Biomedical subjects

Oliver Dittrich-Breiholz

Publications and source records attributed to Oliver Dittrich-Breiholz.

16 recordsLinked to original sources

PubMed2026

Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality.

BACKGROUND: Gene therapy (GT) using retroviral vectors (RVs) is efficacious in treating monogenic diseases. However, there is an inherent risk for severe adverse effects due to insertional mutagenesis. Preclinical safety assessment and patient monitoring are inevitable in GT. To assess the genotoxic risk of novel RV vectors, mainly murine hematopoietic stem and progenitor cells (HPSCs) are routinely used, because human HSPCs cannot be immortalized in vitro using mutagenic vectors. In this study, we aim to identify early signs of clonal outgrowth by performing integration site analyses (ISA). METHODS: The small molecules A83-01, pomalidomide, and UM171 (APU) were used for the ex vivo expansion, lentiviral transduction, and long-term cultivation of umbilical cord blood-derived HSPCs. We determined the influence of APU on the stemness of HSPCs and their differentiation capacity via single-cell RNA sequencing (scRNA seq) and in xenotransplantation studies. To track vector insertion site dynamics, we transduced 7-day expanded HSPCs with a mutagenic or a safer RV. ISA was conducted in human HSPCs over a 5-week cultivation in vitro and compared to the bone marrow of xenotransplanted mice to assess clonal skewings. RESULTS: APU supported the expansion of CD34+CD38-CD45RA-CD90+EPCR+ HSPCs. scRNA seq confirmed the enrichment of HSC signature genes in APU-expanded HSPCs compared to the clinically used medium SFT3 (SCF, FLT3-L, TPO, IL-3). After RV transduction, APU still maintained around 30% of CD34+ cells for 5 more weeks. Without the compounds, already 2 weeks post-transduction, less than 10% of cells were CD34+. The long-term culture allowed the detection of high-risk integrations of the mutagenic SIN-LV.SF in MEIS1 or SUSD6 due to their increasing abundance over time. Bone marrow of xenotransplanted mice was less clonal but did not support the outgrowth of insertional mutants. Overall, APU increased clonal diversity. CONCLUSIONS: Our findings propose that long-term cultivation of transduced HSPC in APU allows for outgrowth of clonal integration sites. The decrease of clonality has been observed in gene therapy patient's years after treatment. Thus, the in vitro model could be used to develop novel human HSPC-based genotoxicity assays that predict insertional mutagenesis, in addition to existing preclinical biosafety assays.

Humans↗

PubMed2025

Deep inflations maintain surfactant function and alveolar fluid balance in lungs with reduced surfactant protein B levels during mechanical ventilation.

Surfactant protein B (SP-B) is essential for surface tension reducing function of pulmonary surfactant and alveolar unfolding processes during inspiration. SP-B is reduced early in acute lung injury. Hence, we hypothesize that 1) reduced SP-B expression increases susceptibility to ventilation-induced lung injury (VILI), and 2) deep inflations (DI) are protective against VILI. Conditional SP-B knockout mice were randomized into OFF (reduced SP-B) and ON groups (normal SP-B) and subjected to mechanical ventilation at zero end-expiratory pressure. Over 4 h of ventilation, either 4 or 16 DI were administered. Lung mechanics were recorded, and pulmonary structure was quantified by design-based stereology. Inflammatory cells and bulk RNA sequencing were measured in bronchoalveolar lavage (BAL) and tissue, respectively. No differences in inflammatory cells in BAL were detected between ON and OFF groups. During ventilation, alveolar derecruitment-related increase in elastance was most pronounced in OFF-4DI but reversible by DI so that lung mechanics did not worsen. Finally, volumes of the alveolar liquid lining layer and the intracellular surfactant were largest, whereas the surface area of the apical plasma membrane of type II pneumocytes was smallest in OFF-4DI, suggesting impaired surfactant secretion. A higher frequency of DI prevented these abnormalities. Electron microscopy revealed disorganized tight junctions between alveolar epithelial cells in OFF-4DI, which was linked with decreased expression of genes relevant to the apical junctional complex. Reduced SP-B resulted in a progressive increase in surface tension and a disturbed fluid balance without triggering definite VILI. Maintenance of residual surfactant function is highly dependent on DI in conditions of reduced SP-B levels.NEW & NOTEWORTHY Surfactant protein B (SP-B) is critical for efficient surfactant function in the lung. Reduced SP-B levels occur at an early stage of acute lung injury and impair alveolar unfolding. In this study, we demonstrate that mechanical ventilation of lungs with reduced SP-B levels does not trigger ventilation-induced lung injury but results in disbalance of alveolar fluid volume and increase in surface tension due to failure of surfactant maintenance. Deep inflations prevent these ventilation-induced effects.

