Search PubMed⌕ Search

Biomedical subjects

D C Hooper

Publications and source records attributed to D C Hooper.

At least 91 records · Page 5Linked to original sources
PubMed1995

Spleen cells from antigen-minimized mice are superior to spleen cells from germ-free and conventional mice in the stimulation of primary in vitro proliferative responses to nominal antigens.

T lymphocytes from mice reared under conditions of differential exposure to food, environmental and microbial antigens were compared for phenotypic shifts that may be associated with prior exposure to antigens as well as functional variations in the ability to respond to antigens de novo. While the intra-epithelial CD8 T cell compartment was found to differ significantly in the type of T cell receptor predominantly expressed, CD4 T cells from various lymphoid organs of conventionally reared specific pathogen-free (CL-SPF) mice showed only subtle phenotypic differences from cells obtained from antigen-minimized germ-free (AF) and germ-free (GF) mice. Cells derived from mice exposed to a reduced antigen load exhibited primary in vitro proliferative responses to antigens such as dinitrophenyl-keyhole limpet hemocyanin which were significantly enhanced when compared with similar responses of cells from conventional mice. In cell mixing experiments, differences in the reactivity of T cells from the spleens of AF, GF and CL-SPF mice were dependent on the source of the spleen cells employed as antigen-presenting cells (APC). Experiments in which the T cell population was held constant revealed that, as APC, spleen cells from AF mice were most often superior to spleen cells from GF mice which were in turn considerably better than a similar population from SPF mice. We conclude that the enhanced primary reactivity of spleen cells from AF mice to nominal antigen in vitro is likely to be the result of a difference in the function and/or regulatory activities of the cell population employed as APC in this investigation.

Animals↗
PubMed1995

Expression of C1q, a subcomponent of the rat complement system, is dramatically enhanced in brains of rats with either Borna disease or experimental allergic encephalomyelitis.

In situ hybridization, RT-PCR and Northern blot analysis as well immunohistochemistry were used to examine the expression of C1q, a subcomponent of the rat complement system, in brains of rats infected with Borna disease virus (BDV) and rats afflicted with experimental allergic encephalomyelitis (EAE) induced by the adoptive transfer of myelin basic protein specific T cells. C1q mRNA, which was not detected in normal brain, became clearly detectable using RT-PCR analysis by d14 post infection (p.i.) with BDV. Maximal levels of C1q mRNA were reached 21 days p.i. when inflammatory reactions in the brain were also at a peak. Similarly, C1q mRNA was elevated when the clinical symptoms of EAE became evident 5 days following cell transfer. C1q positive cells, as identified by immunohistology, were preferentially localized in grey and white matter of the hippocampus and basolateral cortex. The C1q positive cells resembled microglial cells in morphology. The correlation of C1q expression with the development of neurological disease as well as the dramatic increase of C1q within brain regions with inflammatory lesions suggest that local biosynthesis of C1q may play a role in the pathogenesis of Borna virus induced and autoimmune encephalomyelitis.

Animals↗
PubMed1995

Expression of the rabies virus glycoprotein in transgenic tomatoes.

We have engineered tomato plants (Lycopersicon esculentum Mill var. UC82b) to express a gene for the glycoprotein (G-protein), which coats the outer surface of the rabies virus. The recombinant constructs contained the G-protein gene from the ERA strain of rabies virus, including the signal peptide, under the control of the 35S promoter of cauliflower mosaic virus. Plants were transformed by Agrobacterium tumefaciens-mediated transformation of cotyledons and tissue culture on selective media. PCR confirmed the presence of the G-protein gene in plants surviving selection. Northern blot analysis indicated that RNA of the appropriate molecular weight was produced in both leaves and fruit of the transgenic plants. The recombinant G-protein was immunoprecipitated and detected by Western blot from leaves and fruit using different antisera. The G-protein expressed in tomato appeared as two distinct bands with apparent molecular mass of 62 and 60 kDa as compared to the 66 kDa observed for G-protein from virus grown in BHK cells. Electron microscopy of leaf tissue using immunogold-labeling and antisera specific for rabies G-protein showed localization of the G-protein to the Golgi bodies, vesicles, plasmalemma and cell walls of vascular parenchyma cells. In light of our previous demonstration that orally administered rabies G-protein from the same ERA strain elicits protective immunity in animals, these transgenic plants should provide a valuable tool for the development of edible oral vaccines.

Agrobacterium tumefaciens↗
PubMed1995

Sequential mutations of gyrA in Escherichia coli associated with quinolone therapy.

