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At least 91 records · Page 5Linked to original sources
PubMed2003

Myeloid differentiation factor 88-dependent post-transcriptional regulation of cyclooxygenase-2 expression by CpG DNA: tumor necrosis factor-alpha receptor-associated factor 6, a diverging point in the Toll-like receptor 9-signaling.

The immune stimulatory unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce expression of cyclooxygenase-2 (cox-2). The present study demonstrates that CpG DNA can up-regulate cox-2 expression by post-transcriptional mechanisms in RAW264.7 cells. To determine the CpG DNA-mediated signaling pathway that post-transcriptionally regulates cox-2 expression, a cox-2 translational reporter (COX2-3'-UTR-luciferase) was generated by inserting sequences within the 3'-untranslated region (UTR) of cox-2 to the 3' end of the luciferase gene under control of the SV40 promoter. CpG DNA-induced COX2-3'-UTR-luciferase activity was completely inhibited by an endosomal acidification inhibitor chloroquine, a Toll-like receptor 9 antagonist inhibitory CpG DNA, or overexpression of a dominant negative (DN) form of MyD88. However, overexpression of DN-IRAK-1 or DN-TRAF6 resulted in substantial, but not complete, inhibition of the CpG DNA-induced COX2-3'-UTR-luciferase activity. Activation of all three MAPKs (ERK, p38, and JNK) was required for optimal COX2-3'-UTR-luciferase activity induced by CpG DNA. Overexpression of DN-TRAF6 suppressed CpG DNA-mediated activation of p38 and JNK, but not ERK, explaining the partial inhibitory effects of DN-TRAF6 on CpG DNA-induced COX2-3'-UTR-luciferase activity. Co-expression of DN-TRAF6 and N17Ras completely inhibited CpG DNA-induced COX2-3'-UTR-luciferase activity, indicating the involvement of Ras in CpG DNA-mediated ERK and COX2-3'-UTR regulation. Collectively, our results suggest that MyD88 and MAPKs play a key regulatory role in CpG DNA-mediated cox-2 expression at the post-transcriptional level and that TRAF6 is a diverging point in the Toll-like receptor 9-signaling pathway for CpG DNA-mediated MAPK activation.

3' Untranslated Regions↗
PubMed2002

Immunostimulatory properties of phosphorothioate CpG DNA containing both 3'-5'- and 2'-5'-internucleotide linkages.

Synthetic oligodeoxyribonucleotides containing CpG-dinucleotides (CpG DNA) in specific sequence contexts activate the vertebrate immune system. We have examined the effect of 3'-deoxy-2'-5'-ribonucleoside (3'-deoxynucleoside) incorporation into CpG DNA on the immunostimulatory activity. Incorporation of 3'-deoxynucleosides results in the formation of 2'-5'-internucleotide linkages in an otherwise 3'-5'-linked CpG DNA. In studies, both in vitro and in vivo, CpG DNA containing unnatural 3'-deoxynucleoside either within the CpG-dinucleotide or adjacent to the CpG-dinucleotide failed to induce immunostimulatory activity, suggesting that the modification was not recognized by the receptors. Incorporation of the same modification distal to the CpG-dinucleotide in the 5'-flanking sequence potentiated the immunostimulatory activity of the CpG DNA. The same modification when incorporated in the 3'-flanking sequence had an insignificant effect on immunostimulatory activity of CpG DNA. Interestingly, substitution of a 3'-deoxynucleoside in the 5'-flanking sequence distal to the CpG-dinucleotide resulted in increased IL-6 and IL-10 secretion with similar levels of IL-12 compared with parent CpG DNA. The incorporation of the same modification in the 3'-flanking sequence resulted in lower IL-6 and IL-10 secretion with similar levels of IL-12 compared with parent CpG DNA. These results suggest that site-specific incorporation of 3'-deoxynucleotides in CpG DNA modulates immunostimulatory properties.

Animals↗
PubMed2006

CpG DNA modulates interleukin 1beta-induced interleukin-8 expression in human bronchial epithelial (16HBE14o-) cells.

