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PubMed1996

Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA.

We have recently shown that oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG motif) can induce B cells to proliferate, differentiate, and secrete cytokines. In this study we demonstrate that CpG motifs contained in ODN as short as 15 bases in length were quite effective at inducing NK cell lytic activity in vitro in both human and murine lymphocytes. Such ODN were also effective at inducing NK lytic activity, in vivo, in mice. Experiments designed to determine the cellular and cytokine requirements for NK cell induction revealed that B and T cells are not necessary, that the ODN do not augment the activity of highly purified NK cells, and that the ODN augment NK cell activity indirectly by inducing the secretion of IL-12, IFN-alpha beta, and TNF-alpha. Various ODN sequences were prepared to determine the optimal ODN length, motif, palindrome, backbone modification, and dose requirements. We found no requirement for a palindromic sequence but a definite requirement for an unmethylated CpG motif. While necessary, however, a CpG motif was not sufficient for NK cell induction. Instead, there appeared to be stringent requirements for the immediate flanking bases at the 5' and 3' ends as well as for flanking sequences outside the immediate 5' and 3' bases. In particular poly(G) ends seemed to exert a complex qualitative and quantitative effect which could be up- or down-modulating depending on whether the ODN backbone was phosphorothioate modified or not.

Animals↗
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↗
PubMed2002

Detection of bacterial DNA in Latin American patients with reactive arthritis by polymerase chain reaction and sequencing analysis.

OBJECTIVE: Bacteria and/or their antigens are thought to play a role in the pathogenesis of reactive arthritis (ReA). Polymerase chain reaction (PCR) using the 16S ribosomal RNA-PCR method was used to identify bacterial DNA in synovial fluid (SF) and tissue (ST) in a well defined group of patients with chronic ReA. In addition, species found were identified by means of sequence analysis. METHODS: We examined 15 ST and 5 SF samples of 15 patients with ReA, 5 ST samples of 5 patients with osteoarthritis (OA), and 8 SF from 8 patients with closed traumatic knee injuries using a nested PCR with universal 16S rRNA primers. In addition, a nested PCR was developed to detect DNA sequences of Salmonella sp. and Mycoplasma sp. Automated sequencing and comparative data analysis (GenBank) were also performed to identify the species. RESULTS: Bacterial DNA was identified in 8 cases, 5 ST and 3 SF; Chlamydia trachomatis (n = 2), Pseudomonas sp. (n = 3), and Bacillus cereus (n = 2) were the most common microorganisms identified. A variety of microorganisms including Clostridium sp., Lactobacillus sp., Pseudomonas migulae, P. fluorescens, and P. putida, and Neisseria meningitidis serogroup B were also identified. In half of the cases (4/8) 2 to 3 bacterial antigens were identified simultaneously. CONCLUSION: Bacterial DNA is present in the joints in patients with chronic ReA. A wide spectrum of bacteria including some not previously associated with ReA were identified. Further studies are needed to establish their exact role in the pathogenesis of ReA and related arthritides.

Adolescent↗
PubMed2003

The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy.

BACKGROUND: DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein). We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. RESULTS: Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. CONCLUSIONS: The technique detailed here minimizes the cost and effort to replicate a PCR-generated DNA gene fragment library and facilitates several downstream processes (e.g. directional cloning of fragments and gene expression as affinity-tagged fusion proteins) beyond the primary objective of producing DNA microarrays for global gene expression profiling.

Cyanobacteria↗
PubMed1975

On the question of the integration of exogenous bacterial DNA into plant DNA.

Extensive studies with pea, tomato, and barley failed to confirm the evidence presented by previous investigators for integration or replication of exogenously applied bacterial DNA in these plants. Labeled DNA of buoyant density in CsCl intermediate between that of high density donor bacterial DNA and of plant DNA was never observed with axenic plants. Intermediate peaks, similar to those used as evidence for recombination by earlier investigators, were observed only when the plants were contaminated with bacteria. Plant DNA prepared by a published procedure [Ledoux, L. & Huart, R. (1969) J. Mol. Biol. 43, 243-262] was found to be contaminated with unidentified impurities. Such DNA was partially protected from the action of DNase and produced aberrant banding patterns in CsCl after shearing. Much of the published evidence for integration of foreign DNA in plants is based upon experiments with plant DNA prepared by this procedure. We conclude that contamination is the likely explanation for what has been interpreted as evidence for integration.

Centrifugation, Density Gradient↗
PubMed1996

Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides.

Microbial products released during bacterial infection induce cytokine-mediated inflammatory responses that can be protective, but excessive release of inflammatory cytokines may promote development of the sepsis syndrome. We examined the ability of bacterial DNA to induce in vivo cytokine release and to potentiate the toxicity of LPS. Intravenous treatment of mice with Escherichia coli (EC) DNA, but not calf thymus (CT) DNA, induced a rapid (within 4 h) dose-dependent increase in serum IFN-gamma and splenic IFN-gamma-forming cells. Over 90% of splenic IFN-gamma-producing cells were identified by surface phenotype as NK cells. Mice also mounted an IFN-gamma response following challenge with 20-base oligonucleotide that contained an internal CG motif (but did not respond to a control oligonucleotide). Treatment of mice with EC DNA followed by a sublethal LPS challenge resulted in a 3-fold increase in the peak serum level of TNF-alpha and a 10-fold increase in the peak level of IL-6 compared with mice that received CT DNA followed by LPS. Mice treated with EC DNA followed by LPS showed 75% mortality, compared with no deaths in mice treated with CT DNA followed by LPS. EC DNA/LPS treatment of mice with disrupted IFN-gamma genes resulted in a 5% mortality while 59% of similarly treated +/+ mice died. Thus, bacterial DNA induces in vivo release of IFN-gamma which, in turn, is associated with an increase in LPS-induced TNF-alpha and IL-6 release, and with increased sensitivity to the toxic effects of LPS.

