RESUMO
The substantial virus lysis was induced by HIV-1-infected patient serum and normal human complement serum in the presence of purified patient IgG. Non-infected CD4+ T cells coated with the whole virus or with a recombinant HIV-1 envelope gp120 and sensitised with patient IgG were also shown to be susceptible to complement-dependent lysis. The serum level of complement regulatory protein in a fluid phase, the C1-esterase inhibitor, was significantly correlated with serum concentration of C1q-circulating immune complexes (P=0.0062), but inversely with CD4+ T cell count (P < 0.0001). Accordingly, the disease progression in HIV-1-infected patients was significantly correlated with the level of complement activation as determined by serum level of C1-esterase inhibitor (P=0.0001), and inversely correlated with CD4+ cell count (P < 0. 0001) and gp120-specific antibody titre (P=0.0086). These results strongly suggest that the complement activation by gp120-specific antibodies play a very important role in virus clearance, but also in depletion of infected as well as gp120-coated non-infected CD4+ bystander T cells during the course of HIV-1 infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas do Sistema Complemento/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Contagem de Linfócito CD4 , Análise por Conglomerados , Proteínas Inativadoras do Complemento 1/análise , Feminino , Citometria de Fluxo , Transcriptase Reversa do HIV/análise , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Coelhos , Contagem de Cintilação , Estatísticas não ParamétricasRESUMO
Representative strains of serogroup A Neisseria meningitidis were chosen from all major meningitis epidemics worldwide since 1960 and subjected to analysis for the electrophoretic variation of 15 cytoplasmic allozymes and four outer membrane proteins. The 290 strains defined 84 unique electrophoretic types which were classified in nine subgroups. Tests with monoclonal antibodies specific for conserved pilin epitopes showed that the class I, IIa, and IIb epitopes were uniform within the subgroups. Similarly, the subgroups were uniform for expression of different variable regions of class 1 outer membrane protein, with a few minor exceptions. Many of the bacteria tested were isolated in the People's Republic of China, and the epidemiology of Chinese epidemics of meningococcal meningitis is described. The analysis approaches a global description of epidemic meningitis caused by serogroup A meningococci in the past 30 years.
Assuntos
Meningite Meningocócica/epidemiologia , Neisseria meningitidis/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/análise , China/epidemiologia , Eletroforese , Humanos , Camundongos , Neisseria meningitidis/enzimologia , Neisseria meningitidis/genética , Estudos Soroepidemiológicos , SorotipagemAssuntos
Vacinas Bacterianas/imunologia , Flagelos/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Anticorpos Antibacterianos/análise , Antígenos de Bactérias/imunologia , Reações Cruzadas , Humanos , Imunização Passiva , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Fagocitose/efeitos dos fármacosRESUMO
A representative collection of meningococci was isolated from cases and healthy carriers in The Gambia between 1982 and 1988, during and after an epidemic of meningococcal meningitis. These bacteria were subjected to a clonal analysis. All serogroup A bacteria from both cases and carriers were of one clone (A IV-1). Several unrelated clones were observed among serogroup 29E and serogroup Y carrier strains. The serogroup A strains were uniform for serotype and subtype antigens (serotype 4, subtype P1.7) and antibiotic sensitivity pattern. Occasional strains varied in their lipopolysaccharide (LPS), DNA fingerprint pattern, and/or the quantitative expression of the class 1 protein. A high degree of strain-specific variation was found for the expression of class 5 proteins, pili, and sulfonamide sensitivity. The frequency of strains expressing reduced amounts of the class 1 protein, altered LPS, and/or increased amounts of capsular polysaccharide rose among case strains obtained after the epidemic had ceased. These strains seem to be generally resistant to antibody-mediated bactericidal activity.