BACKGROUND: Recognition of repeat unmethylated CpG motifs from bacterial DNA through Toll-like receptor (TLR-9) has been shown to induce interleukin (IL)-8 expression in immune cells. We sought to investigate the role of CpG oligodeoxynucleotides (ODN) on a human bronchial epithelial cells. METHODS: RT-PCR and Western blot analysis were used to determine expression of TLR-9 in human bronchial epithelial cells (16HBE14o-). Cells were treated with CpG ODN in the presence or absence of IL-1beta and IL-8 protein was determined using ELISA. In some cases cells were pretreated with chloroquine, an inhibitor of TLR-9 signaling, or SB202190, an inhibitor of the mitogen activated protein kinase p38, prior to treatment with IL-1beta and CpG. TLR9 siRNA was used to silence TLR9 prior to treatment with IL-1beta and CpG. IkappaBalpha and p38 were assessed by Western blot, and EMSA's were performed to determine NF-kappaB activation. To investigate IL-8 mRNA stability, cells were treated with IL-1beta in the absence or presence of CpG for 2 h and actinomycin D was added to induce transcriptional arrest. Cells were harvested at 15 min intervals and Northern blot analysis was performed. RESULTS: TLR-9 is expressed in 16HBE14o- cells. CpG synergistically increased IL-1beta-induced IL-8 protein abundance, however treatment with CpG alone had no effect. CpC (a control ODN) had no effect on IL-1beta-induced IL-8 levels. In addition, CpG synergistically upregulated TNFalpha-induced IL-8 expression. Silencing TLR9 using siRNA or pretreatment of cells with chloroquine had little effect on IL-1beta-induced IL-8 levels, but abolished CpG-induced synergy. CpG ODN had no effect on NF-kappaB translocation or DNA binding in 16HBE14o- cells. Treatment with CpG increased phosphorylation of p38 and pretreatment with the p38 inhibitor SB202190 attenuated the synergistic increase in IL-8 protein levels. Analysis of the half-life of IL-8 mRNA revealed that IL-8 mRNA had a longer half-life following the co-treatment of CpG and IL-1beta compared to treatment with IL-1beta alone. CONCLUSION: Together, these data demonstrate that CpG modulates IL-8 synthesis in the presence of a pro-inflammatory mediator utilizing TLR9 and post-transcriptional mechanisms involving the activation of p38 and stabilization of IL-8 mRNA.

Bronchi↗
PubMed2003

CpG-A and B oligodeoxynucleotides enhance the efficacy of antibody therapy by activating different effector cell populations.

Immunostimulatory CpG oligodeoxynucleotides (ODNs) can enhance the therapeutic effect of monoclonal antibodies (mAbs) by enhancing antibody-dependent cell-mediated cytotoxicity (ADCC). Distinct classes of CpG ODNs have been found recently to stimulate different effector cell populations. We used murine cancer models to explore the role of various effector cell populations in the antitumor activity seen with mAbs combined with CpG ODNs of the A and B classes. In the 38C13 syngeneic murine lymphoma model, both CpG A and CpG B enhanced the efficacy of murine antilymphoma mAb. Depletion of natural killer (NK) cells alone markedly decreased the efficacy of therapy with mAbs plus CpG A. In contrast, depletion of both NK cells and granulocytes was required to decrease the efficacy of mAb plus CpG B. A human (h) Fc gamma receptor I (FcgammaRI)-expressing transgenic (Tg) mouse model was used to explore the role of FcgammaRI in therapy with mAb and CpG ODN. CpG B induced up-regulation of FcgammaRI in hFcgammaRI Tg mice, whereas CpG A did not. In vitro CpG B also enhanced ADCC of HER-2/neu-expressing tumor cells by the FcgammaRI-directed bispecific antibody MDX-H210 using hFcgammaRI-positive effector cells. In a solid tumor model, tumor growth was inhibited in Tg mice treated with a combination of MDX-H210 and CpG B. These data suggest that CpG A enhance ADCC largely by activating NK cells. In contrast, other effector cell populations, including granulocytes, contribute to the antitumor activity of CpG B and mAbs. FcgammaRI plays an important role in this activity.

Adjuvants, Immunologic↗
PubMed2006

Decreased intracellular TLR9 confers hyporesponsiveness of RAW264.7 cells to subsequent CpG ODN challenge.

