Complex evolution of tandem-repetitive DNA in the Chironomus thummi species group.

The subspecies Chironomus thummi thummi and C. t. piger display dramatic differences in the copy number and chromosomal localization of a tandemly repeated DNA family (Cla elements). In order to analyze the evolutionary dynamics of this repeat family, we studied the organization of Cla elements in the related outgroup species C. luridus. We find three different patterns of Cla element organization in C. luridus, showing that Cla elements may be either strictly tandem-repetitive or be an integral part of two higher-order tandem repeats (i.e., Hinf[lur] elements, Sal[lur] elements). All three types of Cla-related repeats are localized in the centromeres of C. luridus chromosomes. This suggest…

Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin.

Strong cell-type-specific promoters are basic tools in gene therapy allowing for novel applications and focused strategies by transcriptionally targeting gene expression to selected cells. In immunotherapy, dendritic cells (DC) are of central importance, since they represent the principal inducers of immune responses. Here we describe isolation and use of the promoter of the murine actin-bundling protein fascin to target transcriptionally gene expression to cutaneous DC. Using the reporter gene enhanced green fluorescent protein (EGFP), we demonstrate that the fascin promoter mediates a strong antigen expression that is restricted to mature DC. DNA vaccination with antigen-encoding expressi…

Differential gene expression analysis identifies murine Cacnb3 as strongly upregulated in distinct dendritic cell populations upon stimulation

Langerhans cells (LCs) represent the dendritic cell (DC) population in the epidermis. Among the set of genes induced in primary mouse LCs in response to stimulation, both isoforms of the voltage-dependent Ca²(+) channel (VDCC) regulatory subunit Cacnb3 as well as the DC maturation marker Fscn1 were upregulated most strongly. Comparable results were obtained for a recently described myeloid DC line (SP37A3). Other antigen presenting cell populations, namely, bone marrow-derived DCs, macrophages and primary B cells, showed no stimulation-associated upregulation of Cacnb3 expression. Pharmacological inhibition of Ca²(+) channel activity during the stimulation of SP37A3 cells enhanced their T c…

Compartmentalized Production of CCL17 In Vivo

Dendritic cells (DCs)**Abbreviations used in this paper: BM, bone marrow; CHS, contact hypersensitivity; cLN, cutaneous lymph node; CRP, C-reactive protein; DC, dendritic cell; DNFB, dinitrofluorobenzene; EGFP, enhanced green fluorescent protein; LC, Langerhans cell; LP, lamina propria; MACS, magnetic-activated cell sorting; mLN, mesenteric lymph node; ODN, oligodeoxynucleotide; PFA, paraformaldehyde; PP, Peyer's patch; TLR, Toll-like receptor; TRITC, tetramethylrhodamine-5-(and-6-)-isothiocyanate. fulfill an important regulatory function at the interface of the innate and adaptive immune system. The thymus and activation-regulated chemokine (TARC/CCL17) is produced by DCs and facilitates t…

Maturation of Epidermal Langerhans Cells In Vitro Is Accompanied by Downregulation of 4F2 (CD98) as Determined by Differential Display

Following short-term culture, Langerhans cells mature morphologically and functionally into potent immunostimulatory cells. As regulation of gene expression accompanies this maturation process, it is likely that differentially expressed genes are involved in the maturation events. Using the recently described method of differential display, we generated cDNA expression patterns starting with mRNA of murine epidermal Langerhans cells isolated either directly (fLC) or following 3 d cultivation (cLC). Five hundred putative differentially expressed cDNA fragments were recovered from the gel. For a part of the fragments differential expression was confirmed by dot blot and Southern hybridization…

Prophylactic and therapeutic intervention in IgE responses by biolistic DNA vaccination primarily targeting dendritic cells.

Background Allergen gene transfer represents an alternative approach to specific immunotherapy with allergen extracts. Gene gun–mediated DNA immunization with plasmid vectors expressing a transgene under control of the promoter of the fascin gene (pFascin) allows for antigen production predominantly by dendritic cells and resulted in the generation of CD8 + cytotoxic T lymphocytes as well as in the development of a type 1 immune response. Objective We compared the in vivo efficiency of biolistic transfection with pFascin and plasmids containing the cytomegalovirus promoter (pCMV) in a mouse model of type I allergy. Methods BALB/c mice were sensitized with the model allergen β-galactosidase …

15. Mainzer Allergie-Workshop 2003

Nonradioactive Detection of Differentially Expressed Genes Using Complex RNA or DNA Hybridization Probes