Animals↗

PubMed2006

Modulation of host gene expression by the K15 protein of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) contains several open reading frames (ORFs) encoding proteins capable of initiating signal transduction pathways. Among them is the K15 ORF, which consists of eight exons encoding a protein with 12 predicted transmembrane domains and a cytoplasmic C terminus. When transiently expressed, the 8-exon K15 transcript gives rise to a protein with an apparent molecular mass of 45 kDa. K15 interacts with cellular proteins, TRAF (tumor necrosis factor receptor-associated factor) and Src kinases, and activates AP-1, NF-kappaB, and the mitogen-activated protein kinases (MAPKs) c-jun-N-terminal kinase and extracellular signal-regulated kinase. This signaling activity of K15 is related to phosphorylation of Y(481) of the K15 SH2-B motif Y(481)EEV. In this study we demonstrate the expression of an endogenous 45-kDa K15 protein in KSHV BAC36-infected epithelial cells. This endogenous K15 protein shows the same intracellular localization as transiently expressed K15, and expression kinetic studies suggest it to be a lytic gene. We have further determined the downstream target genes of K15 signaling using DNA oligonucleotide microarrays. We demonstrate that K15 is capable of inducing expression of multiple cytokines and chemokines, including interleukin-8 (IL-8), IL-6, CCL20, CCL2, CXCL3, and IL-1alpha/beta, as well as expression of Dscr1 and Cox-2. In epithelial cells, K15-induced upregulation of most genes was dependent on phosphorylation of Y(481), whereas in endothelial cells mutation of Y(481) did not result in a complete loss of Dscr1 and Cox-2 expression and NFAT-activity. Our study establishes K15 as one of the KSHV lytic genes that are inducing expression of multiple cytokines, which have been shown to play an important role in KSHV-associated pathogenesis.

Animals↗

PubMed2006

Induction of C3 and CCL2 by C3a in keratinocytes: a novel autocrine amplification loop of inflammatory skin reactions.

The complement fragment-3a (C3a) acts via a G protein-coupled C3aR and is of importance in allergic and inflammatory diseases. Recent studies suggest the presence of complement proteins in the epidermal compartment and synthesis of some of these proteins (C3, factor B, and factor H) by human primary keratinocytes (KCs) during inflammation. However, expression of C3aR and its role in human KCs is not elucidated thus far. In this study, we demonstrate the expression of C3aR on KCs as detected by quantitative real-time RT-PCR and flow cytometry. IFN-gamma and IFN-alpha strongly up-regulated the surface expression of C3aR on KCs among all other cytokines tested. After up-regulation of C3aR by IFN-gamma and IFN-alpha, we observed the induction of five genes (CCL2, CCL5, CXCL8, CXCL10, and C3) after stimulation of KCs with C3a in microarray analysis. We confirmed the induction of C3 and CCL2 at RNA and protein levels. Furthermore, incubation of C3 with skin mast cells tryptase resulted in the generation of C3 fragments with C3a activity. In conclusion, our data illustrate that epidermal KCs express functional C3aR. The increases of C3 and CCL2 synthesis by C3a and C3 activation by skin mast cell tryptase delineates a novel amplification loop of complement activation and inflammatory responses that may influence the pathogenesis of allergic/inflammatory skin diseases.

Autocrine Communication↗

PubMed2006

Host chemokine (C-C motif) ligand-2 (CCL2) is differentially regulated in HIV type 1 (HIV-1)-infected individuals.