Low-dose CpG ODN pretreatment is known to induce effective protective immunity against acute infectious diseases. In the present study, using primary murine peritoneal macrophages and the macrophage-like cell line, RAW264.7, we investigated whether low-dose CpG ODN pretreatment would induce hyporesponsiveness in response to a subsequent high-dose CpG ODN challenge and further investigated the molecular mechanisms underlying this event. Our results showed that pretreatment with a low dose of CpG ODN inhibits TNF-alpha production stimulated by later high-dose CpG ODN stimulation in a dose- and time-dependent manner. Interestingly, anti-mouse TLR9 blocking antibody added prior to CpG ODN pretreatment did not affect TNF-alpha release, but antibody added after CpG ODN pretreatment augmented the pretreatment effect of CpG ODN. This difference suggests that cell-surface TLR9 is indeed functional on activated cells. Flow cytometry revealed that low-dose CpG ODN pretreatment decreased cell-surface binding and internalization of a subsequent high-dose stimulation, suggesting that decreased internalization of succeeding CpG ODN is associated with reduced TNF-alpha release. Although both intracellular and cell-surface TLR9 expression are observed, low dose of CpG ODN pretreatment increased only cell-surface TLR9 levels. Importantly, low-dose CpG ODN pretreatment also significantly inhibited the activation of NF-kappaB, an important downstream regulator of various proinflammatory cytokines. In summary, our results demonstrate that suppression of TNF-alpha production by low dose of CpG ODN pretreatment correlates with decreased binding and internalization of subsequent CpG ODN, decreased intracellular content of TLR9, and inhibition of NF-kappaB activation.

Adjuvants, Immunologic↗
PubMed2001

Enhancement of antigen-presenting ability of B lymphoma cells by immunostimulatory CpG-oligonucleotides and anti-CD40 antibody.

Immunostimulatory oligodeoxynucleotides containing the CpG motifs (CpG-ODN) can activate antigen-presenting cells including dendritic cells, macrophages, B cells, and enhance production of Thl cytokines. So, CpG-ODN has been regarded as a promising immune adjuvant. Using the A20 B lymphoma cell model, we investigated the effect of CpG-ODN on the immunogenicity of B lymphoma cells and whether CpG-ODN could enhance the antigen-presenting ability of B lymphoma cells. After incubation with CpG-ODN, proliferation of A20 cells remained unchanged. But CpG-ODN stimulation up-regulated the expression of MHC-I, MHC-II, CD40, ICAM-1 molecules in A20 cells, enhanced the antigen uptake ability of A20 cells, and promoted A20 cell production of IgM and IgG. More importantly, A20 cells activated by CpG-ODN could stimulate allogeneic T cells in MLR and antigen-primed T cells to proliferate more efficiently, suggesting the antigen-presenting ability of A20 B lymphoma cells could be enhanced by CpG-ODN stimulation and CpG-ODN-activated B lymphoma cells might be used as a potent cellular vaccine. Although anti-CD40 mAb was as effective as CpG-ODN at activating A20 cells and A20 cells expressed more CD40 molecules after CpG-ODN stimulation, a combination of CpG-ODN and anti-CD40 mAb had no synergistic effect on A20 cell activation. Our data expanded the potential application of CpG-ODN as an immunotherapeutic agent in cancer treatment.

Animals↗
PubMedDate not supplied

Conjugation of ligands at the 5'-end of CpG DNA affects immunostimulatory activity.

Bacterial DNA and synthetic oligonucleotides containing unmethylated CpG dinucleotides (CpG DNA) activate the vertebrate immune system and promote Th1-like immune responses. Several CpG DNAs are currently being tested in clinical trials as either alone or in combination with vaccines, antibodies, and allergens separately or as conjugates for a number of disease indications including cancers, allergies, and asthma. In this paper, we show that conjugation of an oligonucleotide and a CpG DNA through their 5'-ends (5'-5'-linked DNA) significantly reduces the immunostimulatory activity of the CpG DNA. In addition, we found that the reduction in immunostimulatory activity of 5'-5'-linked CpG DNA depends on the size of the oligonucleotide conjugated to CpG DNA. Conjugation of a smaller group or molecule, such as a phosphorothioate group, at the 5'-end of CpG DNA has an insignificant effect on immunostimulatory activity. However, conjugation of a mononucleotide, tetra- or longer oligonucleotide or a fluorescein molecule to the 5'-end of a CpG DNA (5'-5'-linked DNA) significantly suppresses the immunostimulatory activity of CpG DNA. Surprisingly, conjugation of an oligonucleotide or a ligand through the 3'-end of CpG DNA (3'-3'-linked DNA) has an insignificant effect on immunostimulatory activity. Studies of cellular uptake and activation of transcription factor NF-kappaB in J774 cells using fluorescein-conjugated CpG DNAs suggest that the differences in the immune stimulation of 5'- and 3'-end-conjugated CpG DNAs is not as a result of differences in their cellular uptake properties. These results suggest that for optimal immunostimulatory activity, ligands should not be attached at the 5'-end of the CpG DNA.

