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By utilizing a baculoviral expression system described previously (Cascio, M., Schoppa, N. E., Grodzicki, R. L., Sigworth, F. J., and Fox, R. O. (1993) J. Biol. Chem. 268, 22135-22142), functional recombinant homomeric human alpha(1)-glycine receptors (GlyR) were overexpressed in insect cell culture, solubilized, purified, and reconstituted into lipid vesicles via gel filtration. Reconstituted GlyR channels were observed to retain native-like activity in single channel recordings of planar bilayers and in flux assays of small unilamellar vesicles, providing evidence that the recombinant homomeric receptor may be functionally reconstituted. This reconstitution is significant in that it indicates that the overexpressed homomeric receptor is an appropriate substrate for subsequent biophysical characterization aimed at the general elucidation of structure-function. Circular dichroism spectroscopy of reconstituted GlyR indicated a low alpha-helical content and a significant fraction of polyproline structure. The small fraction of observed alpha-helix is insufficient to accommodate the four helical transmembrane domains proposed in models for this receptor. By inference, other members of the homologous ligand-gated channel superfamily, which include the ionotropic gamma-aminobutyric acid, acetylcholine, and serotonin receptors, may also be erroneously modeled, and alternate models should be considered.

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The human alpha 1 glycine receptor (GlyR) was expressed in Sf9 insect cells infected with a recombinant Autographa californica nuclear polyhedrosis baculovirus. Previous studies had indicated that transient expression of this subunit in Xenopus oocytes or human kidney cell lines is sufficient to form active agonist-gated chloride channels. Expression of the alpha 1 GlyR protein resulted in functional channels present on the cell surface of infected Sf9 cells as evidenced by whole-cell patch-clamping and single-channel recordings. These channels were gated by glycine, but not in the presence of strychnine. An immunoreactive 48-kDa protein could be easily visualized on Coomassie-stained sodium dodecyl sulfate-polyacrylamide gels of whole-cell lysates with maximal expression 3 days postinfection. The alpha 1 GlyR protein was solubilized from a membrane fraction of infected Sf9 cells in 1% digitonin and 0.1% deoxycholate and purified by affinity chromatography using aminostrychnine agarose, yielding 0.33 mg/liter of cells. Given the low natural abundance of the native channel, the development of this expression system now provides sufficient purified channel protein for future biochemical and biophysical characterization. Since the glycine receptor shares sequence and structural homology with other members of a ligand-gated channel superfamily, further characterization may establish general rules governing the structure and mechanism of these membrane protein channels.

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We compared immunoblotting and indirect enzyme-linked immunosorbent assay (ELISA) using different antigen preparations to test for antibody to Borrelia burgdorferi in patients with early Lyme disease. With immunoblotting, 16 (53%) of 30 patients had positive tests in acute-phase sera and 25 (83%) had them in convalescent-phase sera. Among 64 controls, false-positive results were obtained in only three individuals with syphilis and in one hospitalized patient with renal allograft rejection. Among the patients with Lyme disease, both IgM and IgG antibodies most commonly bound to the 41-kilodalton (kDa) flagellar antigen, but many patients had binding to other components, particularly those of 25, 55, 58, or 66 kDa, and the order of their appearance was variable. Compared with indirect ELISA (using sonicated whole spirochetes or a flagellin-enriched fraction as the antigen preparation), more patients with Lyme disease had positive tests by immunoblotting, and fewer control subjects had false-positive results. Our results indicate that immunoblotting is superior to indirect ELISA for diagnosing early Lyme disease.

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The diagnostic value of clinical, culture, and serologic findings was studied prospectively in 41 patients with early Lyme disease. Fifteen patients had erythema chronicum migrans alone, and 26 had clinical evidence of disseminated infection, most commonly affecting the brain or meninges, other skin sites, lymph nodes, or joints. Of 40 blood cultures, only one, from a patient with disseminated infection, yielded spirochetes. One of 10 patients tested with localized infection had an elevated IgM response to the Lyme spirochete (200 units or greater) during acute disease. Two to three weeks after beginning antibiotic therapy, four of the 10 patients had elevated specific IgM or IgG responses (200 units or greater). Of the 22 patients tested with disseminated disease, 10 initially had elevated levels of specific IgM or IgG, and 12 had such responses by convalescence. Because of the low yield of cultures and the delay in the specific antibody response, recognition of the clinical picture remains very important in diagnosing early Lyme disease.

