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Biomedical subjects

D C Hooper

Publications and source records attributed to D C Hooper.

At least 37 records · Page 2Linked to original sources
PubMed2001

The role of uric acid in protection against peroxynitrite-mediated pathology.

Peroxynitrite, the product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of inflammatory CNS disorders. Uric acid, an effective scavenger of peroxynitrite, is a purine metabolite present at high levels in the serum of hominoids relative to lower-order animals due to the functional deletion of urate oxidase. Raising the normally low levels of uric acid in mice is therapeutic for experimental allergic encephalomyelitis, an animal model of multiple sclerosis. This therapeutic activity of uric acid is associated with the inhibition of peroxynitrite-induced tissue damage, blood-CNS barrier permeability changes, and CNS inflammation. Based on these findings we have concluded that peroxynitrite has an important role in promoting enhanced vascular permeability and inflammatory cell extravasation. We hypothesize that higher uric acid levels in hominoids evolved to protect against this process.

Animals↗
PubMed2001

Contamination of gowns, gloves, and stethoscopes with vancomycin-resistant enterococci.

OBJECTIVE: [corrected] To measure directly the rate of contamination, during routine patient examination, of gowns, gloves, and stethoscopes with vancomycin-resistant enterococci (VRE). SETTING: A large, academic, tertiary-care hospital. PATIENTS: Between January 1997 and December 1998, 49 patients colonized or infected with VRE were entered in the study. DESIGN: After routine examination, the examiner's glove fingertips, gown (the umbilical region and the cuffs), and stethoscope diaphragm were pressed onto Columbia colistin-nalidixic acid (CNA) agar plates with 5% sheep blood plus vancomycin 6 pg/mL. The stethoscope diaphragm was sampled again after cleaning with a 70% isopropanol wipe. RESULTS: VRE were isolated from at least 1 examiner site (gloves, gowns, or stethoscope) in 33 (67%) of 49 cases. Gloves were contaminated in 63%, gowns in 37%, and stethoscopes in 31%. All three items were positive for VRE in 24%. One case each had stethoscope and gown contamination without glove contamination. Only 1 (2%) of 49 stethoscopes was positive after wiping with an alcohol swab. Contamination at any site was more likely when the patient had a colostomy or ileostomy. Patients identified by rectal-swab culture alone were as likely to contaminate their examiners as were those identified by clinical specimens. CONCLUSIONS: Our study revealed a high rate of examiner contamination with VRE. The similar risk of contamination identified by surveillance and clinical cases reinforces concerns that patients not known to be colonized with VRE could serve as sources for dissemination. Wiping with alcohol is effective in decontaminating stethoscopes.

Academic Medical Centers↗
PubMed2001

Mechanisms and frequency of resistance to gatifloxacin in comparison to AM-1121 and ciprofloxacin in Staphylococcus aureus.

Gatifloxacin, an 8-methoxyfluoroquinolone, was found to be two- to fourfold more active against wild-type Staphylococcus aureus ISP794 than its desmethoxy derivative, AM-1121, and ciprofloxacin, another desmethoxy fluoroquinolone. Single grlBA mutations caused two- to fourfold increases in the MIC of gatifloxacin, and a single gyrase mutation was silent. Double mutations in gyrA and grlA or grlB caused a 32-fold increase in the MIC of gatifloxacin, in contrast to a 128-fold increase for ciprofloxacin and AM-1121. Overexpression of the NorA efflux pump had minimal effect on the MIC of gatifloxacin. The bactericidal activity of the three quinolones at four times the MIC differed only for a double mutant, with gatifloxacin exhibiting a killing pattern similar to that for ISP794, whereas ciprofloxacin and AM-1121 failed to show any killing. With gatifloxacin, selection of resistant mutants at twice the MIC was 100- to 1,000-fold less frequent than with the comparison quinolones, and mutants could rarely be selected at four times the MIC. The limit resistance in ISP74 was 512 times the MIC of gatifloxacin and 1,024 times the MICs of ciprofloxacin and AM-1121. Novel mutations in topoisomerase IV were selected in five of the six single-step mutants, three of which were shown to cause quinolone resistance by genetic studies. In conclusion, topoisomerase IV is the primary target of gatifloxacin. In contrast to comparison quinolones, mutations in both topoisomerase IV and gyrase are required for resistance to gatifloxacin by clinical breakpoints and do not abolish bactericidal effect, further supporting the benefit of the 8-methoxy substituent in gatifloxacin.

Anti-Infective Agents↗
PubMed2001

A mutation in the 5' untranslated region increases stability of norA mRNA, encoding a multidrug resistance transporter of Staphylococcus aureus.