Animals↗
PubMed2003

Comparison of rapid methods for the extraction of bacterial DNA from colonic and caecal lumen contents of the pig.

AIMS: The increasing uses of DNA methodologies to study the micro flora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from the intestinal sample. Thus, the objective of this study was to determine which DNA extraction methods are most effective for luminal samples from pigs. Several routinely used nucleic acid extraction procedures were compared based upon quantity and purity of extracted DNA. METHODS AND RESULTS: DNA was extracted from pig colonic and caecal lumen samples using 19 methods for bacterial DNA extraction. The quantity of total DNA recovered by each extraction method was determined and compared. Two methods using extraction with polyvinylpolypyrrolidone (PVPP) or phenol and two methods involving bead mill homogenization were found to provide the greatest quantity of extracted DNA for both colonic and caecal lumen. Extracted DNA from these four methods was further analysed for purity based upon the presence of PCR inhibitors, which was ascertained by determining the efficiency of amplification of a segment of the 16S rDNA. PCR amplification could be readily achieved with DNA extracted by each of these four methods, but efficiency of amplification tended to be higher with DNA from two of the methods (one extracted with PVPP and one with bead mill homogenization). CONCLUSIONS: Four extraction methods proved to be significantly superior in quantity of DNA extracted from luminal samples. Of these four, no strong inhibitors of PCR amplification were detected in any of the extracted DNA. However, the efficiency of amplification tended to be lower in DNA samples from two of the methods, suggesting the presence of low levels of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study provide a basis for choosing which DNA extraction procedures are most effective for use with samples of pig lumen.

Animals↗
PubMed2006

The detection of bacterial DNA in blood of rats with CCl4-induced cirrhosis with ascites represents episodes of bacterial translocation.

Bacterial DNA (bactDNA) is present in blood and ascitic fluid (AF) in a third of patients with cirrhosis and ascites, but whether this phenomenon represents episodes of bacterial translocation (BT), strictly considered when culture of mesenteric lymph nodes (MLNs) are positive, remains unknown. This study assessed the relationship between bactDNA detection in biological fluids and MLNs and went on to investigate the local and systemic inflammatory status according to its presence. Cirrhosis was induced in rats by ingestion of CCL4. A subgroup of five animals with cirrhosis received norfloxacin (5 mg/kg/day) for 7 days. MLNs and ascitic and pleural fluids were collected at laparotomy and cultured; samples were collected for identification of bactDNA and measurement of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO). BactDNA was detected in MLNs in 12 of 19 animals (63.1%), corresponding in seven cases to culture-positive MLNs, and in five to culture-negative MLNs. BactDNA was detected in biological fluids in 11 of 19 animals (57.9%), and in all cases the same bacteria spp. detected in samples was present in MLNs. BactDNA was not detected in any biological sample from animals receiving norfloxacin. Tumor necrosis factor alpha (TNF-alpha), IL-6, and NO were similar in culture-positive and culture-negative/bactDNA-positive samples, and significantly higher than those observed in animals with culture-negative/bactDNA-negative MLNs, animals with cirrhosis that were receiving norfloxacin, and controls. In conclusion, the presence of bactDNA in biological fluids in rats with cirrhosis constitutes a marker of BT, and it is associated with a marked inflammatory response, independent of the result of the culture.

Animals↗
PubMed2000

A Toll-like receptor recognizes bacterial DNA.

DNA from bacteria has stimulatory effects on mammalian immune cells, which depend on the presence of unmethylated CpG dinucleotides in the bacterial DNA. In contrast, mammalian DNA has a low frequency of CpG dinucleotides, and these are mostly methylated; therefore, mammalian DNA does not have immuno-stimulatory activity. CpG DNA induces a strong T-helper-1-like inflammatory response. Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseases. Despite its promising clinical use, the molecular mechanism by which CpG DNA activates immune cells remains unclear. Here we show that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9. TLR9-deficient (TLR9-/-) mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. TLR9-/- mice showed resistance to the lethal effect of CpG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9-/- mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Lack of detection of enteroviral RNA or bacterial DNA in magnetic resonance imaging-directed muscle biopsies from twenty children with active untreated juvenile dermatomyositis.

OBJECTIVE: To investigate for the presence of increased titers of circulating antibody to putative infectious agents and for detectable viral RNA or bacterial DNA in children with active recent-onset juvenile dermatomyositis (DM). METHODS: Magnetic resonance imaging-directed muscle biopsies were performed in 20 children with active, untreated, recent-onset juvenile DM and in age-matched children with neurologic disease. Sera were tested for complement-fixing antibody to Coxsackievirus B (CVB), influenza A and B, parainfluenza 1 and 3, Mycoplasma pneumoniae, mumps, respiratory syncytial virus, and Reovirus; and by immunofluorescence for IgG antibody to Toxoplasma gondii cytomegalovirus and IgM antibody to Epstein-Barr virus. Muscle from juvenile DM patients and control children, CD-1 Swiss mice with and without CVB1 infection, and viral stock positive for CVB1-6 were tested using reverse-transcriptase polymerase chain reaction with 5 primer sets, 4 probes (1 Coxsackievirus, 3 Enterovirus), and universal primers for DNA. RESULTS: No increased antibody, viral RNA, or bacterial DNA was present in the juvenile DM patients or the control children. CONCLUSION: Juvenile DM may be triggered by unidentified agent(s) in the genetically susceptible host.

Adolescent↗