3' Flanking Region↗
PubMed2002

Immunomodulatory effects of CpG oligodeoxynucleotides on established th2 responses.

CpG oligodeoxynucleotides (CpG ODNs) are known to induce type 1 T-helper-cell (Th1) responses. We have previously demonstrated that CpG ODNs administered during sensitization prevent Th2-mediated eosinophilic airway inflammation in vivo. We also reported that key Th1 cytokines, gamma interferon (IFN-gamma) and interleukin 12 (IL-12), are not necessary for this protection. Recent in vivo data suggest that CpG ODNs might also reverse established pulmonary eosinophilia. In order to clarify how CpG ODNs can inhibit established Th2 responses, we evaluated the cytokine production from splenocytes from antigen- and alum-immunized mice. Restimulation with antigen induced IL-5, which was clearly inhibited by coculture with CpG ODNs in a concentration-dependent manner. CpG ODNs also induced IFN-gamma, but in a concentration-independent manner. The inhibition of IL-5 production was not mediated through natural killer cells or via CD8(+) T lymphocytes. Although IFN-gamma plays an important role in inhibition of antigen-induced IL-5 production by CpG ODNs, IFN-gamma was not the sole factor in IL-5 inhibition. CpG ODNs also induced IL-10, and this induction correlated well with IL-5 inhibition. Elimination of IL-10 reduced the anti-IL-5 effect of CpG ODNs, although incompletely. This may be because IFN-gamma, induced by CpG ODNs, is also inhibited by IL-10, serving as a homeostatic mechanism for the Th1-Th2 balance. Overproduction of IFN-gamma was downregulated by CpG ODN-induced IL-10 via modulation of IL-12 production. These data suggest that CpG ODNs may inhibit established Th2 immune responses through IFN-gamma and IL-10 production, the latter serving to regulate excessive Th1 bias. These properties of CpG ODNs might be a useful feature in the development of immunotherapy adjuvants against allergic diseases such as asthma.

Adjuvants, Immunologic↗
PubMed2002

[CpG-ODN is a potential candidate adjuvant for human vaccines].

OBJECTIVE: To evaluate the adjuvanticity of CpG-ODN for human vaccines in animal models. METHODS: To find suitable animal models, the human CpG-ODN were examined for their in vitro immunostimulatory activities for murine and Rhesus monkey immune cells. Then by using recombinant HBsAg as a model antigen, the adjuvanticity of human CpG-ODN was evaluated in the animal models. RESULTS: Rhesus monkey B cells responded well to all the human CpG-ODN, similarly as that of human B cells. In contrast, only the human CpG-ODN with the CpG motif 5'GTCGTT 3' (CpG2006 etc) could induce murine splenocytes to secret IgM and IFN-gamma, while those with the CpG motif 5'GTCGTC 3' (CpGT7 etc) had less or no effects. The results suggested that Rhesus monkeys and mice could be used as animal models to evaluate the in vivo activities of different human CpG-ODN. Immunized with HBsAg combined with various human CpG-ODN, the mice elicited a stronger Th1 humoral immunity. Consistent with the in vitro findings, CpG-ODN with the CpG motif 5'GTCGTT 3' were more potent than those with the CpG motif 5'GTCGTC 3'. But of note, all the sequences had the same ability for modulation of Th1/Th2 immune response, with the ratio of IgG2a/IgG1 around 1. However, human CpG-ODN had less adjuvanticity for HBsAg in Rhesus monkeys; only CpGT7 increased the antibody titers by 2 times, while CpG2006 had no effect. CONCLUSION: The preliminary results derived from animal models showed that CpG-ODN was a potential candidate Th1 adjuvant for human vaccines.

Adjuvants, Immunologic↗
PubMed1996

Rapid immune activation by CpG motifs in bacterial DNA. Systemic induction of IL-6 transcription through an antioxidant-sensitive pathway.

