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CpG Motifs (cpg + motif)
Selected AbstractsCellular activation by plasmid DNA in various macrophages in primary cultureJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2008Hiroyuki Yoshida Abstract Macrophages are an important group of cells responsible for the inflammatory response to unmethylated CpG dinucleotide (CpG motif) in plasmid DNA (pDNA) via Toll-like receptor 9 (TLR9). This finding is primarily based on in vitro studies. Previous in vivo studies also have suggested that tissue macrophages are involved in inflammatory cytokine release in the circulation following intravenous administration of pDNA to mice. However, the relationship between the in vitro and in vivo studies has not been sufficiently clarified. To gain insight into which types of cells are responsible for the production of cytokines upon interaction with pDNA, peritoneal macrophages, splenic macrophages, hepatic nonparenchymal cells (NPCs) including Kupffer cells and mesangial cells were isolated from mice. All types of primary cultured cells, except for mesangial cells, express TLR9 at varying levels. Splenic macrophages and hepatic NPCs were activated to produce tumor necrosis factor-, (TNF-,) by naked pDNA, whereas peritoneal macrophages and mesangial cells were not. pDNA complexed with N -[1-(2,3-dioleyloxy)propyl]- N,N,N -trimethyl-ammonium chloride/cholesterol liposome induced TNF-, in the splenic macrophages but not in the other cell types. These results indicate that splenic macrophages and hepatic NPCs are closely involved in TNF-, production in response to pDNA. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:4575,4585, 2008 [source] A transcriptomic and proteomic analysis of the effect of CpG-ODN on human THP-1 monocytic leukemia cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2005Cheng-Chin Kuo Abstract The CpG motif of bacterial DNA (CpG-DNA) is a potent immunostimulating agent whose mechanism of action is not yet clear. Here, we used both DNA microarray and proteomic approaches to investigate the effects of oligodeoxynucleotides containing the CpG motif (CpG-ODN) on gene transcription and protein expression profiles of CpG-ODN responsive THP-1 cells. Microarray analysis revealed that 2,h stimulation with CpG-ODN up-regulated 50,genes and down-regulated five genes. These genes were identified as being associated with inflammation, antimicrobial defense, transcriptional regulation, signal transduction, tumor progression, cell differentiation, proteolysis and metabolism. Longer stimulation (8,h) with CpG-ODN enhanced transcriptional expression of 58,genes. Among these 58,genes, none except one, namely WNTI inducible signaling pathway protein,2, was the same as those induced after 2,h stimulation. Proteomic analysis by two-dimensional gel electrophoresis, followed by mass spectrometry identified several proteins up-regulated by CpG-ODN. These proteins included heat shock proteins, modulators of inflammation, metabolic proteins and energy pathway proteins. Comparison of microarray and proteomic expression profiles showed poor correlation. Use of more reliable and sensitive analyses, such as reverse transcriptase polymerase chain reaction, Western blotting and functional assays, on several genes and proteins, nonetheless, confirmed that there is indeed good correlation between mRNA and protein expression after CpG-ODN treatment. This study also revealed that several anti-apoptotic and neuroprotective related proteins, not previously reported, are activated by CpG-DNA. These findings have extended our knowledge on the activation of cells by CpG-DNA and may contribute to further understanding of mechanisms that link innate immunity with acquired immune response(s). [source] Direct role of NF-,B activation in Toll-like receptor-triggered HLA-DRA expressionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2006Keun-Wook Lee Abstract Microbial components, such as DNA containing immunostimulatory CpG motifs (CpG-DNA) and lipopolysaccharides (LPS), elicit the cell surface expression of MHC class II (MHC-II) through Toll-like receptor (TLR)/IL-1R. Here, we show that CpG-DNA and LPS induce expression of the HLA-DRA in the human B cell line, RPMI 8226. Ectopic expression of the dominant negative mutant of CIITA and RNA interference targeting the CIITA gene indicate that CIITA activation is not enough for the maximal MHC-II expression induced by CpG-DNA and LPS. Additionally, nuclear factor (NF)-,B activation is required for the CpG-DNA-activated and LPS-activated HLA-DRA expression, whereas IFN-,-induced MHC-II expression depends on CIITA rather than on NF-,B. Comprehensive mutant analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays, reveal that the functional interaction of NF-,B with the promoter element is necessary for the TLR-mediated HLA-DRA induction by CpG-DNA and LPS. This novel mechanism provides the regulation of MHC-II gene expression with complexity and functional diversity. [source] Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cellsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2003Karen Manon Lammers Abstract A new therapeutic approach for inflammatory bowel diseases is based on the administration of probiotic bacteria. Prokaryotic DNA contains unmethylated CpG motifs which can activate immune responses, but it is unknown whether bacterial DNA is involved in the beneficial effects obtained by probiotic treatment. