RESUMO
Pili were isolated from two different strains of gonococci (33 and 2686) and demonstrated to be pure by the criteria of electron microscopy and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Each purified pili preparation was studied for its ability to inhibit; (a) binding of 124-I-labeled purified 2686 pili by antibody to 2686 pili, and (b) binding of 125-I-labeled purified 33 pili by antibody to 33 pili. In each instance progressive inhibition of binding was produced by homologous pili type, but no significant inhibition was observed using comparable weights of the heterologous pili type. These results indicate that the pili of strains 2686 and 33 are antigenically different.
Assuntos
Antígenos Heterófilos , Neisseria gonorrhoeae/imunologia , Reações Antígeno-Anticorpo , Eletroforese em Gel de Poliacrilamida , Radioisótopos do Iodo , Métodos , Microscopia Eletrônica , Neisseria gonorrhoeae/ultraestrutura , RadioimunoensaioRESUMO
Comparable numbers of types 1, 2, 3, and 4 gonococci were placed on the intact chorioallantoic membrane of 236, 10-day old chick embryos. Types 1 and 2 organisms produced infection and could be cultured from chorioallantoic fluid 2 days later significantly more often (69%) than types 3 and 4 organisms (12%, P < 0.001). This confirms in an animal model the same correlation between colony types and infectivity observed in human volunteers and suggests that types 1 and 2 gonococci possess a fundamental virulence characteristic which is absent from types 3 and 4 organisms. Gonococcal infection of the chick embryo chorioallantoic cavity remains a useful model somewhat analogous to localized gonococcal infection in man.
Assuntos
Modelos Animais de Doenças , Gonorreia/fisiopatologia , Neisseria gonorrhoeae/patogenicidade , Animais , Sangue/microbiologia , Embrião de Galinha , Membranas Extraembrionárias/microbiologia , Gonorreia/etiologia , Humanos , Neisseria gonorrhoeae/citologia , Neisseria gonorrhoeae/isolamento & purificação , VirulênciaRESUMO
Although the immunologic basis of protective immunity in human immunodeficiency virus type 1 (HIV-1) infection has not yet been defined, virus-specific cytotoxic T lymphocytes (CTL) are likely to be an important host defense and may be a critical feature of an effective vaccine. These observations, along with the inclusion of the HIV-1 envelope in the majority of vaccine candidates presently in clinical trials, underscore the importance of the precise characterization of the cellular immune responses to this protein. Although humoral immune responses to the envelope protein have been extensively characterized, relatively little information is available regarding the envelope epitopes recognized by virus-specific CTL and the effects of sequence variation within these epitopes. Here we report the identification of two overlapping CTL epitopes in a highly conserved region of the HIV-1 transmembrane envelope protein, gp41, using CTL clones derived from two seropositive subjects. An eight-amino acid peptide was defined as the minimum epitope recognized by HLA-B8-restricted CTL derived from one subject, and in a second subject, an overlapping nine-amino acid peptide was identified as the minimal epitope for HLA-B14-restricted CTL clones. Selected single amino acid substitutions representing those found in naturally occurring HIV-1 isolates resulted in partial to complete loss of recognition of these epitopes. These data indicate the presence of a highly conserved region in the HIV-1 envelope glycoprotein that is immunogenic for CTL responses. In addition, they suggest that natural sequence variation may lead to escape from immune detection by HIV-1-specific CTL. Since the region containing these epitopes has been previously shown to contain an immunodominant B cell epitope and also overlaps with a major histocompatibility complex class II T cell epitope recognized by CD4+ CTL from HIV-1 rgp160 vaccine recipients, it may be particularly important for HIV-1 vaccine development. Finally, the identification of minimal CTL epitopes presented by class I HLA molecules should facilitate the definition of allele-specific motifs.
Assuntos
Produtos do Gene env/imunologia , Antígenos HIV/química , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Antígenos CD8/análise , Análise Mutacional de DNA , Epitopos , Proteína gp41 do Envelope de HIV/química , Soropositividade para HIV/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Relação Estrutura-AtividadeRESUMO
A phenolic glycolipid from Mycobacterium leprae was purified and used as antigen in an enzyme-linked immunosorbent assay. Antibodies directed against the lipid were seen in serums from leprosy patients but not in serums from uninfected controls or patients infected with other mycobacteria, including Mycobacterium tuberculosis. The antibody response distinguished between the Mycobacterium leprae lipid and the structurally related phenolic glycolipid from Mycobacterium kansasii. This assay has considerable potential as a specific serodiagnostic test for leprosy infection.