A clinical isolate of Escherichia coli HM73 (MIC norfloxacin 2 mg/L) was isolated during norfloxacin therapy from an urinary tract infection in a patient who had been previously treated with pipemidic acid and infected by E. coli HM72 (norfloxacin 0.25), known to harbour a substitution Ser 83-->Leu in the gyrA gene. No difference in accumulation of norfloxacin was found between the two strains. DNA gyrases were isolated by affinity chromatography and assayed for supercoiling activity in the presence of norfloxacin. The minimal effective doses (MEDs) were 20 mg/L, for HM72 and 80 for HM73. DNA sequencing identified in HM73, two mutations leading to substitutions Ser 83 to Leu and Asp 87 to Gly.

Aged↗
PubMed1995

Detection of gyrA and gyrB mutations in quinolone-resistant clinical isolates of Escherichia coli by single-strand conformational polymorphism analysis and determination of levels of resistance conferred by two different single gyrA mutations.

Twelve quinolone-resistant clinical isolates of Escherichia coli (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.25 to 8 micrograms/ml) were transformed with plasmid pJSW101 carrying the gyrA+ gene and with plasmid pJB11 carrying the gyrB+ gene to examine the proportion of gyrA and gyrB mutations. Transformation with pJSW101 resulted in complementation (nalidixic acid MICs, 4 to 32 micrograms/ml; norfloxacin MICs, 0.06 to 0.25 micrograms/ml). In contrast, no change in MICs were observed after transformation with pJB11. A 418-bp fragment of gyrA from the 12 strains was amplified by PCR. Direct DNA sequencing of that fragment identified the causes of quinolone resistance in eight strains as a single point mutation leading to a substitution of the serine at position 83 (Ser-83) to Leu and in four strains as a single point mutation leading to a substitution of Asp-87 to Gly. Exchange of the fragment from one of these strains with that of gyrA+ and transformation of resistance with the hybrid gyrA plasmid indicated the contribution of Gly-87 to resistance and the stabilities of mutants containing GyrA (Gly-87). Thus, gyrA gene mutations are probably encountered more often than gyrB gene mutations in clinical isolates of E. coli. In addition, the substitution of Asp-87 to Gly can be encountered in such strains. On the basis of the level of resistance found in the fragment exchange experiment, the quinolone resistance attributable to Gly-87 appears to be comparable to that attributable to Leu-83. The levels of resistance found in the clinical isolates shown to have a Gly-87 mutation (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.5 to 4 micrograms/ml) suggest that the Gly-87 mutation causes resistance at the level of the nalidixic acid MIC (64 micrograms/ml) or the norfloxacin MIC (0.5 micrograms/ml or less) and that the additional increments in resistance seen in the other strains with higher levels of resistance may be attributable to additional mutations. The single-strand conformational polymorphism analysis with PCR products readily detected te Leu-83 and Gly-87 mutations.

Amino Acid Sequence↗
PubMed1995

Quinolone mode of action.

Physical studies have further defined interactions of quinolones with their principal target, DNA gyrase. The binding of quinolones to the DNA gyrase-DNA complex suggests 2 possible binding sites of differing affinities. Mutations in either the gyrase A gene (gyrA) or the gyrase B gene (gyrB) that affect quinolone susceptibility also affect drug binding, with resistance mutations causing decreased binding and hypersusceptibility mutations causing increased binding. Combinations of mutations in both GyrA and GyrB have further demonstrated the contribution of both subunits to the quinolone sensitivity of intact bacteria and purified DNA gyrase. A working model postulates initial binding of quinolones to proximate sites on GyrA and GyrB. This initial binding then produces conformational changes that expose additional binding sites, possibly involving DNA. Quinolones also inhibit the activities of Escherichia coli topoisomerase IV (encoded by the parC and parE genes), but at concentrations higher than those inhibiting DNA gyrase. The patterns of resistance mutations in gryA and parC suggest that topoisomerase IV may be a secondary drug target in E. coli and Neisseria gonorrhoeae. In contrast, in Staphylococcus aureus these patterns suggest that topoisomerase IV may be a primary target of quinolone action. Regulation of expression of membrane efflux transporters may contribute to quinolone susceptibility in both Gram-positive and Gram-negative bacteria. The substrate profile of the NorA efflux transporter of S. aureus correlates with the extent to which the activity of quinolone substrates is affected by overexpression of NorA. In addition, the Emr transporter of E. coli affects susceptibility to nalidixic acid, and the MexAB OprK transport system of Pseudomonas aeruginosa affects susceptibility to ciprofloxacin.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Quinolones↗
PubMed1994

Rabies ribonucleocapsid as an oral immunogen and immunological enhancer.