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with pure DNA of eight probiotic strains and with total bacterial DNA from human feces collected before and after probiotic ingestion. Cytokine production was analyzed in culture supernatants. Modification of human microflora after probiotic administration was proven by polymerase chain reaction analysis. Here we show that Bifidobacterium genomic DNA induced secretion of the antiinflammatory interleukin-10 by PBMC. Total bacterial DNA from feces collected after probiotic administration modulated the immune response by a decrease of interleukin-1, and an increase of interleukin-10. [source] Priming of immune responses to hepatitis B surface antigen in young mice immunized in the presence of maternally derived antibodiesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2001Risini D Weeratna Abstract Early vaccination is necessary to protect infants from various infectious diseases. However, this is often unsuccessful largely due to the immaturity of the neonatal immune system. Furthermore, maternally derived antibodies can interfere with active immunization. We have previously shown in young mice that immune responses against several different antigens can be improved by the addition of oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN). In this study we have evaluated immunization of newborn (1,7-day-old) BALB/c mice against hepatitis B surface antigen (HBsAg), with alum and/or CpG ODN, in the presence of high levels of maternal antibody against HBsAg (anti-HBs). Seroconversion rates and anti-HBs titers were compared to those induced by a HBsAg-expressing plasmid, since other studies had suggested DNA vaccines to be superior to protein vaccines in young mice with maternal antibody. HBsAg/alum/CpG ODN was superior to DNA vaccine in inducing HBsAg-specific CTL responses in young mice in the presence of maternally transferred anti-HBs antibodies. However, B cell responses to both HBsAg/alum/CpG ODN and DNA vaccines remained weak in the presence of maternally transferred anti-HBs antibodies. [source] Interferon-, priming is involved in the activation of arginase by oligodeoxinucleotides containing CpG motifs in murine macrophagesIMMUNOLOGY, Issue 1pt2 2009Miriam V. Liscovsky Summary Recognition of microbial products by macrophages (M,) stimulates an inflammatory response and plays a critical role in directing the host immune response against infection. In the present work, we showed for the first time that synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG) are able to stimulate, in the presence of interferon-, (IFN-,), both arginase and inducible nitric oxide synthase (iNOS) in murine M,. Unexpectedly, IFN-,, a cytokine believed to be an inhibitor of arginase activity, intervened in the activation of this enzyme. A significant increase in arginase activity was observed upon a short pre-incubation (1 hr) with IFN-, and subsequent CpG stimulation. Therefore, a very interesting observation of this study was that the CpG-mediated arginase activity is dependent on IFN-, priming. The increase in arginase activity as a result of stimulation with CpG plus IFN-,was correlated with augmented expression of the arginase II isoform. The use of pharmacological specific inhibitors revealed that arginase activity was dependent on p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated protein kinase (ERK), but independent of c-Jun N-terminal kinase (JNK) activation. This report reveals a singular effect of the combination of CpG and IFN-,, one of the mayor cytokines produced in response to CpG administration in vivo. [source] Adjuvant effects of CpG oligodeoxynucleotides on responses against T-independent type 2 antigensIMMUNOLOGY, Issue 1 2001J. Kovarik Summary Oligodeoxynucleotides containing CpG motifs (CpG-ODN) are potent in vitro B-cell activators and they have been successfully used to increase in vivo antibody responses to T-dependent peptide and protein antigens. In contrast, the use of CpG-ODN to enhance in vivo antibody responses to various T-independent type 2 (TI-2) antigens has recently generated contradictory results. In this study, we compared the CpG-ODN stimulatory effect on antibody responses of adult and young BALB/c mice to trinitrophenylaminoethyl-carboxymethyl (TNP) -Ficoll and to polysaccharides (PS) from several distinct serotypes of Streptococcus pneumoniae (SPn). CpG-ODN co-administration significantly enhanced antigen-specific immunoglobulin M (IgM), IgG, IgG1 and IgG2a titres to TNP-Ficoll. The depletion of