NorA, a multidrug efflux pump in Staphylococcus aureus, protects the cell from multiple drugs, including quinolones. The flqB mutation (T-->G) in the 5' untranslated region upstream of norA causes norA overexpression of 4.9-fold in cis, as measured in norA::blaZ fusions. The transcriptional initiation site of norA was unchanged in mutant and wild-type strains, but the half-life of norA mRNA was increased 4.8-fold in the flqB mutant compared to the wild-type strain. Computer-generated folding of the first 68 nucleotides of the norA transcript predicts an additional stem-loop and changes in a putative RNase III cleavage site in the flqB mutant.

5' Untranslated Regions↗
PubMed2001

Overexpression of cytochrome C by a recombinant rabies virus attenuates pathogenicity and enhances antiviral immunity.

The pathogenicity of individual rabies virus strains appears to correlate inversely with the extent of apoptotic cell death they induce and with the expression of rabies virus glycoprotein, a major inducer of an antiviral immune response. To determine whether the induction of apoptosis by rabies virus contributes to a decreased pathogenicity by stimulating antiviral immunity, we have analyzed these parameters in tissue cultures and in mice infected with a recombinant rabies virus construct that expresses the proapoptotic protein cytochrome c. The extent of apoptosis was strongly increased in primary neuron cultures infected with the recombinant virus carrying the active cytochrome c gene [SPBN-Cyto c(+)], compared with cells infected with the recombinant virus containing the inactive cytochrome c gene [SPBN-Cyto c(-)]. Mortality in mice infected intranasally with SPBN-Cyto c(+) was substantially lower than in SPBN-Cyto c(-)-infected mice. Furthermore, virus-neutralizing antibody (VNA) titers were significantly higher in mice immunized with SPBN-Cyto c(+) at the same dose. The VNA titers induced by these recombinant viruses paralleled their protective activities against a lethal rabies virus challenge infection, with SPBN-Cyto c(+) revealing an effective dose 20 times lower than that of SPBN-Cyto c(-). The strong increase in immunogenicity, coupled with the marked reduction in pathogenicity, identifies the SPBN-Cyto c(+) construct as a candidate for a live rabies virus vaccine.

Animals↗
PubMed2001

Uric acid levels in patients with multiple sclerosis: analysis in mono- and dizygotic twins.

Presence of nitrotyrosine in cells surrounding plaques indicates that peroxynitrite may be the cause of brain lesions in multiple sclerosis. Low levels of uric acid, a natural scavenger of peroxynitrite, were demonstrated in blood of patients with multiple sclerosis in comparison with control individuals. These observations were now extended to 132 sets of twins with one sibling affected by multiple sclerosis. In blood of both mono- and dizygotic twins the uric acid levels were lower in the twin with the disease than in the healthy twin.

Female↗
PubMed2001

Inactivation of peroxynitrite in multiple sclerosis patients after oral administration of inosine may suggest possible approaches to therapy of the disease.

Peroxynitrite has been implicated in the pathogenesis of multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE). Previously, we have shown that administration of uric acid (UA), a peroxynitrite scavenger, is therapeutic in EAE We have also shown that MS patients have lower levels of serum uric acid than healthy individuals or those with other neurological diseases. The aim of this investigation was therefore to raise serum UA levels in MS patients. Oral administration of UA failed to increase low serum UA levels, evidently due to its degradation by gastrointestinal bacteria. However, serum UA could be raised and maintained at elevated levels for a year and more without reported side-effects by oral administration of its precursor inosine. Three of 11 patients given inosine showed some evidence of clinical improvement and there was no sign of disease progression in the remaining patients. Gadolinium-enhanced lesions, observed in two patients before receiving inosine, could not be detected after either 10 or IS months inosine treatment These data provide evidence that serum UA levels can be readily manipulated and that the benefit of higher levels to individuals with MS should be studied further in greater number of patients.

Administration, Oral↗
PubMed2000

The peroxynitrite scavenger uric acid prevents inflammatory cell invasion into the central nervous system in experimental allergic encephalomyelitis through maintenance of blood-central nervous system barrier integrity.