The analysis of differential gene expression has become increasingly important in recent years. Typically, differentially expressed genes are identified in a primary screening procedure, yielding candidate genes whose differential expression has to be verified. We provide a highly sensitive, efficient and nonradioactive differential screening procedure to analyze numerous candidate genes in a single step. This comprises labeling of poly(A)+ RNA of the cell types analyzed with DIG Chem-Link and differential hybridization to the candidate genes fixed on dot blots. DIG Chem-Link allows, to our knowledge, for the first time efficient and direct nonradioactive labeling of RNA in vitro. Advantag…

Isolation of Differentially Expressed Genes in Epidermal Langerhans Cells

Epidermal Langerhans cells (LC) represent immature dendritic cells (DC) resident in the skin, which are not yet able to prime naive T cells1. In vitro cultivation of LC in the presence of keratinocytes, supplying survival and differentiation signals, induces maturation events in LC2. These are highlighted by the downregulation of the biosynthesis of MHC class II molecules3, by the upregulation of the surface expression of adhesion and costimulatory molecules like CD80, CD86, CD54 and CD584,5, and by the acquisition of a potent immunostimulatory capacity for T cells6. Mature LC are potent inducers of naive T cells. Thus LC represent an ideal model system to investigate the maturation of DC (…

Expression of the actin-bundling protein fascin in cultured human dendritic cells correlates with dendritic morphology and cell differentiation.

Dendritic cells are key players of the immune system as they efficiently induce primary immune responses by activating naive T cells. We generated human dendritic cells from CD14+ blood precursors and investigated expression of the actin-bundling protein fascin during maturation by western blotting, immunofluorescence, and cytofluorometry. Cells obtained by culture of CD14+ blood precursors in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, which were only weakly positive for the maturation marker CD83, expressed low amounts of fascin. Addition of a cytokine cocktail including tumor necrosis factor alpha, interleukin-1beta, interleukin-6, and prostaglandi…

The role of NO in contact hypersensitivity

Contact dermatitis or contact hypersensitivity (CHS) is a common T lymphocyte-mediated allergic disease characterized by local inflammatory skin reactions following contact with small reactive compounds called haptens. In common with other allergic processes, the development of contact dermatitis proceeds in two phases: a sensitization phase which occurs on first exposure to allergen, and an elicitation phase which occurs on subsequent exposure when the clinical manifestations of the disease are observed. This process is hapten-specific. While the pathophysiology of the sensitization phase is well characterized, our understanding of the elicitation phase is still incomplete, including the r…

Mouse langerhans cells differentially express an activated T cell-attracting CC chemokine.

Epidermal Langerhans cells represent an immature population of dendritic cells, not yet able to prime naive T cells. Following in vitro culture Langerhans cells mature into potent immunostimulatory cells. We constructed a representative cDNA library of in vitro matured murine Langerhans cells. Applying a differential screening procedure 112 differentially expressed cDNA clones were isolated. Thirty-six clones represented cDNA fragments of the same gene, identifying it to be the most actively expressed gene induced in maturing Langerhans cells. A full-length cDNA was sequenced completely. The open reading frame codes for a protein of 92 amino acids containing a leader peptide of 24 amino aci…

Transcriptional targeting of dendritic cells in gene gun-mediated DNA immunization favors the induction of type 1 immune responses

Cutaneous dendritic cells (DC) are pivotal for the elicitation of antigen-specific immune responses following gene gun-mediated biolistic transfection of the skin. We transcriptionally targeted transgene expression to DC using vectors containing the murine fascin promoter (pFascin) to control antigen production and compared the immune response elicited with conventional DNA immunization using plasmid constructs with the ubiquitously active CMV promoter (pCMV). Biolistic transfection with pFascin initiated a marked type 1 immune response characterized by the occurrence of a large population of IFN-gamma-producing T helper (Th) cells in spleen and draining lymph nodes. Consistently, immunoglo…

The human fascin gene promoter is highly active in mature dendritic cells due to a stage-specific enhancer.

Abstract Dendritic cells (DC), regarded as the most efficient APCs of the immune system, are capable of activating naive T cells. Thus, DC are primary targets in immunotherapy. However, little is known about gene regulation in DC, and for efficient transcriptional targeting of human DC, a suitable promoter is still missing. Recently, we successfully used the promoter of the murine actin-bundling protein fascin to transcriptionally target DC by DNA vaccination in mice. In this study, we report on isolation of the human fascin promoter and characterization of its regulatory elements. The actively expressed gene was distinguished from a conserved inactive genomic locus and a continuous region …

Maturation of epidermal Langerhans cells: increased expression of beta- and gamma-actin isoforms as a basis of specialized cell functions.