CD4+ cells by monclonal antibodies (GK1.5) identified their essential role in CpG-ODN-mediated enhancement of antibody responses. In contrast to TNP-Ficoll, CpG-ODN failed to enhance IgM and IgG responses to any of the 18 SPnPS serotypes tested. Providing T-cell epitopes by the conjugation of SPnPS to the carrier protein tetanus toxoid again allowed CpG-ODN to mediate enhancement of IgG, IgG2a and IgG3 responses to most SPnPS serotypes. Thus, antigen-presenting cell/T-cell interaction appears to largely mediate the in vivo influence of CpG-ODN on antibody responses to TI-2 antigens. In early life, additional factors limit CpG-ODN modulation of antibody responses to TI-2 antigens. [source] CpG oligodeoxynucleotides accelerate reovirus type 2-triggered insulitis in DBA/1 suckling miceINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 5 2002T. Hayashi Summary. We reported previously that reovirus type-2 (Reo-2) triggers T-helper (Th) 1-mediated autoimmune insulitis resulting in temporal impaired glucose tolerance (IGT) approximately 10 days post infection (d.p.i) in suckling DBA/1 mice. We hypothesized that CpG motifs in bacteria may enhance virus-induced insulitis through its content of unmethylated CpG motifs. In the infected mice, the intraperitoneal treatment of synthetic 20-base oligodeoxynucleotides with CpG motifs (CpG ODN) caused increase in cumulative incidence of insulitis with IGT, increased serum interferon (IFN)-, concentration, and high frequency of autoantibody against pancreatic islet cells, compared to the infected mice without CpG ODN at 17 d.p.i. Also CD4+ and CD8+ lymphocytes infiltrated in and/or around pancreatic islets in the CpG ODN-treated mice. This evidence suggests that CpG ODN may contribute to accelerate Reo-2-induced autoimmune reaction against pancreatic islet cells via additional effects of Th1 cytokines especially IFN-,. [source] Effects of inflammatory response on in vivo transgene expression by plasmid DNA in miceJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2008Keiko Kako Abstract To examine the effects of inflammatory response to plasmid DNA (pDNA) on transgene expression, serum tumor necrosis factor-, (TNF-,) was measured after intravenous injection of pDNA or calf thymus DNA (CT DNA) in the naked or complexed form with cationic liposomes (lipoplex). pDNA with many CpG motifs induced TNF-, production regardless of the forms. No significant TNF-, production was detected when CT DNA or methylated pDNA was injected. Clodronate liposomes and dexamethasone were used to deplete phagocytes or to inhibit inflammatory responses, respectively. Transient depletion of phagocytes, such as liver Kupffer cells and splenic macrophages, by clodronate liposomes slightly altered the tissue distribution of 32P-pDNA lipoplex, but significantly reduced the TNF-, production and transgene expression. Dexamethasone significantly inhibited the initial transgene expression, but increased the duration of the expression slightly. Use of NF-,B activity-dependent plasmid vector suggested that the inhibition of NF-,B activation is involved in the reduced expression by these treatments. These findings indicate that tissue macrophages are closely involved in the CpG motif-dependent TNF-, production. It is also suggested that TNF-, activates NF-,B and increases transgene expression by pDNA having many NF-,B binding sites, but TNF-, also reduces transgene expression at later time periods, leading to short-term transgene expression. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97: 3074,3083, 2008 [source] Bifidobacterium and Lactobacillus DNA in the human placentaLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2009R. Satokari Abstract Aims:, Bifidobacteria and lactobacilli are part of the human normal intestinal microbiota and may possibly be transferred to the placenta. It was hypothesized that intestinal bacteria or their components are present in the placenta and that the foetus may be exposed to them. We investigated the presence of bifidobacteria and lactobacilli and their DNA in the human placenta. Methods and Results:, We studied 34 human placentae (25 vaginal and nine caesarean deliveries) for the presence Bifidobacterium spp. and Lactobacillus rhamnosus. Cultivation was used for the detection of viable cells and genus and species-specific PCR for the detection of DNA. No bifidobacteria or lactobacilli were found by cultivation. Bifidobacterial DNA was detected in 33 and L. rhamnosus DNA in 31 placenta samples. Conclusions:, DNA from intestinal bacteria was found in most placenta samples. The results suggest that horizontal transfer of bacterial DNA from mother to foetus may occur via placenta. Significance and Impact of the Study:, Bacterial DNA contains unmethylated CpG oligodeoxynucleotide motifs which induce immune effects. Specific CpG motifs activate Toll-like receptor 9 and subsequently trigger Th-1-type immune responses. Although the newborn infant is considered immunologically immature, exposure by