Several cytokines and chemokines including chemokine (C-C motif) ligand-2 (CCL2) are induced in HIV-1 infection. However, the impact of HIV-1 viremia on CCL2 regulation is largely unknown. We utilized a DNA oligonucleotide microarray covering 110 inflammatory genes. Five genes were induced by at least 2-fold in PBMCs of HIV-1 viremic (>100,000 RNA copies ml(-1)) as compared with aviremic (<50 RNA copies ml(-1)) individuals. These genes were CCL2, CXC chemokine ligand-10, IFN-gamma, GTP-cyclohydrolase-1 and C-C chemokine receptor-1. In addition to microarray data verification by real-time PCR, analysis of independent patient samples revealed a similar expression pattern. CCL2 was the most strongly regulated gene at mRNA level and its serum concentration was significantly elevated in viremic compared with aviremic and HIV-1 seronegative controls, indicating a positive correlation between viremia and CCL2. Flow cytometric studies demonstrated a higher percentage of CCL2-expressing CD14(+) monocytes in viremic compared with aviremic individuals. These results suggest a highly restricted modulation of host inflammatory gene response by HIV. Genes up-regulated in the viremic state, in particular CCL2, presumably serve as potential enhancing factors in HIV-1 replication, represented by high viral load in HIV-1 viremic patients. Inhibition of increased CCL2 production could provide a new therapeutic intervention in HIV-1 infection.

Antiretroviral Therapy, Highly Active↗

PubMed2006

Small interfering RNAs generated by recombinant dicer induce inflammatory gene expression independent from the TAK1-NFkappaB-MAPK signaling pathways.

Generation of mixtures of small interfering (si) RNAs by recombinant dicer avoids selection of efficient target sites within mRNAs but little is known about off-target effects of this approach. Using recombinant human dicer we generated siRNA mixtures (dsiRNA) directed against the protein kinase TAK1 and its subunit TAB1, important upstream molecules in the pathways activated by IL-1, TNF, and toll-like receptors (TLR). dsiRNA against TAK1 or TAB1 significantly suppressed their target proteins as well as TAK1-mediated activation of NFkappaB, p38 MAPK, and JNK, and of IL-8 transcription. However, microarray analysis of 136 endogenous inflammatory genes revealed that dsiRNA against TAB1 or TAK1 did not suppress IL-1 or TNF-induced genes but rather induced a broader range of 15 inflammatory genes as well as seven known interferon-response genes. The same genes were induced by dsiRNA directed against luciferase but not by a synthetic control siRNA molecule. Hence, our results show that complex mixtures of siRNA induce an inflammatory gene response that is independent from TAK1-mediated signal transduction. In the light of the increasing usage of enzymatically prepared libraries of siRNA these results provide important insight into potential off-target effects of this approach.

Gene Expression↗

PubMed2006

Identification of mouse genes with highly specific expression patterns in differentiated intestinal epithelium.

BACKGROUND & AIMS: Few genes that regulate intestinal epithelium development, homeostasis, or function are known. We reasoned that potential candidate regulators of these processes could be identified based on their activation during intestinal epithelium development and their subsequent specific and restricted expression. METHODS: Genes were identified by differential display and microarray analyses, further selected according to sequence and UniGene expression profiles, and analyzed by RNA in situ hybridization of mouse fetal and adult intestines and in intestinal polyp tissue. RESULTS: Five genes with unknown physiological function predominantly or exclusively expressed in the intestinal epithelium were identified. Their expression is activated at distinct times during intestinal development and maturation and is maintained in highly specific, spatially distinct patterns in the adult intestinal epithelium. Two of the genes were up-regulated in intestinal tumors, 1 was down-regulated, and 2 were apparently unaltered. CONCLUSIONS: Based on sequence and expression, the identified genes represent good candidates for regulators of intestinal epithelium integrity or function. Their expression patterns suggest a morphologically not obvious molecular regionalization of the intestinal epithelium along the crypt villus axis. This approach should be an efficient means to identify novel genes required for intestinal epithelium homeostasis and function.

Animals↗

PubMed2006

Cell target genes of Epstein-Barr virus transcription factor EBNA-2: induction of the p55alpha regulatory subunit of PI3-kinase and its role in survival of EREB2.5 cells.

