RESUMO
For the development of a third generation of pertussis vaccine that could improve the control of the disease, it was proposed that the immune responses induced by the classic whole cell vaccine (wP) or after infection should be used as a reference point. We have recently identified a vaccine candidate based on outer membrane vesicles (OMVs) derived from the disease etiologic agent that have been shown to be safe and protective in mice model of infection. Here we characterized OMVs-mediated immunity and the safety of our new candidate. We also deepen the knowledge of the induced humoral response contribution in pertussis protection. Regarding the safety of the OMVs based vaccine (TdapOMVsBp,) the in vitro whole blood human assay here performed, showed that the low toxicity of OMVs-based vaccine previously detected in mice could be extended to human samples. Stimulation of splenocytes from immunized mice evidenced the presence of IFN-γ and IL-17-producing cells, indicated that OMVs induces both Th1 and Th17 response. Interestingly TdapOMVsBp-raised antibodies such as those induced by wP and commercial acellular vaccines (aP) which contribute to induce protection against Bordetella pertussis infection. As occurs with wP-induced antibodies, the TdapOMVsBp-induced serum antibodies efficiently opsonized B. pertussis. All the data here obtained shows that OMVs based vaccine is able to induce Th1/Th17 and Th2 mixed profile with robust humoral response involved in protection, positioning this candidate among the different possibilities to constitute the third generation of anti-pertussis vaccines.
Assuntos
Imunidade Humoral , Vacina contra Coqueluche/imunologia , Coqueluche/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Bordetella pertussis , Células Cultivadas , Feminino , Humanos , Soros Imunes/imunologia , Interferon gama/imunologia , Interleucina-17/imunologia , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose , Células RAW 264.7 , Baço/citologia , Baço/imunologia , Células Th17/imunologia , Vacinas Acelulares/imunologiaRESUMO
Although Bordetella pertussis contains and transcribes loci encoding type III secretion system (TTSS) homologues, expression of TTSS-associated proteins has been reported only for non-laboratory-adapted Irish clinical isolates. Here we confirm such a result for clinical isolates obtained from patients treated in Argentinean hospitals. Moreover, we demonstrate that the expression of TTSS-associated proteins is independent both of the year in which the isolate was obtained and of the types of polymorphic alleles for other virulence factors but is dependent on environmental growth conditions. Interestingly, we observed that TTSS-associated protein expression is lost after successive in vitro passages but becomes operative again when bacteria come into contact with the host. This in vivo activation of TTSS expression was observed not only for clinical isolates previously adapted to the laboratory after successive in vitro passages but also for vaccine strains that did not express the system in vitro. The reversibility of TTSS expression, demonstrated by its switching off-on when the bacterium comes into contact with the host, appears to be an adaptive response of this pathogen.
Assuntos
Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Bordetella pertussis/genética , Bordetella pertussis/patogenicidade , Fatores de Virulência/genética , Alelos , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Meio Ambiente , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Polimorfismo Genético , Fatores de Virulência/metabolismo , Coqueluche/microbiologia , Coqueluche/patologiaRESUMO
OBJECTIVES: Pertussis continues causing significant morbidity and mortality worldwide. Although its epidemiology has been studied in many developed countries, the current pertussis situation in South America is scarcely known. This review summarizes the most important recent data concerning pertussis in a country of South America, Argentina. METHODS: CDC criteria were used for pertussis diagnosis. Proportion of pertussis cases by age, immunization status, and immunization coverage rate evaluated at the Argentinean National Pertussis Reference Centers was reported. Bordetella pertussis isolates were characterized and compared with vaccine strains. RESULTS: From 2002 to nowadays, a steady increase of pertussis cases was observed. Most of these cases correspond to patients younger than six months old that received less than three doses of vaccine. However, cases in adolescent and adults have also been detected. For this situation, which is not peculiar to Argentina, several explanations have been proposed. Among them, the inability of current vaccines to induce long-lasting immunity is the most widely accepted as a cause of pertussis resurgence. Furthermore, antigenic divergence between local clinical isolates and vaccine strains may have aggravated the effect of waning immunity. CONCLUSIONS: Pertussis is an important problem for public health in Argentina. Divergence between vaccine strains and local isolates could contribute to the described pertussis epidemiology.
Assuntos
Coqueluche/epidemiologia , Adolescente , Argentina/epidemiologia , Bordetella pertussis/classificação , Bordetella pertussis/isolamento & purificação , Criança , Pré-Escolar , Impressões Digitais de DNA , Humanos , Imunoterapia Ativa/estatística & dados numéricos , Incidência , Lactente , Recém-Nascido , Coqueluche/diagnósticoRESUMO
The phospholipid and fatty acid composition of the brain and spinal cord lipids of fresh water fish Prochilodus lineatus ("sábalo") were studied. The fluorescence anisotropy of phospholipids labelled with 1.6-diphenyl 1.3.5-hexatriene, was also investigated. Phosphatidyl-choline was the most abundant phospholipid followed by phosphatidyl-ethanolamine, phosphatidyl-serine, phosphatidyl-inositol and sphingomyelin. The fatty acid composition of all phospholipids except sphingomyelin showed the presence of n-9, n-6 and n-3 series of unsaturated acids. The presence of n-6 polyunsaturated fatty acid was represented by arachidonic acid that was found preferentially in phosphatidyl-inositol. N-3 fatty acids were represented by 20:5n-3, 22:5n-3 and 22:6n-3. Docosa 4,7,10,13,16,19 hexaenoic acid was the most abundant of n-3 fatty acids and was preferentially found in phosphatidyl ethanolamine and phosphatidyl-serine. The percentage of 22:6n-3 acid decreased with age. The simultaneous presence of n-6 fatty acids (arachidonic acid) and n-3 fatty acids in fresh water fishes contrast with the absence of n-6 fatty acids in nervous tissue of marine fishes. Phosphatidyl choline was the most fluid of all phospholipids from brain and spinal cord.