Assuntos
Glicolipídeos/imunologia , Hanseníase/diagnóstico , Mycobacterium leprae/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Testes SorológicosRESUMO
Mycobacterium leprae induces T cell reactivity and protective immunity in the majority of exposed individuals, but the minority that develop leprosy exhibit various types of immunopathology. Thus, the definition of epitopes on M. leprae antigens that are recognized by T cells from different individuals might result in the development of an effective vaccine against leprosy. A sequence from the 65-kD protein of this organism was recognized by two HLA-DR2-restricted, M. leprae-specific helper T cell clones that were derived from a tuberculoid leprosy patient. Synthetic peptides were used to define this epitope as Leu-Gln-Ala-Ala-Pro-Ala-Leu-Asp-Lys-Leu. A similar peptide that was derived from the third hypervariable region of the HLA-DR2 chain, Glu-Gln-Ala-Arg-Ala-Ala-Val-Asp-Thr-Tyr, also activated the same clones. The unexpected cross-reactivity of this M. leprae-specific DR2-restricted T cell epitope with a DR2 peptide may have to be considered in the design of subunit vaccines against leprosy.
Assuntos
Antígenos de Bactérias/imunologia , Epitopos/imunologia , Antígenos HLA-DR/imunologia , Mycobacterium leprae/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Antígeno HLA-DR2 , Hanseníase/imunologia , Dados de Sequência Molecular , Peptídeos/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Gonoccocal pili facilitate attachment of virulent Neisseria gonorrhoeae to human cells. To characterize this attachment function, purified gonococcal pili isolated from four strains possessing antigenically distinct pili were radiolabeled with 125I and used to measure the attachment of pili to various human cells in vitro. Human buccal and cervical-vaginal mucosal epithealial cells, fallopian tube mucosa, and sperm bound pili in greater numbers per micrometer2 of surface area (1--10) than fetal tonsil fibroblasts, HeLa M cells, erythrocytes, or polymorphonuclear leukocytes. This cell specificity of attachment suggests a greater density of membrane pili binding sites on cells similar or identical to cells from natural sites of infection. The pili binding sites were quantitated as 1 X 10(4) per cervical-vaginal squamous cell. Pili of all antigenic types attached equally to a given cell type, implying that the attachment moiety of each pilus was similar. Attachement of gonoccocal pili to human cells occurred quickly with saturation of presumed receptor sites within 20--60 min. Attachment was temperature dependent (37 degrees greater than 20 degrees greater than 4 degrees C), and pH dependent (3.5 less than 4.5 less than 5.5 less than 7.5). Attachment was inhibited by antibody to pili (homologous pili Ab greater than heterologous Ab). The extent of possible protection against gonococcal infection due to inhibition of pili-mediated attachment might prove limited as a result of the considerable antigenic heterogeneity among pili and the observation that blockage of pili attachment is maximal only with antibody to pili of the infecting strain.
Assuntos
Neisseria gonorrhoeae/fisiologia , Anticorpos Antibacterianos , Colo do Útero/microbiologia , Eritrócitos/microbiologia , Tubas Uterinas/microbiologia , Feminino , Células HeLa/microbiologia , Humanos , Mucosa Bucal/microbiologia , Neisseria gonorrhoeae/ultraestrutura , Neutrófilos/microbiologia , Tonsila Palatina/microbiologia , Vagina/microbiologiaRESUMO
The susceptibility of strains of Neisseria gonorrhoeae to the bactericidal action of normal human sera was determined for isolates from patients with disseminated gonococcal infection and uncomplicated gonorrhea. Serum susceptibility was correlated with penicillin susceptibility and auxotype. 38 of 39 strains (97%) of N. gonorrhoeae from Seattle patients with disseminated gonococcal infection were resistant to the complement-dependent bactericidal action of normal human sera. 36 of these were inhibited by less than or equal to mug/ml of penicillin G and required arginine, hypoxanthine, and uracil for growth on chemically defined medium (Arg-Hyx-Ura- auxotype). 12 of 43 isolates from patients with uncomplicated gonorrhea were also of the Arg-Hyx-Ura-auxotype, inhibited by less than or equal to 0.030 mug/ml of penicillin G, and serum resistant. Of the 31 remaining strains of other auxotypes isolated from patients with uncomplicated gonorrhea, 18 (58.1%) were sensitive to normal human sera in titers ranging from 2 to 2,048. The bactericidal action of normal human sera may prevent the dissemination of serum-sensitive gonococci. However, since only a small proportion of individuals infected by serum-resistant strains develop disseminated gonococcal infection, serum resistance appears to be a necessary but not a sufficient virulence factor for dissemination. Host factors such as menstruation and pharyngeal gonococcal infection may favor the dissemination of serum-resistant strains. Since serum-resistant Arg-Hyx-Ura strains are far more frequently isolated from patients with disseminated gonococcal infection than serum-resistant strains of other auxotypes, Arg-Hyx-Ura-strains may possess other virulence factors in addition to serum resistance.