Uric acid (UA), a product of purine metabolism, is a known scavenger of peroxynitrite (ONOO(-)), which has been implicated in the pathogenesis of multiple sclerosis and experimental allergic encephalomyelitis (EAE). To determine whether the known therapeutic action of UA in EAE is mediated through its capacity to inactivate ONOO(-) or some other immunoregulatory phenomenon, the effects of UA on Ag presentation, T cell reactivity, Ab production, and evidence of CNS inflammation were assessed. The inclusion of physiological levels of UA in culture effectively inhibited ONOO(-)-mediated oxidation as well as tyrosine nitration, which has been associated with damage in EAE and multiple sclerosis, but had no inhibitory effect on the T cell-proliferative response to myelin basic protein (MBP) or on APC function. In addition, UA treatment was found to have no notable effect on the development of the immune response to MBP in vivo, as measured by the production of MBP-specific Ab and the induction of MBP-specific T cells. The appearance of cells expressing mRNA for inducible NO synthase in the circulation of MBP-immunized mice was also unaffected by UA treatment. However, in UA-treated animals, the blood-CNS barrier breakdown normally associated with EAE did not occur, and inducible NO synthase-positive cells most often failed to reach CNS tissue. These findings are consistent with the notion that UA is therapeutic in EAE by inactivating ONOO(-), or a related molecule, which is produced by activated monocytes and contributes to both enhanced blood-CNS barrier permeability as well as CNS tissue pathology.

Animals↗
PubMed2000

Protection of myelin basic protein immunized mice from free-radical mediated inflammatory cell invasion of the central nervous system by the natural peroxynitrite scavenger uric acid.

Peroxynitrite (ONOO(-)), the product of nitric oxide (NO(radical)) and superoxide (O(2)(-radical)), is believed to be a major contributor to immunotoxicity when produced by activated cells expressing inducible nitric oxide synthase (iNOS). Uric acid (UA) is a natural scavenger of ONOO(-) that is present at high levels in the sera of humans and other higher order primates relative to most lower mammals. We have previously shown that UA treatment is therapeutic in experimental allergic encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). In this study we have examined the effect of UA therapy on the dynamics of the appearance of iNOS-positive cells in central nervous system (CNS) tissue of mice subjected to the stimuli that cause EAE. The results indicate that UA prevents activated monocytes from entering CNS tissue where they may contribute to the pathogenesis of MS and other CNS diseases.

Animals↗
PubMed2000

The development of monoclonal human rabies virus-neutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure.

To provide a more defined and safer replacement for the human rabies immune globulin (HRIG) from pooled serum which is currently used for treatment of exposure to rabies virus we have developed a series of human rabies virus-specific monoclonal antibodies. Mouse-human heterohybrid myeloma cells producing rabies virus-specific human monoclonal antibodies were prepared using B cells obtained from volunteers recently-immunized with a commercial rabies virus vaccine (HDCV). Cell lines producing antibody which neutralized the Evelyn-Rokitnicki-Abelseth (ERA) rabies virus strain in vitro were cloned and the resulting monoclonal antibodies characterized for isotype, specificity against a variety of rabies virus isolates, and neutralization capacity. The ability of the monoclonal antibodies to neutralize a variety of rabies virus strains in vitro correlated with their binding specificity for these viruses in an enzyme-linked immunoadsorbant assay (ELISA). A number of these antibodies have proven suitable for the formulation of a prophylactic human monoclonal antibody-based reagent which would provide significant advantages to the HRIG in having defined, reproducible specificity, lessened possibility of contamination with viral pathogens, and consistent availability.

Antibodies, Monoclonal↗
PubMed2000

Mechanisms of action and resistance of older and newer fluoroquinolones.

The fluoroquinolones interact with 2 bacterial targets, the related enzymes DNA gyrase and topoisomerase IV, both of which are involved in DNA replication. Quinolones form complexes of these enzymes with DNA, complexes that block movement of the DNA-replication fork and thereby inhibit DNA replication. Many older quinolones differ in their relative activities against gyrase and topoisomerase IV in a bacterial cell, having greater potency against gyrase than against topoisomerase IV in many gram-negative bacteria and greater potency against topoisomerase IV than against gyrase in many gram-positive bacteria. Several newer quinolones appear to have more closely balanced activity against these enzymes. Resistance to fluoroquinolones occurs as a result of mutational amino acid substitutions in the subunits of the more sensitive (or primary-target) enzyme within the cell. If, however, both enzymes are similarly susceptible to a fluoroquinolone, then the level of resistance caused by a primary-target mutation may be low and may be limited by the sensitivity of the secondary target. Fluoroquinolones also differ in the extent to which common bacterial multidrug efflux pumps affect their activity, with some compounds being unaffected by resistance mechanisms because of overexpression of such pumps. Newer fluoroquinolone interaction with dual targets and avoidance of efflux-resistance mechanisms may each contribute to the lower frequencies of selection of resistant mutants in the laboratory.

