Specific inhibition of PCR by non-extendable oligonucleotides using a 5' to 3' exonuclease-deficient DNA polymerase.

The Stoffel fragment of Taq DNA polymerase lacks the 5' to 3' exonuclease activity that hydrolyzes potentially blocking DNA strands during primer extension. We there-fore asked whether by using this fragment in the PCR, non-extendable, base-paired oligonucleotides could inhibit amplification in a sequence-dependent manner. Model targets were chosen from the partially conserved ribosomal 16S rDNA of three bacterial species: E. coli, Bacillus subtilis and Neisseria gonorrhoea. A single pair of primers was capable of amplifying a homologous 240-bp region from all three. Two non-extendable "blocking" oligonucleotides were synthesized with sequences complementary to the inter-primer regions of E. coli and B. subtilis, respectively. Both blockers were shown specifically to prevent amplification of their complementary targets, but not of the reciprocal control targets or of the reciprocal control targets or of the non-complementary N. gonorrhea. Specificity was further confirmed by an internal positive control. Similar inhibition was seen with mixtures of targets in a single reaction. With intact Taq DNA polymerase, no blocking was observed. Primers and blockers targeting specific regions of N. gonorrhoea rDNA were used to confirm the requirement that blockers be directed to the inter-primer region. Sequence-dependent amplification inhibition, such as that demonstrated here, would be applicable to PCR-related strategies using primers capable of using multiple targets, where such selective inhibition could be useful.

Soluble oligonucleosomal complexes in synovial fluid from inflamed joints.

OBJECTIVE: To determine whether soluble oligonucleosomal DNA, typical of that released during apoptotic cell death, is present in synovial fluids from inflamed joints and, if so, whether it is present in sufficient concentrations to have pathophysiologic significance. METHODS: Fifty synovial fluid specimens from 46 patients were studied, 41 from joints with a variety of inflammatory disorders and 9 from osteoarthritic joints. DNA from freshly collected synovial fluid was isolated and quantitated by microfluorometry, and the oligonucleosomal fraction was measured by radiolabeling, gel electrophoresis, and autoradiography. Specific immunoprecipitation with monoclonal antihistone antibody, after DNA radiolabeling in whole synovial fluid, was used to detect histone binding. RESULTS: DNA with a typical oligonucleosomal ladder was observed in most specimens. The mean +/- SD oligonucleosomal DNA concentration was 14.1 +/- 18.5 microg/ml in synovial fluids from inflamed joints, considerably higher than that in osteoarthritic synovial fluids. Additionally, the DNA was shown to be complexed with histone, as would be expected. Control experiments were performed to show that the oligonucleosomal DNA was present in soluble form and did not arise due to in vitro artifact. The DNA concentrations were found to correlate significantly with the concentrations of synovial fluid leukocytes, most of which were neutrophils. CONCLUSION: Synovial fluids from inflamed joints contain oligonucleosomal DNA typical of that released during apoptotic cell death. The probable source is fluid-phase neutrophils undergoing apoptotic cell death, although this was not directly demonstrated. The concentrations are sufficient to have biologic activity similar to that shown in vitro, including lymphoproliferation and stimulation of interleukin-6 secretion. A mechanism by which oligonucleosomal DNA may contribute to perpetuation of rheumatoid synovitis is proposed. If it is generalizable to other sites of inflammation, as seems probable, similar oligonucleosomal DNA release accompanying inflammation may play a pathogenetic role in other disorders, including systemic lupus erythematosus.

Domain-directed polymerase chain reaction capable of distinguishing bacterial from host DNA at the single-cell level: characterization of a systematic method to investigate putative bacterial infection in idiopathic disease.