Assuntos
Atividade Bactericida do Sangue , Gonorreia/microbiologia , Neisseria gonorrhoeae , Doença Aguda , Anticorpos Antibacterianos , Proteínas do Sistema Complemento , Convalescença , Meios de Cultura , Gonorreia/sangue , Gonorreia/imunologia , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/crescimento & desenvolvimento , Penicilina G/farmacologia , VirulênciaRESUMO
We examined the vaginal washings from patients with nonspecific vaginitis (NSV) to seek biochemical markers and possible explanations for the signs and symptoms of this syndrome. Seven amines were identified including methylamine, isobutylamine, putrescine, cadaverine, histamine, tyramine, and phenethylamine. These amines may contribute to the symptoms of NSV and may contribute to the elevated pH of the vaginal discharge. They may also be partly responsible for the "fishy" odor that is characteristic of vaginal discharges from these patients. Among the seven amines, putrescine and cadaverine were the most abundant and were present in all vaginal discharges from each of ten patients before treatment. These amines are produced in vitro during growth of mixed vaginal bacteria in chemically defined medium, presumably by decarboxylation of the corresponding amino acids. We hypothesize the anaerobic vaginal organisms, previously shown to be quantitatively increased in NSV, are responsible for the amine production, because metronidazole inhibited the production of amines by vaginal bacteria in vitro, and Haemophilus vaginalis did not produce amines. H. vaginalis did release high concentrations of pyruvic acid and of amino acids during growth in peptone-starch-dextrose medium, whereas, other vaginal flora consumed both pyruvic acid and amino acids in the same medium during growth. These findings suggest that a symbiotic relationship may exist between H. vaginalis and other vaginal flora in patients with NSV.
Assuntos
Aminas/análise , Vagina/metabolismo , Vaginite/metabolismo , Aminoácidos/análise , Ampicilina/uso terapêutico , Líquidos Corporais/análise , Butilaminas/análise , Cadaverina/análise , Cromatografia em Camada Fina , Eletroforese , Estolato de Eritromicina/uso terapêutico , Feminino , Histamina/análise , Humanos , Metilaminas/análise , Metronidazol/uso terapêutico , Fenetilaminas/análise , Putrescina/análise , Espermidina/análise , Espermina/análise , Tiramina/análise , Vaginite/tratamento farmacológicoRESUMO
Gonococcal pili, pure by the criteria of electron microscopic examination and polyacrylamide gel electrophoresis in sodium dodecyl sulfate, have been prepared by repeated cycles of precipitation with 0.1 M MgCl(2), followed by dissolution in 0.01 M Tris pH 8, 0.01 M NaN(3). Using a fluorescein-conjugated antibody prepared to pili from a single strain, pilar antigen(s) was found to be present in each of 18 strains of gonococci tested, and absent from strains of pilated meningococci, nonpathogenic Neisseria sp., and Escherichia coli. Purified pili, labeled with (125)I were used in an antigen binding assay to quantitatively measure antibody to pili in rabbit sera and in 561 human sera. The range of antibody activity for 133 persons unlikely to have experienced gonorrhea was 0.1-1.6 mug/ml with a geometric mean of 0.5 mug/ml. This geometric mean antibody activity was significantly lower than the geometric mean for asymptomatically infected males (1.0 mug/ml, P < 0.002), males with symptomatic gonococcal anterior urethritis (1.6 mug/ml, P < 0.001), or asymptomatically infected females (4.2 mug/ml, P < 0.001). Antibody appeared in elevated levels (> 1.6 mug/ml) 2-3 wk after infection and returned toward control levels 1 or more months after treatment. Antibody levels higher than 1.6 mug/ml were found in 26 (50%) of 52 males with gonococcal anterior urethritis, in 10 (33%) of 30 males with asymptomatic urethral infection and in 50 (89%) of 56 asymptomatically infected females. In a high-risk group of 103 females for whom culture results and antibody to pili were compared, 58 (57%) had elevated antibody levels to pili and 86% of the infected females were within this seropositive group. The antigen binding assay may provide a means to detect asymptomatic gonococcal infection in women.