Microarray analysis covering most of the annotated RNAs in the human genome identified a panel of genes induced by the Epstein-Barr virus (EBV) EBNA-2 transcription factor in the EREB2.5 human B-lymphoblastoid cell line without the need for any intermediate protein synthesis. Previous data indicating that PIK3R1 RNA (the alpha regulatory subunit of PI3-kinase) was induced were confirmed, but it is now shown that it is the p55alpha regulatory subunit that is induced. Several EBV-immortalized lymphoblastoid cell lines were shown to express p55alpha. Expression of PI3-kinase p85 regulatory and p110 catalytic subunits was not regulated by EBNA-2. Proliferation of EREB2.5 lymphoblastoid cells was inhibited by RNAi knock-down of p55alpha protein expression, loss of p55alpha being accompanied by an increase in apoptosis. p55alpha is thus a functional target of EBNA2 in EREB2.5 cells and the specific regulation of p55alpha by EBV will provide an opportunity to investigate the physiological function of p55alpha in this human cell line.

Apoptosis↗

PubMed2006

Inhibition of mRNA deadenylation and degradation by different types of cell stress.

We have previously observed rapid and strong inhibition of mRNA deadenylation and degradation in response to UV-B light [Gowrishankar et al., Biol. Chem. 386 (2005), pp. 1287-1293]. Expression analysis using a microarray for inflammatory genes showed that UV-B light induces stabilization of all short-lived mRNAs assayed. Stabilization was observed in HeLa cells, as well as in the keratinocyte line HaCaT. It affected constitutively expressed mRNA species, as well as species induced by the inflammatory cytokine IL-1. Many of the latter encode proteins involved in inflammation, suggesting that stress-induced inhibition of mRNA deadenylation contributes to changes in inflammatory gene expression. Deadenylation and degradation of tet-off-expressed mRNAs were also inhibited upon exposure to H2O2. However, scavengers of reactive oxygen species did not interfere with UV-B-induced inhibition of degradation, arguing against the involvement of UV-induced H2O2 in these effects of UV-B light. Heat shock and hyperosmolarity also inhibited mRNA deadenylation and degradation, whereas gamma-radiation did not. Thus, inhibition of mRNA deadenylation and degradation is a cellular response elicited by several but not all inducers of cell stress.

Adenine↗

PubMed2005

Simultaneous blockade of NFkappaB, JNK, and p38 MAPK by a kinase-inactive mutant of the protein kinase TAK1 sensitizes cells to apoptosis and affects a distinct spectrum of tumor necrosis factor [corrected] target genes.

The inflammatory response is characterized by the induction (or repression) of hundreds of genes. The activity of many of these genes is controlled by MAPKs and the IkappaB kinase-NFkappaB pathway. To reveal the effects of blocking these pathways simultaneously, fibroblasts were infected with retroviruses encoding TAK1K63W, an inactive mutant of the protein kinase TAK1. Expression of this protein inhibited tumor necrosis factor (TNF)-induced activation of NFkappaB, JNK, and p38 MAPK and sensitized the cells to TNF-induced apoptosis. 23 different microarray experiments were used to analyze the expression of >7000 genes in these cells. We identified 518 genes that were regulated by TNF in both TAK1K63W-expressing cells and control cells, 37 genes induced by TNF only when TAK1K63W was present, and 48 TNF-induced genes that were suppressed by TAK1K63W. The TNF-inducible genes that were most strongly suppressed by TAK1K63W, ccl2, ccl7, ccl5, cxcl1, cxcl5, cxcl10, saa3, and slpi also had much lower basal levels of expression, indicating that TAK1 also played a role in their normal expression. Chromatin immunoprecipitation studies on four of these genes suggested that inactivation of TAK1 activity led to direct suppression of expression at the transcriptional level because of impaired recruitment of RNA polymerase II to their promoters. ccl2 induction by TNF or interleukin-1 was also suppressed in cells that expressed TAK1 antisense RNA or that were genetically deficient in JNK1/2 or p65 NFkappaB. These data suggest that regulation of the expression of a selected group of inflammation-related genes is funneled through TAK1, making it a potentially useful target for more specific anti-inflammatory drug development.