Assuntos
Portador Sadio/microbiologia , Surtos de Doenças , Meningite Meningocócica/microbiologia , Neisseria meningitidis/classificação , Sepse/microbiologia , Anticorpos Monoclonais , Proteínas da Membrana Bacteriana Externa/análise , Portador Sadio/epidemiologia , DNA Bacteriano/análise , Eletroforese em Gel de Poliacrilamida , Gâmbia , Variação Genética , Humanos , Lipopolissacarídeos/análise , Meningite Meningocócica/epidemiologia , Nasofaringe/microbiologia , Neisseria meningitidis/efeitos dos fármacos , Neisseria meningitidis/genética , Mapeamento por Restrição , Sepse/epidemiologia , Sorotipagem , Fatores de TempoRESUMO
Methods published for the purification of P.II proteins from Neisseria gonorrhoea have been modified to allow the purification of class 5 proteins from Neisseria meningitidis serogroup A bacteria. The five class 5 protein electrophoretic variants detected within an epidemic in the Gambia (a, b, c, d, and e) and three other variants (f, g, and h) found within other isolates of the same clone in West Africa have been purified with yields of 6-28 mg. The NH2-terminal amino acid sequence for variant c differs from those of the other class 5 proteins, whereas the latter are very similar to the sequence predicted for two class 5 proteins from DNA analyses of serogroup C meningococci and determined for 8 P.II proteins from gonococci. Numerous other regulatory, chemical, and serological differences were found between the c protein and the other class 5 proteins such that we recommend that the class 5 proteins be subdivided into two subclasses. mAbs have been isolated that distinguish between these two protein subclasses and Western blotting with these antibodies enabled us to conclude that both protein subclasses were found in bacteria isolated from different epidemics and pandemics of the last 50 yr.
Assuntos
Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Neisseria meningitidis/classificação , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Epitopos/análise , Variação Genética , Dados de Sequência Molecular , Peso Molecular , Neisseria meningitidis/genética , SorotipagemAssuntos
Meningite Meningocócica/epidemiologia , Neisseria meningitidis/classificação , Animais , Proteínas da Membrana Bacteriana Externa/análise , Atividade Bactericida do Sangue , Criança , Proteínas do Sistema Complemento/imunologia , Surtos de Doenças/história , Gâmbia , História do Século XX , Humanos , Meningite Meningocócica/história , Neisseria meningitidis/análise , Neisseria meningitidis/imunologia , Coelhos , SorotipagemRESUMO
Two new monoclonal antibodies (MN14G21.17 and MN14E9.15) specific for the serotype 4 antigen of meningococci were isolated. The antibodies were raised against a previously non-typable serogroup B strain from the Netherlands and were shown to react with the serotype antigen of the prototype reference strains for serotype 4 and serogroup A as well as with that of the homologous strain. Further screening of 290 serogroup A and B case isolates with the monoclonal antibodies indicated that the serotype 4 epitope was present on all 100 serogroup A strains tested, including representative isolates from 28 epidemics, and on most isolates from a recent serogroup B epidemic (1981-1983) in Cuba. In addition, 68 isolates from recent sporadic cases (1980-1986) and/or clusters of cases of serogroup B disease in many parts of Europe, the USA, the USSR and Australia were also of this serotype.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/análise , Neisseria meningitidis/imunologia , Especificidade de Anticorpos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/análise , Humanos , Neisseria meningitidis/classificação , SorotipagemRESUMO
We have formerly described a clonal analysis of 423 serogroup A isolates of Neisseria meningitidis which distinguished 21 clones and 34 electrophoretic types. The clones were found to be uniform within distinct epidemiological situations and distinct between different sets of epidemics. Strains representative of the 34 electrophoretic types have now been analyzed by restriction endonuclease digestion of their genomic DNA's. The results correlated partially with the clonal assignments; the discrepancies were epidemiologically more consistent with the interpretation of the clonal analysis than with that of the restriction endonuclease analysis.
Assuntos
DNA Bacteriano/análise , Neisseria meningitidis/classificação , Técnicas de Tipagem Bacteriana , Clonagem Molecular , Eletroforese em Gel de Ágar , Neisseria meningitidis/análise , Mapeamento por RestriçãoRESUMO
A bacterial strain collection has been established consisting of 423 strains of Neisseria meningitidis serogroup A isolated from 23 epidemics or outbreaks that have occurred since 1960 as well as from earlier epidemics and from numerous nonepidemic situations. A combination of starch gel electrophoresis of seven cytoplasmic enzymes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of two outer membrane proteins was used to resolve the clonal population structure of these bacteria. Fifty electrophoretic types were assigned to 21 clones on the basis of a cluster analysis. The clones were separated into four distinct serogroup A subgroups, all of which were isolated from cases as recently as 1983. Most epidemics or outbreaks were characterized by their association with a single or predominant clone, although some epidemics were apparently of mixed etiology and others yielded rare isolates belonging to other clones. Seven predominant clones were recognized that have caused sets of epidemics since 1915. At least two of these sets can be considered to represent mutually exclusive pandemics first detected in 1967 and 1973, respectively. The results define a new typing scheme, which can be used for a comprehensive description of former and future epidemics. A list of strains and their epidemiologic data is appended.