bacterial DNA may programme the infant's immune development during foetal life earlier than previously considered. [source] Nasal CpG oligodeoxynucleotide administration induces a local inflammatory response in nonallergic individualsALLERGY, Issue 9 2009A. Mĺnsson Background:, We have previously demonstrated the presence of toll-like receptor 9 in the nasal mucosa of both healthy and allergic individuals. CpG motifs, found in bacterial and viral DNA, elicit strong immunostimulatory effects via this receptor. CpG is known to skew the immune system towards a T helper 1 (Th1) profile, thereby suppressing Th2-driven allergic responses. This study was designed to examine the effects of CpG administration in the human nose. Methods:, Twenty subjects, of whom 10 suffered from seasonal allergic rhinitis (AR), were challenged intranasally with CpG outside pollen season. Symptom scores, nasal airway resistance (NAR), and nasal and pulmonary nitric oxide (NO) levels were assayed prior to challenge and 30 min, 6, 24 and 48 h post challenge. The presence of leukocytes and various cytokines were analyzed in nasal lavage (NAL) fluids before and after CpG exposure. Results:, Increased NAR, nasal NO production and secretion of interleukin (IL)-1,, IL-6, and IL-8 were seen after CpG exposure. Further analysis revealed that this inflammatory response was more marked in healthy subjects than among patients with AR, although a higher basal inflammatory response was recorded in the allergic group. In vitro experiments suggest that the effects induced by CpG are mediated by epithelial cells and neutrophils. Conclusion:, Nasal administration of CpG induces a local airway inflammation, more distinct among healthy than allergic individuals. The reduced responsiveness to CpG in allergic patients might be related to the ongoing minimal persistent inflammation. Results from cytokine analyses reflect the ability of CpG to induce a pro-inflammatory Th1-like immune response. [source] CpG-containing ODN has a limited role in the protection against Toxoplasma gondiiPARASITE IMMUNOLOGY, Issue 2 2004R. Saavedra SUMMARY Bacterial DNA containing immunostimulatory motifs (CpG) induces the development of a TH1 immune response. Since protection against Toxoplasma gondii is correlated with this type of response, the aim of this work was to determine if a synthetic oligodeoxynucleotide (ODN) containing CpG sequences could be useful as adjuvant for the induction of a long-lasting protective immune response against T. gondii. BALB/c mice immunized with a total soluble antigen of T. gondii (TSA2) mixed with ODN-containing CpG sequences developed a typical TH1 response, as determined by antibody isotypes and interferon-, (IFN-,) and interleukin-4 (IL-4) production by spleen cells. However, they did not resist a challenge with the virulent RH strain of the parasite. Absence of protection paralleled with lower levels of IFN-,, when compared with mice vaccinated with the live tachyzoites of the attenuated ts.4 strain of the parasite, which resisted this challenge. Intraperitoneal injection of ODN alone to mice induced a high degree of resistance to a lethal challenge inoculated by the same route. Nevertheless, this nonspecific protection was transient. Thus, the use of ODN containing CpG motifs as adjuvant is of limited value for the induction of a protective immune response against T. gondii. [source] DNA vaccination against tumorsTHE JOURNAL OF GENE MEDICINE, Issue 1 2005Gérald J. Prud'homme Abstract DNA vaccines have been used to generate protective immunity against tumors in a variety of experimental models. The favorite target antigens have been those that are frequently expressed by human tumors, such as carcinoembryonic antigen (CEA), ErbB2/neu, and melanoma-associated antigens. DNA vaccines have the advantage of being simple to construct, produce and deliver. They can activate all arms of the immune system, and allow substantial flexibility in modifying the type of immune response generated through codelivery of cytokine genes. DNA vaccines can be applied by intramuscular, dermal/epidermal, oral, respiratory and other routes, and pose relatively few safety concerns. Compared to other nucleic acid vectors, they are usually devoid of viral or bacterial antigens and can be designed to deliver only the target tumor antigen(s). This is likely to be important when priming a response against weak tumor antigens. DNA vaccines have been more effective in rodents than in larger mammals or humans. However, a large number of methods that might be applied clinically have been shown to ameliorate these vaccines. This includes in vivo electroporation, and/or inclusion of various immunostimulatory molecules, xenoantigens (or their epitopes), antigen-cytokine fusion genes, agents that improve antigen uptake or presentation, and molecules that activate innate immunity mechanisms. In addition, CpG motifs carried by plasmids can overcome the negative effects of regulatory T cells. There have been