Unmethylated CpG dinucleotides (CpG motif) in bacterial DNA or synthetic oligodeoxynucleotides (CpG DNA) rapidly activate murine B cells to secrete IL-6 and IgM, as well as to proliferate. Within 30 min after CpG DNA stimulation in vivo, IL-6 mRNA levels were increased in liver, spleen, and thymus cells. Serum IL-6 protein was markedly increased within 1 h of stimulation. Treatment of a B cell line with CpG DNA led to an increase in the transcriptional activity of the IL-6 promoter. This CpG DNA-induced IL-6 production was not mediated via either a protein kinase C (PKC)-, protein kinase A (PKA)-, or nitric oxide (NO.)-dependent pathway but was inhibited by an antioxidant. In addition, the level of intracellular reactive oxygen species was increased within 20 min after CpG DNA, but not control non-CpG DNA, treatment. These results suggest that CpG DNA-induced IL-6 production is mediated through a reactive oxygen intermediate-dependent pathway. CpG DNA-mediated IL-6 production was enhanced by simultaneous signals delivered through the Ag receptor. The addition of neutralizing Abs against IL-6 to B cell cultures along with CpG oligodeoxynucleotides essentially abolished the CpG DNA-induced increased IgM secretion but had no significant effect on the B cell proliferation induced by the CpG motif. Our results suggest that the induction of IL-6 expression in response to CpG motifs in bacterial DNA may be an important immune defense mechanism that facilitates a rapid response to microbial infection.

Animals↗
PubMed2001

Identification and characterization of the DNA binding domain of CpG-binding protein.

CpG-binding protein is a transcriptional activator that exhibits a unique DNA binding specificity for unmethylated CpG motifs. CpG-binding protein contains a cysteine-rich CXXC domain that is conserved in DNA methyltransferase 1, methyl binding domain protein 1, and human trithorax. In vitro DNA binding assays reveal that CpG-binding protein contains a single DNA binding domain comprised of the CXXC domain and a short carboxyl extension. Specific mutation to alanine of individual conserved cysteine residues within the CXXC domain abolishes DNA binding activity. Denaturation/renaturation experiments in the presence of various metal cations demonstrate that the CXXC domain requires zinc for efficient DNA binding activity. Ligand selection of high affinity binding sites from a pool of degenerate oligonucleotides reveals that CpG-binding protein interacts with a variety of sequences that contains the CpG dinucleotide with a consensus binding site of (A/C)CpG(A/C). Mutation of the CpG motif(s) present within ligand-selected oligonucleotides ablates the interaction with CpG-binding protein, and mutation to thymine of the nucleotides flanking the CpG motifs reduces the affinity of CpG-binding protein. Hence, a CpG motif is necessary and sufficient to comprise a binding site for CpG-binding protein, although the immediate flanking sequence affects binding affinity.

Alanine↗
PubMed2001

CpG DNA induces cyclooxygenase-2 expression and prostaglandin production.

Unmethylated CpG motifs found in bacterial DNA are potent activators of the innate and acquired immune systems, and rapidly induce the production of proinflammatory cytokines. We hypothesized that CpG DNA may also elicit the production of prostaglandins (PG), which are central lipid mediators of the immune and inflammatory response. To test our hypothesis, we stimulated murine spleen cells and RAW 264.7 murine macrophage cells with CpG DNA and assessed the effects on the PG synthesis pathway. Compared to control, DNA-containing CpG motifs induced >5-fold increase in PGE (2) production and rapidly up-regulated cyclooxygenase-2 (COX-2) at both the mRNA and protein level. CpG DNA was an extremely strong inducer of COX-2 as concentrations as low as 3 ng/ml induced COX-2 protein expression. The CpG DNA-induced PGE (2) down-regulated the immune response elicited by CpG. Blockade of PGE (2) production with selective COX-2 inhibitors or neutralizing anti-PGE (2) antibody markedly enhanced IFN-gamma secretion in vitro from CpG DNA-stimulated spleen cells. Moreover, selective COX-2 inhibition increased CpG DNA-induced IFN-gamma secretion in vivo. Inhibition of COX-2 also increased CpG DNA-induced lytic activity of NK cells. Taken together, these data indicate that DNA containing CpG motifs is a potent inducer of COX-2 and PGE (2) production. CpG-induced PG may subsequently down-regulate the immune and inflammatory responses elicited by the CpG DNA.

Adjuvants, Immunologic↗
PubMed1999

Progressive region-specific de novo methylation of the p16 CpG island in primary human mammary epithelial cell strains during escape from M(0) growth arrest.