Assuntos
Meningite Meningocócica/microbiologia , Neisseria meningitidis/classificação , Proteínas da Membrana Bacteriana Externa/análise , Surtos de Doenças , Eletroforese em Gel de Poliacrilamida , Neisseria meningitidis/isolamento & purificação , Sorotipagem/métodos , Fatores de TempoRESUMO
A typing scheme has recently been developed for Neisseria meningitidis serogroup A based on the clonal population structure of these bacteria. An international strain collection consisting of 423 group A strains isolated from 23 epidemics or outbreaks since 1963, as well as from older epidemics and numerous non-epidemic situations was used in the analysis. Strains were first segregated into electrophoretic types, depending on the combined score for the electrophoretic mobilities of 7 cytoplasmic isoenzymes resolved by starch gel electrophoresis and of 2 outer membrane proteins resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The bacteria were subsequently assigned to one of 21 clones after numerical analysis of their electrophoretic types. The epidemiological value of the typing scheme was assessed by examining case and carrier strains isolated during (1982-83) and subsequent to (1984-85) an epidemic in the Gambia, West Africa. The case isolates, all of which were serogroup A, were of a single clonal type. All serogroup A carrier isolates were also of this clone, while carrier strains of other serogroups showed greater clonal diversity. These results indicate that case strains during an epidemic show little clonal diversity and thus that the typing scheme is of value in distinguishing the etiology of epidemics. A retrospective epidemiological analysis of the strains in the international collection showed that most serogroup A epidemics were associated with a single or predominant clone, although some epidemics were of mixed etiology.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Portador Sadio/microbiologia , Surtos de Doenças , Meningite Meningocócica/microbiologia , Neisseria meningitidis/classificação , África , Proteínas da Membrana Bacteriana Externa/análise , Eletroforese em Gel de Poliacrilamida , Eletroforese em Gel de Amido , Humanos , Isoenzimas/análise , Meningite Meningocócica/epidemiologia , Neisseria meningitidis/análise , Neisseria meningitidis/enzimologia , Neisseria meningitidis/genética , Estudos RetrospectivosRESUMO
Low-temperature electron spin resonance spectroscopy was used to investigate the redox centres of Micrococcus luteus membranes. Three different types of iron-sulphur centres were distinguished. Two of these, a [4Fe-4S]3+-type cluster giving rise to a signal at g = 2.01 in the oxidized state and a [2Fe-2S] cluster with a spectrum at g = 2.03 and 1.93 in the reduced state, were attributable to succinate dehydrogenase. Another, generating signals in the reduced state at g = 2.027, 1.90 and 1.78 was identified as a 'Rieske' iron-sulphur centre. This latter cluster had a mid-point potential (pH 7.0) of +130 mV. In addition, signals characteristic of high-spin ferric haem (g = 6.20), low-spin ferric haem (g = 3.67, 3.36 and 3.01) and Cu2+ (g = 2.18 and 2.02) were also detected. The ferric-haem features, together with the Cu2+ and 'Rieske' centres, were enriched in membrane residues insoluble in Triton X-100, which are known from difference spectroscopy to contain cytochromes b-560, c-550 and a-601 (aa3 oxidase). The signals demonstrated by electron spin resonance for M. luteus membranes showed marked similarities to those documented for the complexes II, III, and IV of mitochondria. However, signals analogous to complex I (NADH-ubiquinone reductase) could not be demonstrated for M. luteus membranes.