The use of a broad-range PCR to target conserved sequences in the bacterial ribosome has long been recognized as an approach to investigating idiopathic human diseases for the presence of bacteria. An important example is rheumatoid arthritis where the hypothesis of a bacterial etiology remains viable despite failure of previous methods to identify a causative organism. Practical implementation of this strategy, however, was impeded by requirements unique to the study of these diseases. Hence, an adequately characterized method for achieving this has not appeared. We now describe such a method based on the use of a broadly reactive pair of deoxyinosine-containing primers. Detailed characterization addressing these requirements provided evidence that DNA from at least 96% of potential prokaryotic pathogens would be detected. The sensitivity was shown to approximate that of a single organism per reaction. Importantly, this sensitivity was shown to be maintained among multiple targets and to be unimpaired by a large excess of human DNA. Similar results were obtained with extracts of inflamed human synovial fluid to which as little as 0.01 pg of bacterial DNA or, alternately, a single organism per reaction was added. This method was also shown correctly to detect the causative bacteria in clinically infected synovial fluids, further documenting its applicability to such specimens. Finally, it was converted to an in situ method by which bacterial sequences were histochemically localized to Michaelis-Guttman bodies in tissue sections from a patient with malakoplakia, a poorly understood infectious disease. The broad reactivity and high sensitivity achieved by this approach translate into a high likelihood of detecting an unknown bacterium if present in a clinical specimen. Conversely, if properly controlled, the failure of this method to detect such organisms can provide evidence supporting rejection of the bacterial etiology hypothesis, an important aspect unique to this approach. For those bacterial sequences that are detected, the ability to localize them in situ would help define their histologic context and hence facilitate their pathogenetic interpretation. The present method should therefore be appropriate for use in systematically studying rheumatoid arthritis as well as a number of other important idiopathic disorders where a bacterial etiology is suspect.

Use of the polymerase chain reaction to study arthritis due to Neisseria gonorrhoeae.

OBJECTIVE: To explore the utility of the polymerase chain reaction (PCR) in the diagnosis, management, and investigation of arthritis due to Neisseria gonorrhoeae. METHODS: The PCR was used to detect DNA from N gonorrhoeae in model systems and in extracts of synovial fluid (SF) from patients with systemic gonococcal infections and objective evidence of arthritis. RESULTS: One N gonorrhoeae organism or its equivalent was detectable in human SF from inflamed joints. Five of 8 patients with systemic N gonorrhoeae infection and arthritis had N gonorrhoeae DNA demonstrated by PCR in at least 1 pretreatment SF specimen that was N gonorrhoeae culture positive. Thirty-seven of 38 control specimens were negative, the exception probably being due to cross-contamination from a positive specimen. All specimens that were positive for N gonorrhoeae by other methods were also positive by PCR. Two others were positive by PCR but negative by other methods. Four pretreatment specimens were negative by all methods, including PCR. This suggests that, for these patients, negative cultures reflected true absence of N gonorrhoeae, and not the presence of unculturable organisms. This group also had a significantly shorter duration of disease than did the patients with N gonorrhoeae DNA found in their SF. All patients had a prompt response to antibiotic treatment. Two of 3 patients whose specimens were previously positive showed marked decreases in SF N gonorrhoeae DNA after treatment. CONCLUSION: The PCR using these or similar oligonucleotide primers can be a useful adjunct in the diagnosis of gonococcal arthritis and can be of value in assessing its response to therapy. In some N gonorrhoeae-associated arthritides, there appears to be a lack of both viable and nonviable N gonorrhoeae organisms in the SF. These observations may have implications regarding the pathogenesis of this form of arthritis.

Design and characterization of PCR primers for detection of pathogenic Neisseriae.

Oligonucleotide polymerase chain reaction (PCR) primers directed at a group of closely related Neisserial species were designed from 16S rDNA sequences even though only a single such sequence from the targeted group was known. The amplifiable group included all Neisserial species considered pathogenic for man, including Neisseria gonorrhoea and Neisseria meningitidis. None of 43 other bacterial DNA specimens were amplified, including five non-Neisserial Neisseriaceae and three non-pathogenic Neisseriae. Another non-pathogenic Neisserial species gave a signal only at high DNA concentrations. DNA specimens from the pathogenic Neisseriae were detectable in amounts as low as 0.01 pg per PCR reaction, the approximate equivalent of a single organism, with equal sensitivity in buffer or in simple extracts of human inflammatory synovial fluids to which Neisserial DNA had been added. Simultaneously studied control specimens lacking added DNA were negative. The approach used to design these group-directed primers using only a single rDNA sequence from the targeted group by exploiting known patterns of sequence conservation among the 16S rDNA genes may prove useful for designing other similar group-directed primers. Polymerase chain reaction primers prepared in this way should prove of value in a number of areas, both investigational and clinical.