CpG island methylation plays an important role in normal cellular processes, such as genomic imprinting and X-chromosome inactivation, as well as in abnormal processes, such as neoplasia. However, the dynamics of de novo CpG island methylation, during which a CpG island is converted from an unmethylated, active state to a densely methylated, inactive state, are largely unknown. It is unclear whether the development of de novo CpG island methylation is a progressive process, in which a subset of CpG sites are initially methylated with a subsequent increase in methylation density, or a single event, in which the initial methylation event encompasses the entire CpG island. The tumor suppressor gene p16/CDKN2a/INK4a (p16) is inactivated by CpG island methylation during neoplastic progression in a variety of human cancers. We investigated the development of methylation in the p16 CpG island in primary human mammary epithelial cell strains during escape from mortality stage 0 (M(0)) growth arrest. The methylation status of 47 CpG sites in the p16 CpG island on individual DNA molecules was determined by sequencing PCR clones of bisulfite-treated genomic DNA. The p16 CpG island was initially methylated at a subset of sites in three discrete regions in association with p16 transcriptional repression and escape from M(0) growth arrest. With continued passage, methylation gradually increased in density and methylation expanded to sites in adjacent regions. Thus, de novo methylation in the p16 CpG island is a progressive process that is neither site specific nor completely random but instead is region specific. Our results suggest that early detection of methylation in the CpG island of the p16 gene will require methylation analysis of the three regions and that the identification of region-specific methylation patterns in other genes may be essential for an accurate assessment of methylation-mediated transcriptional silencing.

Breast↗
PubMed2004

Liposome-encapsulated CpG oligodeoxynucleotides as a potent adjuvant for inducing type 1 innate immunity.

Unmethylated cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs) exhibit potent immunostimulating activity by binding with Toll-like receptor 9 (TLR9) expressed on antigen-presenting cells. Here, we show that CpG-ODN encapsulated in cationic liposomes (CpG-liposomes) improves its incorporation into CD11c(+) dendritic cells (DCs) and induces enhanced serum interleukin (IL)-12 levels compared with unmodified CpG-ODN. CpG-liposome potently activated natural killer (NK) cells (84.3%) and NKT cells (48.3%) to produce interferon-gamma (IFN-gamma), whereas the same dose of unmodified CpG-ODN induced only low numbers of IFN-gamma-producing NK cells (12.7%) and NKT cells (1.6%) to produce IFN-gamma. In contrast with the NKT cell agonist alpha-galactosylceramide, which induces both IFN-gamma and IL-4 production by NKT cells, CpG-liposome only induced IFN-gamma production by NKT cells. Such potent adjuvant activities of CpG-liposome were absent in TLR9-deficient mice, indicating that CpG-liposome was as effective as CpG-ODN in stimulating type 1 innate immunity through TLR9. In addition to TLR9, at least two other factors, IL-12 production by DCs and direct contact between DCs and NK or NKT cells, were essential for inducing type 1 innate immunity by CpG-liposome. Furthermore, ligation of TLR9 by CpG-liposome coencapsulated with ovalbumin (OVA) caused the induction of OVA-specific CTLs, which exhibited potent cytotoxicity against OVA-expressing tumor cells. These results indicate that CpG-liposome alone or combined with tumor antigen protein provides a promising approach for the prevention or therapy of tumors.

Adjuvants, Immunologic↗
PubMed2003

The CpG island searcher: a new WWW resource.

Clusters of CpG dinucleotides in GC rich regions of the genome called "CpG islands" frequently occur in the 5' ends of genes. Methylation of CpG islands plays a role in transcriptional silencing in higher organisms in certain situations. We have established a CpG-island-extraction algorithm, which we previously developed [Takai and Jones, 2002], on a web site which has a simple user interface to identify CpG islands from submitted sequences of up to 50kb. The web site determines the locations of CpG islands using parameters (lower limit of %GC, ObsCpG/ExpCpG, length) set by the user, to display the value of parameters on each CpG island, and provides a graphical map of CpG dinucleotide distribution and borders of CpG islands. A command-line version of the CpG islands searcher has also been developed for larger sequences. The CpG Island Searcher was applied to the latest sequence and mapping information of human chromosomes 20, 21 and 22, and a total of 2345 CpG islands were extracted and 534 (23%) of them contained first coding exons and 650 (28%) contained other exons. The CpG Island Searcher is available on the World Wide Web at http://www.cpgislands.com or http://www.uscnorris.com/cpgislands/cpg.cgi.