Animals↗

PubMed2005

Death receptor-induced signaling pathways are differentially regulated by gamma interferon upstream of caspase 8 processing.

FasL and gamma interferon (IFN-gamma) are produced by activated T cells and NK cells and synergistically induce apoptosis. Although both cytokines can also elicit proinflammatory responses, a possible cross talk of these ligands with respect to nonapoptotic signaling has been poorly addressed. Here, we show that IFN-gamma sensitizes KB cells for apoptosis induction by facilitating death-inducing signaling complex (DISC)-mediated caspase 8 processing. Moreover, after protection against death receptor-induced apoptosis by caspase inhibition or Bcl2 overexpression, IFN-gamma also sensitized for Fas- and TRAIL death receptor-mediated NF-kappaB activation leading to synergistic upregulation of a variety of proinflammatory genes. In contrast, Fas-mediated activation of JNK, p38, and p42/44 occurred essentially independent from IFN-gamma sensitization, indicating that the apoptosis- and NF-kappaB-related FasL-IFN-gamma cross talk was not due to a simple global enhancement of Fas signaling. Overexpression of FLIP(L) and FLIP(S) inhibited Fas- as well as TRAIL-mediated NF-kappaB activation and apoptosis induction in IFN-gamma-primed cells suggesting that both responses are coregulated at the level of the DISC.

Apoptosis↗

PubMed2004

MEK1-dependent delayed expression of Fos-related antigen-1 counteracts c-Fos and p65 NF-kappaB-mediated interleukin-8 transcription in response to cytokines or growth factors.

Binding sites for the dimeric transcription factor activator protein (AP)-1 are found in numerous immunoregulatory and inflammatory genes. The precise mechanisms by which AP-1 activates or represses immune response genes and in particular the roles of individual AP-1 subunits in inflammatory responses are largely unknown. We report here that c-Fos and Fos-related antigen-1 (Fra-1), two inducible components of AP-1, are recruited to the endogenous interleukin (IL)-8 promoter in an IL-1-dependent manner. c-Fos activates IL-8 transcription and synergizes in this effect with p65 NF-kappaB. In contrast, Fra-1 strongly inhibits inducible IL-8 transcription. Fra-1 activation involves its stabilization, ubiquitination, and interaction with histone deacetylase-1. Blockade of MEK1 by PD98059 suppresses c-Fos and Fra-1 expression and, thus, affects two counteractive signals for IL-8 mRNA synthesis simultaneously. This disturbs the inducible recruitment of TATA box-binding protein and RNA polymerase II to the IL-8 promoter. Additional experiments reveal that, in conjunction with p65 NF-kappaB, the MEK1-ERK-dependent synthesis of c-Fos and Fra-1 serves to adjust the overall expression level of IL-8 in response to two of its physiological inducers, IL-1 and epidermal growth factor. Relative to c-Fos, the delayed recruitment of Fra-1 to the IL-8 promoter provides an example how AP-1 subunits may dampen excessive chemokine synthesis.

Cell Line↗

PubMed2004

NFkappaB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP.

Fas (APO-1/CD95) is the prototypic death receptor, and the molecular mechanisms of Fas-induced apoptosis are comparably well understood. Here, we show that Fas activates NFkappaB via a pathway involving RIP, FADD, and caspase-8. Remarkably, the enzymatic activity of the latter was dispensable for Fas-induced NFkappaB signaling pointing to a scaffolding-related function of caspase-8 in nonapoptotic Fas signaling. NFkappaB was activated by overexpressed FLIPL and FLIPS in a cell type-specific manner. However, in the context of Fas signaling both isoforms blocked FasL-induced NFkappaB activation. Moreover, down-regulation of both endogenous FLIP isoforms or of endogenous FLIPL alone was sufficient to enhance FasL-induced expression of the NFkappaB target gene IL8. As NFkappaB signaling is inhibited during apoptosis, FasL-induced NFkappaB activation was most prominent in cells that were protected by Bcl2 expression or caspase inhibitors and expressed no or minute amounts of FLIP. Thus, protection against Fas-induced apoptosis in a FLIP-independent manner converted a proapoptotic Fas signal into an inflammatory NFkappaB-related response.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Comparative analysis of T-cell costimulation and CD43 activation reveals novel signaling pathways and target genes.