Anti-Infective Agents↗
PubMed2000

Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis.

Peroxynitrite (ONOO(-)), a toxic product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of CNS inflammatory diseases, including multiple sclerosis and its animal correlate experimental autoimmune encephalomyelitis (EAE). In this study we have assessed the mode of action of uric acid (UA), a purine metabolite and ONOO(-) scavenger, in the treatment of EAE. We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease. In mice with active EAE, exogenously administered UA penetrates the already compromised blood-CNS barrier, blocks ONOO(-)-mediated tyrosine nitration and apoptotic cell death in areas of inflammation in spinal cord tissues and promotes recovery of the animals. Moreover, UA treatment suppresses the enhanced blood-CNS barrier permeability characteristic of EAE. We postulate that UA acts at two levels in EAE: 1) by protecting the integrity of the blood-CNS barrier from ONOO(-)-induced permeability changes such that cell invasion and the resulting pathology is minimized; and 2) through a compromised blood-CNS barrier, by scavenging the ONOO(-) directly responsible for CNS tissue damage and death.

Animals↗
PubMed2000

Mechanisms and frequency of resistance to premafloxacin in Staphylococcus aureus: novel mutations suggest novel drug-target interactions.

Premafloxacin is a novel 8-methoxy fluoroquinolone with enhanced activity against Staphylococcus aureus. We found premafloxacin to be 32-fold more active than ciprofloxacin against wild-type S. aureus. Single mutations in either subunit of topoisomerase IV caused a four- to eightfold increase in the MICs of both quinolones. A double mutation (gyrA and either grlA or grlB) caused a 32-fold increase in the MIC of premafloxacin, while the MIC of ciprofloxacin increased 128-fold. Premafloxacin appeared to be a poor substrate for NorA, with NorA overexpression causing an increase of twofold or less in the MIC of premafloxacin in comparison to a fourfold increase in the MIC of ciprofloxacin. The frequency of selection of resistant mutants was 6.4 x 10(-10) to 4.0 x 10(-7) at twofold the MIC of premafloxacin, 2 to 4 log(10) less than that with ciprofloxacin. Single-step mutants could not be selected at higher concentrations of premafloxacin. In five single-step mutants, only one previously described uncommon mutation (Ala116Glu), and four novel mutations (Arg43Cys, Asp69Tyr, Ala176Thr, and Pro157Leu), three of which were outside the quinolone resistance-determining region (QRDR) were found. Genetic linkage studies, in which incross of grlA(+) and outcross of mutations were performed, showed a high correlation between the mutations and the resistance phenotypes, and allelic exchange experiments confirmed the role of the novel mutations in grlA in resistance. Our results suggest that although topoisomerase IV is the primary target of premafloxacin, premafloxacin appears to interact with topoisomerase IV in a manner different from that of other quinolones and that the range of the QRDR of grlA should be expanded.

Alleles↗
PubMed2000

Induction of fibronectin-binding proteins and increased adhesion of quinolone-resistant Staphylococcus aureus by subinhibitory levels of ciprofloxacin.

We recently reported that strain EN1252a, a fluoroquinolone-resistant derivative of Staphylococcus aureus NCTC8325 with mutations in grlA and gyrA, expressed increased levels of fibronectin-binding proteins (FnBPs) and showed a significantly higher attachment to fibronectin-coated polymer surfaces after growth in the presence of subinhibitory concentrations of ciprofloxacin. The present study evaluated the occurrence and frequency of fluoroquinolone-induced FnBP-mediated adhesion in clinical isolates of fluoroquinolone-resistant methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). Eight of ten MRSA isolates and four of six MSSA isolates with grlA and gyrA mutations exhibited significant increases in attachment to fibronectin-coated surfaces after growth in the presence of one-quarter the MIC of ciprofloxacin. Fluoroquinolone-induced FnBP-mediated adhesion of one clinical MRSA strain and the double mutant strain EN1252a also occurred on coverslips removed from the subcutaneous space of guinea pigs. For strain EN1252a, the regulation of fnb transcription by sub-MICs of ciprofloxacin was studied on reporter plasmids carrying fnb-luxAB fusions. One-quarter of the MIC of ciprofloxacin significantly increased fnbB, but not fnbA, promoter activity of the fluoroquinolone-resistant mutant but not its fluoroquinolone-susceptible parent ISP794. This response was abolished by pretreatment with rifampin, indicating an effect at the level of transcription. Activation of the fnbB promoter was not due to an indirect effect of ciprofloxacin on growth rate and still occurred in an agr mutant of strain EN1252a. These data suggest that sub-MIC levels of ciprofloxacin activate the fnbB promoter of some laboratory and clinical isolates, thus contributing to increased production of FnBP(s) and leading to higher levels of bacterial attachment to fibronectin-coated or subcutaneously implanted coverslips.