few studies in humans, but recent clinical trials suggest that plasmid/virus, or plasmid/antigen-adjuvant, prime-boost strategies generate strong immune responses, and confirm the usefulness of plasmid-based vaccination. Copyright © 2004 John Wiley & Sons, Ltd. [source] Epigenetic regulation of the expression of the novel stem cell marker CDCP1 in cancer cellsTHE JOURNAL OF PATHOLOGY, Issue 1 2006J-i Ikeda Abstract CDCP1 is a novel stem cell marker that is expressed in several types of cancer. The mechanisms by which CDCP1 expression is regulated, and the clinical implications of this marker, have not been clarified. In this report, we examine the epigenetic regulation of CDCP1 expression in cell lines and clinical samples from patients with breast cancer. Many CpG sequences were localized around the transcription initiation site of CDCP1. These CpG motifs were found to be poorly methylated in cell lines with high levels of CDCP1 expression and heavily methylated in cell lines with low levels of CDCP1 expression. The in vitro methylation of CpG sites decreased CDCP1 promoter activity, and the addition of a demethylating reagent restored activity. In 25 breast cancer samples, an inverse correlation was noted between the CDCP1 expression level and the proportion of methylated to non-methylated CpG sites. Tumours with high-level CDCP1 expression showed higher levels of proliferation, as revealed by immunohistochemical detection of the MIB-1 antigen, than tumours with low-level CDCP1 expression. These findings indicate that the expression of CDCP1 is regulated by methylation of its promoter region in tumours. CDCP1 expression may prove to be useful in the further characterization of cancers. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Anti-HBV effects of CpG oligodeoxynucleotide-activated peripheral blood mononuclear cells from patients with chronic hepatitis B,APMIS, Issue 10 2005NING LI Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) are known as a potent Th1-like immune enhancer in vertebrates. Chronic hepatitis B is the immunocompromising condition. We therefore investigated the effects of CpG ODN on cultured cells from chronic hepatitis B patients and healthy controls. The inhibitory effects of CpG ODN on hepatitis B virus (HBV) were also studied. The secretion of IFN-, by CpG ODN-activated peripheral blood mononuclear cells (PBMCs) from chronic hepatitis B patients and healthy controls was significantly increased when compared with PBMCs alone or GpC ODN-stimulated PBMCs. After activation with CpG ODN, the IFN-, secretion by chronically HBV-infected patient PBMCs is less than that by healthy control PBMCs. Treatment of HepG2 2.2.15 cells with culture supernatants of PBMCs activated by CpG ODN can significantly suppress the secretion of HBsAg, HBeAg and HBV DNA as compared with that of PBMCs without CpG ODN activation under the same conditions. No inhibitory effect on the replication of HBV was found for CpG ODN treatment alone. Our results indicated that CpG ODN could efficiently enhance the immune response of chronic hepatitis B patients. Moreover, the CpG ODN-activated PBMCs from chronic hepatitis B patients were able to significantly inhibit HBV replication in vitro, suggesting that CpG ODN may be a potential immunoregulator against HBV infection in the future. [source] Enhancement of immunity and resistance in mice by pig IL-6 gene and CpG motifs encapsulated in chitosan nanoparticleBIOTECHNOLOGY JOURNAL, Issue 2 2008Qian Chen Abstract This study was conducted to explore the synergetic effect of a novel plasmid containing a porcine IL-6 gene and CpG motifs on immunity of mice in order to develop an effective adjuvant to boost resistance against infection. The synthetic oligodeoxynucleotide containing 11 CpG motifs was inserted into the reconstructed VR1020 plasmid containing the pig IL-6 gene (VRPIL6), designated VRIL6C, and then encapsulated in chitosan nanoparticles (CNP) prepared by ionic cross linkage, designated VRIL6C-CNP. The 3-week old mice were injected, respectively, with VRIL6C-CNP, VRIL6-CNP, CpG-CNP and VR1020-CNP to detect the changes of immunity. At 28 days post inoculation, the mice were challenged with virulent hemolytic serotype 2 Streptococcus to test their resistance against infection. The results showed that there was a significant increase in immunoglobulins and interleukins in mice receiving VRIL6C-CNP compared with the control groups, as well as an increase in the lymphocytes and monocytes in the inoculated mice, so that the immunity was remarkably improved in the VRIL6C-CNP group. The challenge provoked stronger immunity and protection against infection in the VRIL6C-CNP group than in the control mice that manifested severe symptoms and lesions. This suggests that VRIL6C-CNP could remarkably enhance the nonspecific immunity of mice, and facilitate the development of an effective immunopotentiator to promote the resistance of the animals against infection. [source] | ||||||||||||||||||||||||||||