Assuntos
Transporte de Elétrons , Micrococcus/metabolismo , Proteínas de Bactérias/análise , Membrana Celular/análise , Cobre/análise , Espectroscopia de Ressonância de Spin Eletrônica , Heme/análise , Ferro/análise , Potenciais da Membrana , Proteínas de Membrana/análise , Oxirredução , Solubilidade , Enxofre/análiseRESUMO
Low-temperature electron spin resonance spectroscopy has been used to study the biophysical properties of succinate dehydrogenase from the gram-positive bacterium Micrococcus luteus. The paramagnetic redox centres of the enzyme were identified in a succinate-dehydrogenase--antigen complex, which had been purified with the aid of monospecific serum from membranes solubilized with Triton X-100. The centres were characterized in further detail using the membrane-bound and Triton-solubilized forms of the enzyme. These studies distinguished two types of iron-sulphur centres, viz. a [4Fe-4S]3+ cluster displaying a narrow signal at g = 2.01 in the oxidized state (conventionally termed centre S-3) and a [2Fe-2S )0 cluster with an axial signal at g = 2.03 and 1.93 in the reduced state (conventionally termed centre S-1). Centre S-3 had a mid-point redox potential of +10 mV, a comparatively low value for this type of cluster. The behaviour of the g = 1.93 signal of centre S-1 was a complex function of the redox potential, microwave power and temperature of measurement. When measured at low power (i.e. non-saturating conditions), the intensities observed for the g = 1.93 signal poised at various critical potentials in the redox titration were similar. However, the corresponding intensities differed markedly at high power, where conditions were saturating. It is proposed that under saturating conditions the spin-lattice relaxation of the [2Fe-2S] cluster S-1 (mid-point potential +70 mV) is enhanced by centre S-3 between the potential range +10-+70 mV and by an ESR-silent centre, termed centre S-2, with a mid-point potential of -295 mV.
Assuntos
Micrococcus/enzimologia , Succinato Desidrogenase/análise , Precipitação Química , Espectroscopia de Ressonância de Spin Eletrônica , Imunoquímica , Potenciais da Membrana , OxirreduçãoRESUMO
Succinate dehydrogenase (EC 1.3.99.1) of Micrococcus luteus was selectively precipitated from Triton X-100-solubilized membranes by using specific antiserum. The precipitated enzyme contained equimolar amounts of four polypeptides with apparent molecular weights of 72,000, 30,000, 17,000, and 15,000. The 72,000 polypeptide possessed a covalently bound flavin prosthetic group and appeared to be strongly antigenic as judged by immunoprinting experiments. Low-temperature absorption spectroscopy revealed the presence of cytochrome b556 in the antigen complex. By analogy with succinate dehydrogenase purified from other sources, the 72,000 and 30,000 polypeptides were considered to represent subunits of the succinate dehydrogenase enzyme, whereas one (or both) of the low-molecular-weight polypeptides was attributed to the apoprotein of the b-type cytochrome. A succinate dehydrogenase antigen cross-reacting with the M. luteus enzyme complex could be demonstrated in membranes of Micrococcus roseus, Micrococcus flavus, and Sarcina lutea, but not in the membranes isolated from a wide variety of other gram-positive and gram-negative bacteria.
Assuntos
Micrococcus/enzimologia , Succinato Desidrogenase/isolamento & purificação , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Imunodifusão , Imunoeletroforese , Substâncias Macromoleculares , Peso Molecular , Octoxinol , PolietilenoglicóisRESUMO
Membrane-bound antigens of the respiratory chain of Micrococcus luteus were analyzed by crossed immunoelectrophoresis after growth of the organism in the presence of 59Fe, the flavin adenine dinucleotide-flavin mononucleotide precursor D-[2-14C]riboflavin, or the heme precursor 5-amino-[4-(14)C]levulinic acid. Using zymograms and procedures of selective extraction in conjunction with autoradiography, it was possible to resolve and partially characterize a number of antigens. Succinate dehydrogenase (EC 1.3.99.1) was shown to possess covalently bound flavin and nonheme iron and was possibly present as a complex with cytochrome. Three other dehydrogenases, namely, NADH dehydrogenase, NAD(P)H dehydrogenase (EC 1.6.99.3), and malate dehydrogenase (EC 1.1.1.37), contained flavin in noncovalent linkage, the NAD(P)H dehydrogenase also possessing nonheme iron. Four other discrete antigens (or antigen complexes) containing both iron and heme centers also resolved, as were two minor immunogens possessing iron as the sole detectable prosthetic group.