Anti-Infective Agents↗
PubMed2000

Selective targeting of topoisomerase IV and DNA gyrase in Staphylococcus aureus: different patterns of quinolone-induced inhibition of DNA synthesis.

The effect of quinolones on the inhibition of DNA synthesis in Staphylococcus aureus was examined by using single resistance mutations in parC or gyrA to distinguish action against gyrase or topoisomerase IV, respectively. Norfloxacin preferentially attacked topoisomerase IV and blocked DNA synthesis slowly, while nalidixic acid targeted gyrase and inhibited replication rapidly. Ciprofloxacin exhibited an intermediate response, consistent with both enzymes being targeted. The absence of RecA had little influence on target choice by this assay, indicating that differences in rebound (repair) DNA synthesis were not responsible for the results. At saturating drug concentrations, norfloxacin and a gyrA mutant were used to show that topoisomerase IV-norfloxacin-cleaved DNA complexes are distributed on the S. aureus chromosome at intervals of about 30 kbp. If cleaved complexes block DNA replication, as indicated by previous work, such close spacing of topoisomerase-quinolone-DNA complexes should block replication rapidly (replication forks are likely to encounter a cleaved complex within a minute). Thus, the slow inhibition of DNA synthesis at growth-inhibitory concentrations suggests that a subset of more distantly distributed complexes is physiologically relevant for drug action and is unlikely to be located immediately in front of the DNA replication fork.

Anti-Infective Agents↗
PubMed2000

A new two-component regulatory system involved in adhesion, autolysis, and extracellular proteolytic activity of Staphylococcus aureus.

A transposition mutant of Staphylococcus aureus was selected from the parent strain MT23142, a derivative of strain 8325. The site of transposition was near the 5' terminus of the gene arlS. ArlS exhibits strong similarities with histidine protein kinases. Sequence analysis suggested that arlS forms an operon with upstream gene arlR. The predicted product of arlR is a member of the OmpR-PhoB family of response regulators. The arlS mutant formed a biofilm on a polystyrene surface unlike the parent strain and the complemented mutant. Biofilm formation was associated with increased primary adherence to polystyrene, whereas cellular adhesion was only slightly decreased. In addition, the arlS mutant exhibited increased autolysis and altered peptidoglycan hydrolase activity compared to the parental strain and to the complemented mutant. As it has been shown for coagulase-negative staphylococci that some autolysins are able to bind polymer surfaces, these data suggest that the two-component regulatory system ArlS-ArlR may control attachment to polymer surfaces by affecting secreted peptidoglycan hydrolase activity. Finally, the arlS mutant showed a dramatic decrease of extracellular proteolytic activity, including serine protease activity, in comparison to the wild-type strain and the complemented mutant, and cells grown in the presence of phenylmethylsulfonyl fluoride (a serine protease inhibitor) showed an increased autolysin activity. Since the locus arlR-arlS strikingly modifies extracellular proteolytic activity, this locus might also be involved in the virulence of S. aureus.

Amino Acid Sequence↗
PubMed2000

Expression of the multidrug resistance transporter NorA from Staphylococcus aureus is modified by a two-component regulatory system.

To dissect genetically the regulation of NorA, a multidrug transporter of Staphylococcus aureus, we analyzed the differential expression of the norA promoter using a transcriptional fusion with a beta-lactamase reporter gene. Expression studies with an arlS mutant revealed that the norA promoter is ArlS dependent. The arlR-arlS locus was shown to code for a two-component regulatory system. The protein ArlR has strong similarity to response regulators, and ArlS has strong similarity to protein histidine kinases. We have also analyzed the 350-bp region upstream of the Shine-Dalgarno sequence of norA by gel mobility shift experiments. It was shown that only the 115-bp region upstream of the promoter was necessary for multiple binding of an 18-kDa protein. From transcriptional fusions, we have localized four different putative boxes of 6 bp, which appear to play a role in the binding of the 18-kDa protein and in the up-regulation of norA expression in the presence of the arlS mutation. Furthermore, the gel mobility shift of the 18-kDa protein was modified in the presence of the arlS mutation, and the arlS mutation altered the growth-phase regulation of NorA. These results indicate that expression of norA is modified by a two-component regulatory system.

Bacterial Proteins↗