Biomedical subjects

P M Rumore

Publications and source records attributed to P M Rumore.

5 recordsLinked to original sources

PubMed1997

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.

Bacterial Infections↗

PubMed1997

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.

DNA↗

PubMed1995

Glomerular uptake of nucleosomes: evidence for receptor-mediated mesangial cell binding.

DNA-containing immune complexes (IC) are believed to have a central causal role in the glomerulonephritis of systemic lupus erythematosus. Extracellular DNA which provides the antigenic source for these ICs circulates as oligonucleosomes (ON). The in vivo glomerular uptake of radiolabeled ON in rats, as well as its binding by cultured rat mesangial cells, was examined. The data show that the binding of ON to kidney, and specifically glomeruli, was almost fourfold greater than that of purified DNA. Uptake appeared dose-dependent and saturable, while there were no differences in hepatic or splenic uptake. Most of the nucleosomal DNA recovered from glomeruli was TCA-precipitable, and on gel electrophoresis was about 100 to 300 bp, a size sufficient to allow formation of large ICs. In vitro studies demonstrated that ON are bound by cultured mesangial cells in a dose-dependent and saturable manner, with a dissociation constant of 1.25 x 10(-10) M/liter and 750 binding sites per cell. Autoradiography of cell cultures incubated with radiolabeled ON showed deposition along the plasma membrane which was inhibited by excess unlabeled ON. The data show that binding of ON to glomeruli exceeds that of purified DNA and may be mediated by histones. ON bind to mesangial cells in a receptor-mediated fashion. The data support the hypothesis of in situ formation of DNA-containing ICs and suggest a role for the mesangial cell in lupus glomerulonephritis.

Animals↗

PubMed1994

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.

Adult↗

PubMed1990

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.

DNA↗