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We determined the antibody response against the Ixodes dammini spirochete in Lyme disease patients by indirect immunofluorescence and an enzyme-linked immunosorbent assay (ELISA). The specific IgM response became maximal three to six weeks after disease onset, and then declined, although titers sometimes remained elevated during later disease. Specific IgM levels correlated directly with total serum IgM. The specific IgG response, often delayed initially, was nearly always present during neuritis and arthritis, and frequently remained elevated after months of remission. Although results obtained by indirect immunofluorescence and the ELISA were similar, the ELISA was more sensitive and specific. Cross-reactive antibodies from patients with other spirochetal infections were blocked by absorption of sera with Borrelia hermsii, but titers of Lyme disease sera were also decreased. To further characterize the specificity of the humoral immune response against the I. dammini spirochete, 35S-methionine-labeled spirochetal antigens were identified by immunoprecipitation with sera from Lyme arthritis patients. These polypeptides had molecular weights of 62, 60, 47, 37, 22, 18, and 15 kDa, and were not recognized by control sera. We conclude that the ELISA, without absorption, is the best method to assay the humoral immune response in Lyme disease, and we have identified methionine-containing spirochetal polypeptides that may be important in Lyme arthritis.

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From three endemic locations of erythema chronicum migrans disease in North Rhine-Westphalia, Germany, we recovered 19 isolates of a spirochete from Ixodes ricinus ticks. The infection rate in adult ticks was 16 percent. The isolated spirochete is immunologically related to the Ixodes dammini spirochete, Borrelia duttoni, and Treponema pallidum. Using indirect immunofluorescence, the sera of 90 patients with erythema chronicum migrans disease showed antibody titers against the isolated spirochete, which correlated with the clinical course. Similarly, antibodies were demonstrated in the sera of 21 patients with acrodermatitis chronica atrophicans. These results suggest an etiologic role for the Ixodes ricinus spriochete in European erythema chronicum migrans disease.

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Since the summer of 1982, we have cultured patient specimens for Lyme disease spirochetes. Of 118 patients cultured, four specimens yielded spirochetes: two from blood, one from a skin biopsy specimen of erythema chronicum migrans (ECM), and one from cerebrospinal fluid. All four isolates appeared identical when examined with a monoclonal antibody. However, attempts to recover the spirochete from synovium or synovial fluid were unsuccessful. In addition, the organism could not be visualized in skin or synovial biopsy specimens using the avidin-biotin peroxidase complex detection system. Thus, the current yield in culturing spirochetes from patients is quite low, and it is not yet known whether the organism is still alive later in the disease when arthritis is present.

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The antibody response to the Ixodes dammini spirochete was determined in 41 serial serum samples from 12 patients with Lyme disease. By enzyme-linked immunosorbent assay (ELISA), 11 of the 12 patients had higher titers of specific IgM antibody (greater than 1:200) during early disease than did 40 control subjects. Specific IgM antibody titers, which correlated with total amounts of IgM antibody (P less than .001), sometimes remained elevated throughout the illness. During neuritis, nine of 10 patients had higher specific IgG antibody titers (greater than 1:200) than did controls, and when arthritis was present, all had such titers, which remained elevated after months of remission. In the ELISA, antibody responses determined by single or serial dilutions were similar, but the ELISA was more sensitive and specific than was immunofluorescence. Adsorption of sera with Borrelia hermsii generally resulted in a fourfold decrease in titers of cross-reactive antibodies, but the titers of sera from patients with Lyme disease were also reduced. Currently, the ELISA, without adsorption, is the best diagnostic test for Lyme disease.