The administration of rabies ribonucleocapsid (RNP) by oral as well as parenteral routes was found to prime specific T cells and elicit N-protein-specific antibodies. per os and intramuscular immunization led to the production of antibodies of the IgA and IgG isotypes, respectively. Mice primed orally with RNP produced significantly enhanced amounts of virus-neutralizing antibody, compared with non-immune controls, upon subsequent parenteral booster immunization with inactivated rabies virus. Thus oral immunization with rabies RNP primed cells capable of mediating a secondary systemic response to rabies virus. The results of experiments in which peptide and protein antigens were administered either physically coupled to or mixed with RNP indicate that RNP has an inherent capacity to enhance immune responses.

Adjuvants, Immunologic↗
PubMed1994

Spontaneous proliferation of Peyer's patch cells in vitro.

Under normal circumstances most lymphoid cell populations do not exhibit strong proliferative reactions in culture unless provoked by antigen or mitogen. The autologous mixed lymphocyte reaction (AMLR) mediated by adult T cells is a relatively weak proliferative response that occurs in the absence of known heterologous stimuli. In this investigation we demonstrate that Peyer's patch (PP) cells possess an inherent capacity to commence dividing in vitro and to display an exceptionally vigorous AMLR. The magnitude and kinetics of this spontaneous proliferation resemble that of a secondary response to a strong mucosal immunogen such as reovirus type 1/Lang. Analysis of the cellular components of the PP cultures implicates CD4+CD8- T cells as the major responding population and dendritic cells (DC) as stimulators. Mixing experiments indicate that spleen contains a cell population which can stimulate PP T cells, albeit to a lesser extent than PP cells. Similarly, splenic T cells have a reduced but significant capacity to respond to PP DC, in comparison to PP T cells. These differences suggest the possibility that there may be a decreasing gradient of antigenicity between the gut and the spleen which is reflected in the spontaneous activity of PP versus splenic T cells in vitro. We propose that PP cells are in fact responding in vitro to heterologous antigens derived from food, enteric microbes and other environmental sources. This notion is supported by the observation that PP cells from antigen-minimized germ-free mice fail to proliferate spontaneously in culture.

Animals↗
PubMed1994

Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome.

We identified a quinolone resistance locus, flqB, linked to transposon insertion omega 1108 and fus on the SmaI D fragment of the Staphylococcus aureus NCTC 8325 chromosome, the same fragment that contains the norA gene. S. aureus norA cloned from flqB and flqB+ strains in Escherichia coli differed only in a single nucleotide in the putative promoter region. There was no detectable change in the number of copies of norA on the chromosomes of flqB strains, but they had increased levels of norA transcripts. Cloned norA produced resistance to norfloxacin and other hydrophilic quinolones and reduced norfloxacin accumulation in intact cells that was energy dependent, suggesting active drug efflux as the mechanism of resistance. Drug efflux was studied by measurement of norfloxacin uptake into everted inner membrane vesicles prepared from norA-containing E. coli cells. Vesicles exhibited norfloxacin uptake after the addition of lactate or NADH, and this uptake was abolished by carbonyl cyanide m-chlorophenylhydrazone and nigericin but not valinomycin, indicating that it was linked to the pH gradient across the cell membrane. Norfloxacin uptake into vesicles was also saturable, with an apparent Km of 6 microM, a concentration between those that inhibit the growth of flqB and flqB+ S. aureus cells, indicating that drug uptake is mediated by a carrier with a high apparent affinity for norfloxacin. Ciprofloxacin and ofloxacin competitively inhibited norfloxacin uptake into vesicles. Reserpine, which inhibits the multidrug efflux mediated by the bmr gene of bacillus subtilis, which is similar to norA, abolished norfloxacin uptake into vesicles as well as the norfloxacin resistance of an flqB mutant, suggesting a potential means for circumventing quinolone resistance as a result of drug efflux in S. aureus. These findings indicate that the chromosomal flqB resistance locus is associated with increased levels of expression of norA and strongly suggest that the NorA protein itself functions as a drug transporter that is coupled to the proton gradient across the cell membrane.

Amino Acid Sequence↗
PubMed1993

Limitations of plasmid complementation test for determination of quinolone resistance due to changes in the gyrase A protein and identification of conditional quinolone resistance locus.