The CD43 lymphocyte surface receptor is involved in the regulation of lymphocyte adhesion and activation. Many CD43 functions remain controversial or unclear, and it is not known to which extent CD43 signaling pathways are shared with or distinct from those used by the T-cell receptor (TCR). Here, we systematically compared signaling events and target gene expression induced by CD43 or T-cell costimulation in primary human peripheral T cells. These studies identify nuclear factor-kappaB (NF-kappaB) p65 serine 468 as a novel inducible phosphorylation site strongly induced by T-cell costimulation and only weakly triggered by CD43 ligation. We also identified CD43 as a novel Jun N-terminal kinase (JNK) activator and a comprehensive analysis of further signaling events suggests that both stimuli use overlapping but also distinct signaling pathways. Microarray analysis of inflammatory genes shows 1 group of genes coregulated by both stimuli and 2 further groups of target genes affected solely by costimulation or primarily by CD43.

Antigens, CD↗

PubMed2003

Disruption of the c-JUN-JNK complex by a cell-permeable peptide containing the c-JUN delta domain induces apoptosis and affects a distinct set of interleukin-1-induced inflammatory genes.

The transcription factor activator protein (AP)-1 plays crucial roles in proliferation, cell death, and the immune response. c-JUN is an important component of AP-1, but only very few c-JUN response genes have been identified to date. Activity of c-JUN is controlled by NH2-terminal phosphorylation (JNP) of its transactivation domain by a family of JUN-NH2-terminal protein kinases (JNK). JNK form a stable complex with c-JUN in vitro and in vivo. We have targeted this interaction by means of a cell-permeable peptide containing the JNK-binding (delta) domain of human c-JUN. This peptide strongly and specifically induced apoptosis in HeLa tumor cells, which was paralleled by inhibition of serum-induced c-JUN phosphorylation and up-regulation of the cell cycle inhibitor p21cip/waf. Application of the c-JUN peptide to interleukin (IL)-1-stimulated human primary fibroblasts resulted in up-regulation of four genes, namely COX-2, MnSOD, I kappa B alpha, and MAIL and down-regulation of 10 genes, namely CCL8, mPGES, SAA1, hIAP-1, hIAP-2, pent(r)axin-3, CXCL10, IL-1 beta, ICAM-1, and CCL2. Only a small group of genes, namely pent(r)axin-3, CXCL10, ICAM-1, and IL-1 beta, was inhibited by both the c-JUN peptide and the JNK inhibitor SP600125. Thereby, and by additional experiments using small interfering RNA to suppress endogenous c-JUN we identify for the first time three distinct groups of inflammatory genes whose IL-1-induced expression depends on c-JUN, on JNK, or on both. These results shed further light on the complexity of c-JUN-JNK-mediated gene regulation and also highlight the potential use of dissecting signaling downstream from JNK to specifically target proliferative diseases or the inflammatory response.

Amino Acid Sequence↗

PubMed2002

Multiple control of interleukin-8 gene expression.

Interleukin (IL)-8, a prototypic human chemokine, was detected more than a decade ago as the founding member of the chemokine superfamily. One of the most remarkable properties of IL-8 is the variation of its expression levels. In healthy tissues, IL-8 is barely detectable, but it is rapidly induced by ten- to 100-fold in response to proinflammatory cytokines such as tumor necrosis factor or IL-1, bacterial or viral products, and cellular stress. Recently, significant advances in the understanding of signaling pathways, which coordinately regulate IL-8 transcription as well as mRNA stabilization in response to external stimuli, have been made. Maximal IL-8 amounts are generated by a combination of three different mechanisms: first, derepression of the gene promoter; second, transcriptional activation of the gene by nuclear factor-kappaB and JUN-N-terminal protein kinase pathways; and third, stabilization of the mRNA by the p38 mitogen-activated protein kinase pathway. In that way, cells are able to rapidly increase and at the same time, to fine-tune the amount of IL-8 secreted and thereby control the extent of leukocytes attracted to sites of tissue injury.

Animals↗