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From ticks of the type Ixodes ricinus, 19 strains of a spirochete were isolated at three places of infection of erythema chronicum migrans disease. The spirochete was immunologically related to Borrelia duttoni, Treponema pallidum and Ixodes dammini spirochete, the causative organism of North American erythema chronicum migrans disease (Lyme disease). The isolated spirochete differed from the North American one in its reaction with monoclonal antibodies and possibly in its electronmicroscopic structure. A corresponding spirochete was isolated from the blood of a woman with erythema chronicum migrans. Of 39 patients with erythema chronicum migrans mostly treated with antibiotics 50% had increased IgG antibody titre (1:64 to 1:1024) against the isolated spirochete, while among 51 untreated patients with tick-transmitted meningopolyneuritis 90% had increased IgG antibody titres. Fourfold antibody titres increases or falls were found on 50 occasions. IgG antibody titres up to 1:64 were demonstrated also in CSF, in 22 instances with significant changes. Increased serum IgM antibody titres of 1:32 to 1:256 were observed in 20% and 68%, respectively, of patients. These findings suggest that the isolated spirochete is the causative agent of erythema chronicum migrans disease in Europe. Its antigen structure and arrangement is similar to that of the causative agent of Lyme disease.

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We recovered a newly recognized spirochete from the blood, skin lesions (erythema chronicum migrans [ECM]), or cerebrospinal fluid of 3 of 56 patients with Lyme disease and from 21 of 110 nymphal or adult lxodes dammini ticks in Connecticut. These isolates and the original one from l. dammini appeared to have the same morphologic and immunologic features. In patients, specific IgM antibody titers usually reached a peak between the third and sixth week after the onset of disease; specific IgG antibody titers rose slowly and were generally highest months later when arthritis was present. Among 40 patients who had early disease only (ECM alone), 90 per cent had an elevated IgM titer (greater than or equal to 1:128) between the ECM phase and convalescence. Among 95 patients with later manifestations (involvement of the nervous system, heart, or joints), 94 per cent had elevated titers of IgG (greater than or equal to 1:128). In contrast, none of 80 control subjects had elevated IgG titers, and only three control patients with infectious mononucleosis had elevated IgM titers. We conclude that the I. dammini spirochete is the causative agent of Lyme disease.

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In infections associated with immune complex disease, microbial antigens have rarely been found in the complexes. Using an enzyme-linked immunoassay, we studied the immune complexes of a patient who had hematogenous Pseudomonas aeruginosa osteomyelitis associated with palpable purpura, arthritis, and microscopic hematuria. After separation of the complexes into high and low molecular weight fractions, a 6-fold selective concentration of P aeruginosa antibody was found in the low molecular weight fraction compared with the concentration of the serum. Following disruption of immunoglobulin, the high molecular weight fraction competed with solid-phase P aeruginosa antigen for P aeruginosa antibody from another source. After successful treatment of the infection, the patient's symptoms resolved, and the complexes disappeared. These findings strongly suggest that immune complexes in this patient contained P aeruginosa antigen and antibody that may have been pathogenetic in his disease.

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Potent inhibitors of cholesterol side chain cleavage were tested for inhibition of 11 beta-hydroxylation of 11-deoxycortisol by bovine adrenal cortex mitochondria. Compounds which inhibited 11 beta-hydroxylation were metyrapone, 4-phenylimidazole, 1-benzylimidazole, 17 beta-ureido - 1, 4- androstadien-3-one. SU-8000, 4-methylaminoglutethimide, and 20 alpha-hydroxycholestrol. Compounds which did not inhibit 11 beta-hydroxylation at concentrations of 0.5 mM were d-aminoglutethimide tartrate, 1-aminoglutethimide tartrate, N-methylaminoglutethimide, 16 alpha-methylpregnenolone, 16 beta-methylpregnenolone, 20-tolylpregnenediol, 16 alpha-chloropregnenolone-3-acetate, 16 alpha-benzyloxpregnenolone-3-acetate and cyanoketone. The results obtained indicate that aminoglutethimide and its congeners, the 16-halogenated and 16-benzoylated derivatives of pregnenolone and cyanoketone are specific inhibitors of cholesterol side chain cleavage enzyme. The two mitochondrial steroid oxyganase systems are linked through their competition for a single electron source.

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