Anti-telomere antibodies in systemic lupus erythematosus: a new ELISA test for anti-DNA with potential pathogenetic implications.

BACKGROUND: Telomeric hexamer repeats (TTAGGG/CCCTAA)n are highly repetitive sequences of DNA. They cap the termini of eukaryotic chromosomes and stabilize them, preventing degradation or fusion. Anti ds-DNA is one of the most specific tests for systemic lupus erythematosus (SLE). Of related importance, a preliminary report has suggested that anti-telomere antibodies are also highly specific for the presence of SLE. METHODS: 220 patients with SLE, 79 with rheumatoid arthritis (RA), 54 with other rheumatic diseases and 99 healthy controls were tested for anti-telomere antibody as measured by enzyme immunoassay detecting 30- and 60-mer telomeric repeats (5-10 hexamers). 48 of the 220 SLE patients charts were abstracted for 90 separate clinical, laboratory and treatment parameters. Comparisons were made between SLE and non-SLE patients, and within the lupus group for telomere positivity and among the latter 48 patients for anti-DNA (Farr) levels and SLEDAI scores. RESULTS: Anti-telomere antibody was present in 48.6% of the overall SLE group (220), 71% of our cohort (48), 11% with primary Sjogren's (2/18), 7. 6% with RA (6/79) and 2% of normal controls (2/99) (P<0.001 comparing SLE to all other groups). In the 48 patient cohort, anti-telomere antibody was more sensitive than anti-dsDNA (Farr) (71% vs 50%), but did not correlate with other clinical parameters, SLEDAI scores, or other autoantibodies. CONCLUSIONS: The detection of anti-telomere antibody appears to be more sensitive and may be as specific as anti-dsDNA (Farr) in SLE. The detection of telomeric repeats may be as accurate as other anti-DNA assay methodologies and more specific for the presence of SLE. The immunogenic potential of telomere biology related to the pathogenesis and/or diagnosis of SLE deserves further investigation.

A computer analysis of primer and probe hybridization potential with bacterial small-subunit rRNA sequences.

Analysis of restriction fragment length polymorphism of bacterial small-subunit (SSU) rRNA sequences represents a potential means for characterizing complex bacterial populations such as those found in natural environments. In order to estimate the resolution potential of this approach, we have examined the SSU rRNA sequences in the Ribosomal Database Project bank using a computer algorithm which simulates hybridization between DNA sequences. Simulated hybridizations between a primer or probe sequence and an SSU rRNA sequence yield a value for each potential hybridization. This algorithm has been used to evaluate sites for PCR primers and hybridization probes used for classifying SSU rRNA sequences. Our analysis indicates that length variation in terminal restriction fragments of PCR products from the SSU rRNA sequences can identify a wide spectrum of bacteria. We also observe that the majority of restriction fragment length variation is the result of insertions and deletions rather than restriction site polymorphisms. This approach is also used to evaluate the relative efficiency and specificity of a number of published hybridization probes.

Molecular technique for rapid identification of mycobacteria.

Identification of mycobacteria through conventional microbiological methods is cumbersome and time-consuming. Recently we have developed a novel bacterial identification method to accurately and rapidly identify different mycobacteria directly from water and clinical isolates. The method utilizes the PCR to amplify a portion of the small subunit rRNA from mycobacteria. The 5' PCR primer has a fluorescent label to allow detection of the amplified product. The PCR product is digested with restriction endonucleases, and an automated DNA sequencer is employed to determine the size of the labeled restriction fragments. Since the PCR product is labeled only at the 5' end, the analysis identifies only the restriction fragment proximal to the 5' end. Each mycobacterial species has a unique 5' restriction fragment length for each specific endonuclease. However, frequently the 5' restriction fragments from different species have similar or identical lengths for a given endonuclease. A set of judiciously chosen restriction enzymes produces a unique set of fragments for each species, providing us with an identification signature. Using this method, we produced a library of 5' restriction fragment sizes corresponding to different clinically important mycobacteria. We have characterized mycobacterial isolates which had been previously identified by biochemical test and/or nucleic acid probes. An analysis of these data demonstrates that this protocol is effective in identifying 13 different mycobacterial species accurately. This protocol has the potential of rapidly (less than 36 h) identifying mycobacterial species directly from clinical specimens. In addition, this protocol is accurate, sensitive, and capable of identifying multiple organisms in a single sample.

A molecular technique for identification of bacteria using small subunit ribosomal RNA sequences.

We have recently developed a novel molecular technique for identification of specific bacterial species within a complex mixture. The technique uses PCR to amplify small subunit ribosomal RNA (SSU rRNA) genes from a mixture of bacteria. One of the PCR primers is labeled with a fluorescent dye to allow detection of the amplified product. The PCR product is then digested with restriction enzymes and a capillary electrophoresis unit equipped with a laser-induced fluorescence detector is employed to analyze the restriction fragments. Only restriction fragments that contain the fluorescent-labeled primer are detected. Generally, the nucleotide sequence of the SSU rRNA genes is unique for each bacterial species. Consequently, the fluorescent-labeled restriction fragments from different bacterial species often have characteristic lengths. Thus, the different fluorescent peaks that appear in a capillary electropherogram correspond to labeled restriction fragments from different bacterial species. This protocol allows us to identify a number of different bacterial species in a complex mixture. Only a minute sample of bacterial DNA and a minimal amount of time (8-10 h) are required for this analysis. The protocol is sensitive, rapid and capable of identifying a broad spectrum of bacterial species.