Plasmid pJSW101 derived from pUC19 and carrying the wild-type gyrA gene was found to be unstable in HM72, a quinolone-resistant (QR) clinical isolate of Escherichia coli, and resulted in no change in quinolone MICs. MICs determined in the presence of ampicillin to ensure plasmid presence, however, resulted in complementation. HM72 was proved to have a gyrA mutation based on the DNA sequence of a 418-bp fragment of gyrA. DNA sequencing identified a common mutation encoding Leu-83 as the cause of QR. To identify loci other than gyrA and nfxB contributing to QR in KF111b, zgh-3075::Tn10 (67 min) in CAG12152 was transduced into KF111b. Sixteen percent of the transductants had a fourfold decrease in norfloxacin MIC, indicating the presence of a locus, nfxD, which contributes to QR. Outcross of nfxD from DH151 (gyrA nfxB nfxD zgh-3075::Tn10) resulted in 8% of the KF130 gyrA, 2% of the EN226-3 gyrA, and none of the KL16 (wild-type) transductants, with a four- to eightfold increase in norfloxacin MIC. In the presence of ampicillin, the resistance of a gyrA nfxD double mutant, DH161 nfxD gyrA (from EN226-3), was fully complemented by gyrA+. Thus, gyrA+ plasmid complementation tests for QR may be falsely negative with plasmid instability, a difficulty which may be circumvented by maintenance of plasmid selection. In addition, if nfxD-like mutations occur in gyrA clinical isolates, a positive test may overestimate the level of resistance attributable to gyrA alone.

4-Quinolones↗
PubMed1993

Antigen presentation of herpes simplex virus by corneal epithelium--an in vitro and in vivo study.

Ia antigen (class II antigen) is a histocompatibility antigen that foreign peptides associate with, before antigen presentation to T cells and subsequent triggering of the CD4 T cells. Although corneal epithelium is normally Ia negative it may become Ia positive under abnormal circumstances but the functional significance of this is uncertain. In this study the expression of Ia antigen on corneal epithelium of mice during in vivo primary and secondary herpes simplex keratitis and the in vitro accessory function of corneal epithelium in the presentation of herpes simplex virus (HSV) antigen to in vivo HSV primed T cells were evaluated. Whole mount preparations of corneal epithelium were found to express Ia antigen on days 3, 5, and 7 following corneal inoculation with live HSV. The intensity of the Ia expression was greater in non-immune mice on day 7 after corneal inoculation compared with immune mice. A cellular suspension of corneal epithelium induced HSV primed T cells to proliferate in the presence of HSV antigen. Induction of Ia antigen on corneal epithelium during herpes simplex keratitis may functionally expand the population of antigen presenting cells in the cornea and contribute to T cell activation.

Animals↗
PubMed1993

Quinolone mode of action--new aspects.

The interactions of quinolones with the complex of DNA gyrase and DNA have been elucidated by the sequencing of additional mutant gyrA and gyrB genes that produce altered quinolone susceptibility. Strong patterns have emerged in Escherichia coli in which amino acids between positions 67 and 106 of the gyrase A subunit (GyrA) and at positions 426 and 447 of the gyrase B subunit (GyrB) have been consistently identified as important for quinolone action. The susceptibility patterns and changes in amino acids 426 and 447 in mutant resistant GyrB proteins suggest direct electrostatic interactions with quinolones at these positions. The small size and the polar nature of the serine at position 83 of the E. coli GyrA protein are particularly important for determining enzyme sensitivity and bacterial susceptibility to quinolones. Norfloxacin and ciprofloxacin bind most stably to a complex of DNA gyrase and DNA rather than to either component alone, and reduction of norfloxacin binding to complexes containing resistant GyrA proteins confirms the biological relevance of this direct measure of quinolone interaction with the gyrase-DNA complex. Although recent crystallographic studies have expanded and refine information about gyrase structure at the atomic level, direct determination of the sites of quinolone binding within the gyrase-DNA complex awaits further studies. Although quinolones have little activity against E. coli topoisomerases I and III, topoisomerase IV, a recently described enzyme thought to be involved in chromosome segregation into daughter cells, has homology with GyrA and GyrB, particularly in regions important for quinolone action, and is inhibited by some quinolones in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Quinolones↗
PubMed1993

Characterization of autoreactive helper T cells in a murine model of autoimmune haemolytic disease.

Repeated immunization of mice with rat red blood cells (RRBC) results in the production of both erythrocyte autoantibodies and anti-RRBC antibodies. The manner in which erythrocyte self-tolerance is broken has been little studied. It has been assumed that help for autoreactive B cells is provided by Th cells specific for the foreign RRBC. We show here that autoreactive Th cells can be recovered from RRBC-immunized mice. The Th cells proliferate in vitro whether stimulated by self or rat erythrocytes. Analysis of the specificity of the proliferating cells revealed extensive cross-reactivity for the two types of erythrocytes. It is therefore surprising that, in initial cultures, a slower response is evident when mouse erythrocytes are used as the antigen. From cytotoxic depletion of T-cell subsets, the phenotype of the proliferating cells was identified as Thy-1.2+, Lyt-1.1+, Lyt-2.1-, L3T4+. During in vitro stimulation of the T cells, growth factors characteristic of Th cells are secreted. Finally, we demonstrate that the responding T cells are able to help primary in vitro antibody responses to self and rat erythrocytes. We conclude that autoreactive Th cells are likely to be involved in the experimental induction of autoimmune haemolytic disease in mice.