Algorithms↗
PubMed1991

CpG islands, genes and isochores in the genomes of vertebrates.

We have shown that human genes associated with CpG islands increase in number as they increase in % of guanine + cytosine (GC) levels, and that most genes associated with CpG islands are located in the GC-richest compartment of the human genome. This is an independent confirmation of the concentration gradient of CpG islands (detected as HpaII tiny fragments, or HTF) which was demonstrated in the genome of warm-blooded vertebrates [Aïssani and Bernardi, Gene 106 (1991) 173-183]. We then reassessed the location of CpG islands using the data currently available and confirmed that CpG islands are most frequently located in the 5'-flanking sequences of genes and that they overlap genes to variable extents. We have shown that such extents increase with the increasing GC levels of genes, the GC-richest genes being completely included in CpG islands. Under such circumstances, we have investigated the properties of the 'extragenic' CpG islands located in the 5'-flanking segments of homologous genes from both warm- and cold-blooded vertebrates. We have confirmed that, in cold-blooded vertebrates, CpG islands are often absent; when present, they have lower GC and CpG levels; the latter attain, however, statistically expected values. Finally, we have shown that CpG doublets increase with the increasing GC of exons, introns and intergenic sequences (including 'extragenic' CpG islands) in the genomes from both warm- and cold-blooded vertebrates. The correlations found are the same for both classes of vertebrates, and are similar for exons, introns and intergenic sequences (including 'extragenic' CpG islands). The findings just outlined indicate that the origin and evolution of CpG islands in the vertebrate genome are associated with compositional transitions (GC increases) in genes and isochores.

Animals↗
PubMed2004

Identification of CpG oligodeoxynucleotide sequences that induce IFN-gamma production in canine peripheral blood mononuclear cells.

Oligodeoxynucleotides containing the cytosine-phosphate-guanine (CpG) motif (CpG-ODNs) have been shown to induce T(H)1 immune responses in animals. Since the sequences of CpG-ODNs that induce T(H)1 responses are considered to vary among animal species, it is necessary to identify effective CpG-ODNs in each animal. In order to identify the sequences of CpG-ODNs that induce T(H)1 responses in dogs, mRNA expression and protein production of IFN-gamma were examined in peripheral blood mononuclear cells (PBMCs) from healthy dogs treated with 11 kinds of synthetic CpG-ODNs. One of the 11 CpG-ODNs (No. 2 CpG-ODN, 5'-GGTGCATCGATGCAGGGGGG-3') was shown to significantly increase mRNA expression and protein production of IFN-gamma in canine PBMCs in a manner dependent on the sequence of the CpG motif. This CpG-ODN also enhanced the expression of IL-12 p40 mRNA in canine PBMCs, whereas expression of IL-12 p35, IL-18, and IL-4 mRNAs was not induced by this CpG-ODN. These results indicate that this CpG-ODN was able to produce IFN-gamma by induction of T(H)1-skewed immune response in dogs. CpG-ODNs may be useful for inducing prophylactic and therapeutic immunity against allergic diseases, viral infection, and tumors in dogs.

Animals↗
PubMed2003

The binding of CpG-oligodeoxynucleotides to cell-surface and its immunostimulatory activity are modulated by extracellular acidic pH.

Both the binding of CpG-oligodeoxynucleotides (CpG-ODNs) to cell-surface and its immunostimulatory activity were modulated by extracellular pH in present study. At neutral pH (pH 7.4), the binding of CpG-ODN to splenocyte-surface, as well as that of non-CpG-ODN, was competitively inhibited by non-specific DNA-Herring sperm DNA in a dose dependent manner, indicating their binding sites have no specificity for CpG-motif. When the extracellular pH shifted to acidic (pH 6.4), however, their binding to cell-surface markedly increased, and only the binding of non-CpG-ODN instead of CpG-ODN was inhibited by Herring sperm DNA, implying such pH change enabled CpG-ODN bind to its specific binding-site. Consistently, lymphocytes appeared more sensitive to the stimulation of CpG-ODN at acidic pH, and Herring sperm DNA inhibited the CpG-ODN-induced TNF production from splenocytes at pH 7.4, but not at pH 6.4. These results suggest the existence of membrane receptor that specifically engages CpG-ODN with high affinity only at acidic pH, and support the hypothesis that the binding CpG-ODN to its specific membrane receptor and subsequently triggering of CpG-related signaling occurred within acidified endosomes.

Adjuvants, Immunologic↗