Haemodialysis as a model for studying endogenous plasma DNA: oligonucleosome-like structure and clearance.

The rate of clearance of extracellular plasma DNA in man has important implications for pathogenetic mechanisms in systemic lupus erythematosus (SLE), as well as for certain other clinical states. Present knowledge of this parameter is derived exclusively from studies of injected, naked DNA in animals. Recent information indicates that the physiologic form of plasma DNA in SLE is that of oligonucleosome-like molecules rather than of naked DNA and consists of multimeric complexes of DNA bound to histone, probably arising from an apoptotic process. In order to study the rate at which these oligonucleosome-like complexes are removed from plasma and to do so in man rather than experimental animals, we exploited the observation that during haemodialysis large amounts of DNA are released, apparently within the dialysis coil, into the patient's plasma. Since this release appears to cease promptly with termination of the procedure, it offered the potential for estimating the rate of removal of such DNA from human plasma. Moreover, if that DNA, as postulated, were shown to possess an oligonucleosome-like structure resembling that found endogenously in human SLE, the relevance of such information to the human disease state would be further enhanced. The present results support the conclusion that DNA released into plasma during haemodialysis possesses such an oligonucleosome-like structure. The plasma half-life of that DNA in man was found not to exceed 4 min. The highly dynamic state thus implied for extracellular endogenous plasma DNA in man has important implications for pathogenetic mechanisms dependent on dsDNA in SLE. Moreover, individuals undergoing chronic haemodialysis, who are thereby exposed to a very large cumulative amount of such DNA, might serve as models for studying its long-term sequelae.

Geometric differences allow differential enzymatic inactivation of PCR product and genomic targets.

The value of the polymerase chain reaction (PCR) is markedly limited by the ease of carry-over contamination. We predicted that the location of the target sequence which spans the linear center of the molecule in PCR products, but not in genomic molecules, would allow digestion by certain exonucleases (Exo). This would eliminate amplifiable targets specifically from PCR products and do so without the need to control either the size of the genomic molecules or the extent of the digestion reaction. Testing with T7 Exo and model targets in phage lambda DNA yielded results consistent with those predicted. By heat inactivating the Exo, complete reaction mixtures could be decontaminated and then amplified in an automatic thermal cycler without reopening the reaction tubes and risking recontamination.

Invasion of the central nervous system by Borrelia burgdorferi in acute disseminated infection.

OBJECTIVE: To determine central nervous system (CNS) involvement in acutely disseminated Borrelia burgdorferi infection by measurement of borrelia-specific DNA using the polymerase chain-reaction (PCR) assay and to compare the results of this with standard serological tests. DESIGN: Prospective study with laboratory investigators blinded to clinical data. SETTING: Multicenter office practice with a central reference laboratory. PATIENTS: Cerebrospinal fluid (CSF) was collected from 12 patients with acute disseminated Lyme borreliosis with less than 2 weeks of active disease. The normal control specimens came from 16 patients whose CSF samples had been sent to the clinical laboratory for tests unrelated to the present study. MAIN OUTCOME MEASURES: Clinical evidence of disease and laboratory abnormalities. RESULTS: Eight of the 12 patients (four of six with multiple areas of erythema migrans and four of six with cranial neuritis without erythema migrans) had B burgdorferi-specific DNA in their CSF. Among the 12 patients studied, nine had acute cranial neuritis and six had multiple erythema migrans lesions. Just four of the eight who were found to have spirochetal DNA in their CSF had complaints suggestive of CNS infection. In three of the PCR-positive CSF samples, no other abnormalities were noted. None of 16 samples from controls were positive in the PCR assay. CONCLUSION: B burgdorferi can invade the CNS early in the course of infection. Careful consideration should be given to choosing antibiotics that achieve adequate CSF levels in patients with disseminated infection.