Epidermal Langerhans cells (LC) represent immature dendritic cells. During in vitro culture in the presence of keratinocytes they mature into potent immunostimulatory cells for naive T cells. This process is thought to simulate in vivo maturation of LC following activation by antigen contact. Maturation of LC is accompanied by morphological alterations. Applying a differential screening procedure we isolated differentially expressed cDNAs involved in the maturation events including cDNAs of the cytoskeletal actin isoforms beta- and gamma-actin. Stronger signals with hybridization probes derived from cultured LC compared with probes derived from freshly isolated LC indicate upregulation of a…

Kontaktallergie (V 58–V 65)

NO synthase II in mouse skeletal muscle is associated with caveolin 3

The inducible-type NO synthase (NOS II; iNOS) is constitutively expressed in slow-twitch skeletal muscle fibres of guinea-pigs [Gath, Closs, Gödtel-Armbrust, Schmitt, Nakane, Wessler and Förstermann (1996) FASEB J. 10, 1614-1620]. Here we studied the expression of NOS II in skeletal muscle of wild-type and NOS II-deficient mice and investigated the molecular basis for the membrane association of this NOS in muscle. A basal expression of NOS II mRNA and protein was detected in skeletal muscle from untreated wild-type mice; expression increased when mice were treated with bacterial lipopolysaccharide (LPS). No NOS II was found in any tissue of untreated or LPS-treated NOS II-deficient mice. I…

A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid

AbstractThe phenotype and function of murine dendritic cells (DCs) are primarily studied using bone-marrow–derived DCs (BM-DCs), but may be hampered by the heterogenous phenotype of BM-DCs due to their differential state of maturation. Here we characterize a newly established murine DC line (SP37A3) of myeloid origin. During maintainance in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and M-CSF, SP37A3 cells resemble immature DCs characterized by low expression of major histocompatibility complex (MHC) II and costimulatory molecules and low T-cell stimulatory capacity. Upon stimulation, SP37A3 cells acquire a mature phenotype and activate naive T cells as potent…

Differential Regulation of CCL22 Gene Expression in Murine Dendritic Cells and B Cells

Abstract The activated T cell-attracting CC chemokine CCL22 is expressed by stimulated B cells and mature dendritic cells (DC). We have cloned and sequenced the complete mouse gene, including 4 kb of the 5′-flanking promoter region, and detected two distinct sites for initiation of transcription by 5′-RACE. Reporter gene assays indicate that the promoter reflects the specificity of the endogenous gene. Within the proximal promoter region, we identified potential binding sites for NF-κB, Ikaros, and a putative GC box. All three regions bind proteins. The NF-κB site was shown to specifically bind NF-κB subunits p50 and p65 from nuclear extracts of LPS-stimulated B cells, B cell line A20/2J, T…

Involvement of NO in contact hypersensitivity.

The NO synthases (NOS) generate NO from L-arginine. High concentrations of NO have been shown to be responsible for tissue injury and cell death, while low concentrations of NO induce vasodilatation and other signaling effects. We have investigated the involvement of NO in contact hypersensitivity (CHS) reactions. CHS induced by treatment of BALB/c mice with the contact allergen 2,4-dinitrofluorobenzene (DNFB) was significantly reduced by the NOS inhibitor N-methyl-L-arginine (L-NMA), but not by the stereoisomer D-NMA, as shown by reduced ear swelling responses and evaluation of ear tissue sections. The CHS response was also reduced by aminoguanidine, which is known to preferentially inhibi…

Diminished Contact Hypersensitivity Response in IL‐4 Deficient Mice at a Late Phase of the Elicitation Reaction

Contact hypersensitivity (CHS) is thought to depend on the activation of T cells of Th1 and/or Tc1 type. The role of Th2/Tc2 cells in the contact allergic reaction is not clear. The aim of this study was to analyse the functional contribution of Th2/Tc2 cells in CHS using the interleukin-4 (IL-4) deficient mouse model. Interleukin-4 deficient (IL4T) and control (wt) mice were sensitized by epicutaneous application of 2,4-dinitrofluorobenzene. The ear swelling response measured 24 h after challenge was similar in IL4T and control mice. However, from 48 h onwards, ear swelling values were significantly reduced in IL4T mice. The stimulatory capacity of freshly isolated as well as 3-day culture…

PCR-Amplified cDNA Probes for Verification of Differentially Expressed Genes

Differential display has proven to be a powerful technique for the detection and isolation of differentially expressed genes. By generating reproducible cDNA expression patterns, it is possible to compare gene expression by two or more cell types, developmental stages or tissues and to isolate as yet unknown differentially expressed genes. A sensitive method is necessary to verify the differential expression of the isolated cDNAs. Here we describe the use of adaptor-ligated, PCR-amplified total cDNA of the two cell types compared as a probe for Southern hybridizations with the isolated cDNAs.