Assuntos
Anticorpos Antibacterianos/análise , Gonorreia/imunologia , Neisseria gonorrhoeae/imunologia , Adolescente , Adulto , Animais , Antígenos de Bactérias/isolamento & purificação , Sítios de Ligação de Anticorpos , Criança , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Escherichia coli/imunologia , Feminino , Imunofluorescência , Humanos , Reação de Imunoaderência , Imunoglobulina G , Radioisótopos do Iodo , Masculino , Infecções Meningocócicas/imunologia , Microscopia Eletrônica , Pessoa de Meia-Idade , Neisseria gonorrhoeae/citologia , Neisseria gonorrhoeae/isolamento & purificação , Neisseria meningitidis/imunologia , Testes de Precipitina , Coelhos/imunologiaRESUMO
Chlamydia trachomatis, Ureaplasma urealyticum (T-mycoplasma), and Hemophilus vaginalis have previously been considered possible etiological agents in nongonococcal urethritis (NGU). In this study, current C. trachomatis infection was confirmed by culture and (or) micro-immunofluorescence serology in 26 of 69 men experiencing afirst episode of NGU, and 1 of 39 with no urethritis. Serum IgM immunofluorescent antibody to chlamydia was demonstrated in 16 of 20 men with chlamydia culture positive NGU, and 3 of 39 with chlamydia culture negative NG, and none of 34 with no urethritis. 9 of 10 culture positive men with less than or equal to 10 days symptoms developed immunofluorescent antibody seroconversion in paired sera. U. realyticum was isolated significantly more often and in significantly higher concentration from first voided urine from chlamydia-negative cases of NGU than from chlamydia-positive NGU. Ureaplasmacidal antibody titers increased fourfold in six men, four of whom had negative cultures for for unreaplasma. H. vaginalis was isolated from c9 of 33 men with no urethritis and 2 of 69 with NGU. C. trachomatis is susceptible, and U. urealyticum is resistant to sulfonamides. A 10-day course of sulfisoxazole therapy produced improvement in 13 of 13 chlamydia-positive, unreaplasma-negative, and only 14 of 29 chlamydia-negative, unreaplasma-positive NGU cases (P less than 0.002). Thus, culture, serology, and response to therapy support the etiologic role of chlamydia in NGU. Quantitative culture and response to therapy suggest U. unrealyticum may cause many cases of chlamydia-netative NGU.
Assuntos
Infecções por Chlamydia , Infecções por Mycoplasma , Uretrite/microbiologia , Adulto , Infecções por Chlamydia/tratamento farmacológico , Chlamydia trachomatis/imunologia , Chlamydia trachomatis/isolamento & purificação , Gardnerella vaginalis/isolamento & purificação , Humanos , Masculino , Infecções por Mycoplasma/tratamento farmacológico , Sulfisoxazol/uso terapêutico , Ureaplasma/imunologia , Ureaplasma/isolamento & purificação , Uretra/microbiologia , Uretrite/tratamento farmacológico , Urina/microbiologiaRESUMO
Monoclonal antibodies were used to analyze the antigenic properties of denatured and native forms of gonococcal outer membrane proteins. The protein samples were only partially dissociated by treatment for 30 min at 40 degrees C with 0.1% (w/v) SDS, 0.5% (v/v) Triton X-100, and then processed by polyacrylamide gel electrophoresis without boiling. The resulting pattern included the native aggregated and trimeric forms of protein I and III as they exist in the gonococcal outer membrane, as well as the denatured monomeric forms. Two methods were compared to analyze these gels: gel immunoradioassay (GIRA), and Western blotting. With GIRA longitudinal 50 micron thin slices, up to 40 identical copies per gel, were produced with a microtome cryostat. These slices were exposed to the monoclonal antibody and antibody binding was detected by 125I-protein A and autoradiography. Serotype-specific, monoclonal antibodies reacted most commonly with the native polymeric form of gonococcal protein I and less frequently recognized the denatured, monomeric form. Monoclonal antibodies that recognized the polymeric form of protein I frequently produced antibody-mediated, complement-dependent, bactericidal activity for gonococci bearing the same protein I serotype. The antigen specificity of these functionally relevant antibodies could not be characterized by the Western blotting procedure, which produced incomplete transfer to nitrocellulose paper of the polymeric, high molecular weight protein aggregates. A third technique, radioimmunoprecipitation using partial dissociating conditions, did not permit differentiation between proteins I- and III-specific monoclonals after analysis of the precipitated material by denaturing SDS electrophoresis.