Endogenous circulating DNA in systemic lupus erythematosus. Occurrence as multimeric complexes bound to histone.

Little is known about endogenous systemic lupus erythematosus (SLE) plasma DNA even though it is the presumed precursor of DNA-containing immune complexes, thought to play a central role in lupus glomerulonephritis. DNA purified from SLE plasma formed discrete bands, corresponding to sizes of about 150-200, 400, 600, and 800 bp, closely resembling the characteristic 200 bp "ladder" found with oligonucleosomal (ON) DNA. By radiolabeling DNA while in whole plasma, the very small amounts present could be further characterized. All of 24 such specimens formed two or more discrete bands on 6% PAGE. Detergent treatment of plasma resulted in a DNA migration pattern similar to that of purified DNA, suggesting disruption of DNA-protein complexes. DNA purified from authentic ON and detergent-treated ON behaved similarly. A significant portion of DNA, labeled in SLE plasma could be specifically immunoprecipitated with monoclonal antihistone antibody as was the case with ON. These immunoprecipitates, when redissolved, exhibited the expected size distribution upon PAGE. It is concluded that DNA in SLE plasma occurs as a series of multimeric complexes, at least a portion of which is noncovalently bound to histone. These results are consistent with an ON-like structure for SLE plasma DNA as had been suggested by theoretical considerations and may have important implications for its immunologic behavior in SLE and perhaps other disorders.

Plasma DNA in systemic lupus erythematosus. Characterization of cloned base sequences.

Little is known about plasma DNA in patients with systemic lupus erythematosus (SLE). Previous studies have suggested that it may, in fact, not be derived from the random nucleolysis that might be expected to accompany cell death, which might have been the simplest explanation of its origin. To extend the results of other studies, plasma DNA, obtained from a subgroup of SLE patients who had large amounts of immunoprecipitable plasma DNA, was cloned into a plasmid vector, and the nucleotide base sequences were studied by nucleic acid hybridization. One possible explanation for the anomalous renaturation kinetics of plasma DNA in SLE--namely, that it contained non-human genomic base sequences--was rendered untenable by the fact that 51 consecutive DNA clones from 2 SLE patients were each shown to contain human genomic DNA sequences. Plasma DNA from SLE patients also differed from human genomic control DNA in that those sequences derived from the highly repetitive fraction of human DNA were specifically enriched in one such sequence, Alu, which constituted 55% of the repeat sequences in the plasma DNA clones as compared with 13% in control DNA clones. There was a decrease in the frequency of non-Alu repeat sequences (9% for plasma DNA compared with 23% in control DNA). These differences were statistically significant, and they indicate that SLE plasma DNA contains a nonrandom selection of human genomic base sequences. Several explanations for these findings are considered.

Broad range DNA probes for detecting and amplifying eubacterial nucleic acids.

In this report we describe and characterize two oligomer probes that are broadly homologous to conserved eubacterial 16S ribosomal RNA (rRNA) sequences not present in human 18 rRNA or human mitochondrial 12S rRNA. One or both of the probes can detect all of 23 phylogenetically diverse eubacterial nucleic acids against which they were tested by dot blot hybridization. A sensitivity of about 1 bacterium per 10 eukaryotic cells was achieved. By using these oligomer sequences or their complements as primers in the polymerase chain reaction (PCR), the equivalent of 1 pg of E. coli DNA was detected in the presence of a large excess of eukaryotic DNA. Information useful for partial phylogenetic classification of detected organisms may be obtained by direct sequence analysis of the amplified DNA and comparison with known sequences or catalogs. Such broadly homologous probes offer advantages over more narrowly specific probes for detecting organisms whose identity is unknown. They could thus be employed for recognizing infection by organisms that cannot be cultured as may occur, for example, in tissue culture or in plant or animal diseases of unknown cause, provided the probes fail to hybridize with host nucleic acids.

