and activator of transcription (STAT)5a, interleukin-13, and CC chemokine receptor 7. Intriguingly, NF-kappaB positively regulates STAT5a expression and signaling pathways in HRS cells, and promotes its persistent activation. In fact, STAT5a overexpression was found in most tumor cells of tested patients with classical HD, indicating a critical role for HD. The gene profile underscores a central role of NF-kappaB in the pathogenesis of HD and potentially of other tumors with constitutive NF-kappaB activation.

Base Sequence↗