Anemia, Hemolytic, Autoimmune↗
PubMed1993

The effect of systemic antibiotic and antibiotic-impregnated polymethylmethacrylate beads on the bacterial clearance in wounds containing contaminated dead bone.

A dorsal muscular wound model was used in 40 New Zealand White rabbits to study the effect of systemic and local antibiotics on the bacterial clearance of contaminated dead bone. Devitalized iliac crest bone preincubated with Staphylococcus aureus was implanted in each deep muscular wound with or without tobramycin-impregnated polymethylmethacrylate beads. Either systemic tobramycin or cefazolin was administered for 7 days. Animals were sacrificed at 7 and 14 days. The wounds containing tobramycin beads had significantly fewer bacteria than those without antibiotic beads (2.0 x 10(2) versus 1.3 x 10(6); p < 0.008). The reduction in bacteria due to the tobramycin beads did not differ significantly with respect to the concurrent systemic antibiotics or to the duration of incubation. We conclude that tobramycin-impregnated beads are effective in reducing bacterial count in contaminated bony wounds treated with systemic antibiotics. Furthermore, the bactericidal effect of the antibiotic beads is independent of and additive to the systemic antibiotic delivered to the wounds by well-perfused muscles.

Animals↗
PubMed1992

Synergism of trimethoprim and ciprofloxacin in vitro against clinical bacterial isolates.

For the first time, the effects of combinations of trimethoprim and a fluoroquinolone (ciprofloxacin) against gram-positive and gram-negative bacterial isolates were evaluated in vitro. Synergism was found in 31% (fractional inhibitory concentration, FIC) and 33% (fractional bactericidal concentration, FBC) of 121 clinical isolates of various bacterial strains, most often in Escherichia coli, staphylococci, and enterococci. Antagonism occurred in 1% (FIC) and 3% (FBC). The combination of trimethoprim and ciprofloxacin merits further evaluation for potential usefulness as a clinical regimen.

Ciprofloxacin↗
PubMed1992

Genetics and regulation of outer membrane protein expression by quinolone resistance loci nfxB, nfxC, and cfxB.

Quinolone resistance mutations (cfxB1, marA1, and soxQ1) that reduce porin outer membrane protein OmpF map near 34 min on the Escherichia coli chromosome. Another such mutation, nfxC1, was found in strain KF131 (nfxB, 19 min). nfxC1 and cfxB1 mutants (selected with quinolones) differed slightly but reproducibly from marA1 (selected with tetracycline) and soxQ1 (selected with menadione) mutants in quinolone resistance and linkage to zdd2208::Tn10kan (33.7 min). For nfxB nfxC1 and cfxB1 mutants, as previously shown for marA mutants, resistance and reduced OmpF required the micF locus encoding an antisense RNA complementary to ompF mRNA and were associated with increased micF expression.

4-Quinolones↗
PubMed1992

Primary and secondary human in vitro T-cell responses to soluble antigens are mediated by subsets bearing different CD45 isoforms.

A culture system has been developed which consistently supports in vitro proliferative responses to conventional soluble antigens by human CD4+ T cells from non-immunized donors. T cells exposed to an antigen in primary cultures could be restimulated in vitro in an antigen-specific manner to give secondary responses with greater magnitudes and a more rapid onset than the initial reaction. To characterize further the responding T-cell population in primary compared with secondary reactions, T cells were depleted of CD45RA+ or CD45RO+ cells and stimulated with recall and non-recall antigens. It was found that the soluble non-recall antigen keyhole limpet haemocyanin did not stimulate CD45RO+ T cells, yet induced strong proliferative responses from CD45RA+ T cells. Conversely, it was confirmed that human CD45RO+ T cells respond to the recall antigen-purified protein derivative from Mycobacterium tuberculosis. Cell mixing experiments indicated that CD45RO+ T cells are unlikely to have any suppressive effect on the reactivity of CD45RA+ cells to non-recall antigens. These data provide new support for the hypothesis that CD45RA+ represents the naive and CD45RO+ the memory phenotype of human CD4+ T cells.

Antigens↗