PD 114,759 and PD 115,028, novel antitumor antibiotics with phenomenal potency. I. Isolation and characterization.

The isolation of two new antibiotics, PD 114,759 and PD 115,028, exhibiting in vivo antitumor activity at extremely low doses is described. The physico-chemical properties of these sulfur-containing compounds show that they represent a novel class of antitumor agents.

Circulating DNA in systemic lupus erythematosus. Isolation and characterization.

Immunoprecipitable double-stranded (dsDNA) was previously shown to persist in the circulation of a clinically recognizable subgroup of patients with systemic lupus erythematosus (SLE). Plasma from 10 such patients was subjected to a DNA isolation procedure that used a combination of proteolysis, phenol extraction, and hydroxylapatite adsorption and elution in the presence of urea. The isolated dsDNA was radiolabeled by nick translation and then characterized by isopyknic ultracentrifugation in CsCl under both neutral and alkaline conditions, as well as after digestion with S1-endonuclease. These experiments demonstrated essential identity in nucleotide base composition between the plasma-derived DNA and human genomic DNA. The presence of specific human base sequences in the plasma DNA was demonstrated by finding that authentic human genomic DNA accelerated the renaturation of plasma DNA when compared with the effect of nonhuman, control DNA. The proportion of such sequences in plasma DNA was estimated by attempting to renature the plasma DNA in the presence of human DNA under conditions shown to result in complete renaturation of human DNA in model experiments. In this way, a minimum of 47% of plasma DNA base sequences could be shown also to be present in human genomic DNA. However, an average of 10-20% of the plasma-derived DNA failed to renature under these conditions, a result that was further confirmed by comparing the renaturation of the tritium-labeled plasma DNA specimens, in double-label experiments, with internal controls consisting of 14C-labeled authentic human DNA. Attempts to drive the reaction to completion with human DNA led to a similar conclusion. The relative nonrenaturability of this fraction of plasma DNA did not appear to be attributable to extensive chain breakage, although adequate analysis of this DNA subfraction was limited by reagent availability. It was therefore concluded that, in this group of SLE patients, persistently circulating DNA consisted largely of base sequences also found in human genomic DNA. The additional presence in plasma of a DNA subfraction that differed in its renaturation behavior from human genomic DNA was recognized, although its significance could not be established with certainty.

Novel antitumor agents CI-920, PD 113,270 and PD 113,271. II. Isolation and characterization.

A complex of structurally related compounds that exhibit in vivo antileukemic activity was isolated from fermentation broths of a new streptomycete. The components of this complex are water soluble phosphate esters containing a conjugated triene system. The isolation and characterization of three of these components are described.

Circulating DNA in polyarteritis nodosa and related syndromes.

Patients with systemic lupus erythematosus (SLE) were previously reported to have a high prevalence of circulating DNA in association with systemic vasculitis. The present study was designed to determine whether circulating DNA also characterizes forms of systemic arterial inflammation other than those associated with SLE. A specific counterimmunoelectrophoresis assay capable of detecting 20-50 ng/ml of double-stranded DNA (dsDNA) in plasma was used. Of a total of 16 patients studied, 15 (94%) had circulating DNA. Similarly, 89% of the 47 plasmas from vasculitis patients contained DNA. In each of the 3 control groups, which consisted of normal individuals, patients with nonvasculitic systemic inflammation, and patients receiving high dose corticosteroid and/or cytostatic agents, no more than 8% had plasma DNA. This difference was significant with P less than 0.001. Relevance of this observation to etiologic, pathogenetic, and clinical considerations in the systemic arteritides is discussed. It is suggested that clinical detection of circulating plasma DNA may be useful for the diagnosis and management of systemic polyarteritis nodosa and related syndromes.