Assuntos
Anticorpos Monoclonais , Proteínas da Membrana Bacteriana Externa/análise , Epitopos/análise , Neisseria gonorrhoeae/análise , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Desnaturação Proteica , Radioimunoensaio/métodosRESUMO
Polysulfone membranes have been used as a solid support for chromatography and immunoblotting of phenolic glycolipid I from Mycobacterium leprae. These membranes have an advantage over other supports such as nitrocellulose and silica gel in that very little non-specific background binding of antibodies occurs and assays can readily be carried out with IgM antibodies from human sera. An example of use of the polysulfone chromatography system for detection of phenolic glycolipid I in sera from leprosy patients is described.
Assuntos
Antígenos de Bactérias/análise , Glicolipídeos/imunologia , Hanseníase/sangue , Mycobacterium leprae/imunologia , Sulfonas , Glicolipídeos/sangue , Humanos , Técnicas de Imunoadsorção , Hanseníase/imunologia , Membranas Artificiais , PolímerosRESUMO
A carbohydrate antigen composed predominantly of arabinomannan has been purified from Mycobacterium smegmatis and used in an enzyme-linked immunosorbent assay to detect anti-mycobacterial antibodies in human sera. Sera from 117 controls, 25 tuberculosis patients, 124 leprosy patients and 256 household contacts of leprosy patients were tested. When compared with the control group, 56% of tuberculosis patients, 27% of patients with tuberculoid leprosy, 77% of borderline leprosy cases, and 95% of patients with lepromatous leprosy had elevated titers. Nine percent of the household contact group had abnormally high levels of antibody. The relevance of these findings to the serodiagnosis of incubating leprosy and the management of household contacts of leprosy patients is discussed.
Assuntos
Anticorpos Antibacterianos/análise , Hanseníase/diagnóstico , Mananas/imunologia , Mycobacterium/imunologia , Polissacarídeos/imunologia , Adolescente , Adulto , Idoso , Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Criança , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hanseníase/imunologia , Masculino , Pessoa de Meia-Idade , Tuberculose Pulmonar/imunologiaRESUMO
Monoclonal antibodies directed to six separate antigen molecules of Mycobacterium leprae have been tested in an antigen-capture assay based on combined use of polyclonal ("capture") and monoclonal ("detector") antibody reagents. This approach provides a potentially versatile, sensitive and specific assay for detection and relative quantitation of M. leprae antigens. Characterisation of monoclonal antibodies to mycobacterial lipoarabinomannan (LAM-B) by the antigen-capture assay indicates that some of the antigenic determinants present on LAM-B from M. leprae may be either absent altogether or present at much lower concentrations on the corresponding LAM-B structure form M. tuberculosis.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Lipopolissacarídeos/imunologia , Mycobacterium leprae/imunologia , Anticorpos Monoclonais/análise , Antígenos de Bactérias/análise , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Humanos , Mycobacterium tuberculosis/imunologia , Especificidade da EspécieRESUMO
Acute treatment of migraine has benefited first from major advances in pharmacological science followed in short order, sometimes preceded, by an improved understanding of pathogenesis, especially of headache. This chapter reviews the mechanisms of migraine that provide an understanding of the pharmacology and therapeutic targets for acute migraine medications. General clinical approaches to acute therapy are reviewed, and indices of acceptable acute therapeutic outcomes are discussed. Currently the serotonin (5-HT) 1B/1D agonist group of drugs, triptans, forms the mainstay of acute therapeutic regimens. Other approaches to acute treatment such as simple analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), ergots, and combination medications are reviewed. Finally, the newest acute treatments that are currently exploratory or under clinical investigation are discussed.