Assuntos
Química Encefálica , Ácidos Graxos/análise , Peixes , Fosfolipídeos/análise , Medula Espinal/química , Animais , Ácido Araquidônico/química , Polarização de Fluorescência , Água Doce , Fosfatidilcolinas/químicaRESUMO
The phospholipid and fatty acid composition of the brain and spinal cord lipids of fresh water fish Prochilodus lineatus ([quot ]sábalo[quot ]) were studied. The fluorescence anisotropy of phospholipids labelled with 1.6-diphenyl 1.3.5-hexatriene, was also investigated. Phosphatidyl-choline was the most abundant phospholipid followed by phosphatidyl-ethanolamine, phosphatidyl-serine, phosphatidyl-inositol and sphingomyelin. The fatty acid composition of all phospholipids except sphingomyelin showed the presence of n-9, n-6 and n-3 series of unsaturated acids. The presence of n-6 polyunsaturated fatty acid was represented by arachidonic acid that was found preferentially in phosphatidyl-inositol. N-3 fatty acids were represented by 20:5n-3, 22:5n-3 and 22:6n-3. Docosa 4,7,10,13,16,19 hexaenoic acid was the most abundant of n-3 fatty acids and was preferentially found in phosphatidyl ethanolamine and phosphatidyl-serine. The percentage of 22:6n-3 acid decreased with age. The simultaneous presence of n-6 fatty acids (arachidonic acid) and n-3 fatty acids in fresh water fishes contrast with the absence of n-6 fatty acids in nervous tissue of marine fishes. Phosphatidyl choline was the most fluid of all phospholipids from brain and spinal cord.
RESUMO
ESR spectra of spin probes were used to monitor lipid-protein interactions in native and cholesterol-enriched microsomal membranes. In both systems composite spectra were obtained, one characteristic of bulk bilayer organization and another due to a motionally restricted population, which was ascribed to lipids in a protein microenvironment. Computer spectral subtractions revealed that cholesterol modulates the order/mobility of both populations in opposite ways, i.e., while the lipid bilayer region gives rise to more anisotropic spectra upon cholesterol enrichment, the spectra of the motionally restricted population become indicative of increased mobility and/or decreased order. These events were evidenced by measurement of both effective order parameters and correlation times. The percentages of the motionally restricted component were invariant in native and cholesterol-enriched microsomes. Variable temperature studies also indicated a lack of variation of the percentages of both spectral components, suggesting that the motionally restricted one was not due to protein aggregation. The results correlate well with the effect of cholesterol enrichment on membrane-bound enzyme kinetics and on the behavior of fluorescent probes [Castuma & Brenner (1986) Biochemistry 25, 4733-4738]. Several hypothesis are put forward to explain the molecular mechanism of the cholesterol-induced spectral changes.
Assuntos
Colesterol/química , Lipídeos de Membrana/química , Proteínas de Membrana/química , Microssomos Hepáticos/química , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Cobaias , Técnicas In Vitro , Fluidez de Membrana , Movimento (Física) , Marcadores de Spin , TemperaturaRESUMO
This work studies the phospholipid and fatty acid composition in hake brain and spinal cord and in sea bass brain. Fluorescence anisotropy of phospholipid vesicles labeled with 1,6-diphenyl hexatriene was measured to investigate the associated dynamic properties. In all tissues studied, phosphatidylcholine and phosphatidylethanolamine were the major constituents with minor contributions of phosphatidylserine, phosphatidylinositol and sphingomyelin. Fatty acids belong to the n-9 and n-3 series exclusively. Phosphatidylinositol from hake spinal cord and phosphatidylethanolamine and phosphatidylserine from hake brain contain the greatest percentages of eicosa-5,8,11,14,17-pentaenoic (20:5) and docosa-4,7,10,13,16,19-hexaenoic (22:6), respectively. For all fractions studied the total content of saturated fatty acids increases in the order of hake spinal cord, hake brain, sea bass brain together with a decrease in the sum of monounsaturated fatty acids. The comparison between fluorescence anisotropy values and fatty acid composition clearly demonstrates that saturated acids and 20:5 and 22:6 exert a rigidizing effect.
Assuntos
Química Encefálica , Ácidos Graxos/análise , Peixes/metabolismo , Fosfolipídeos/análise , Medula Espinal/química , Animais , Bass/metabolismo , Encéfalo/metabolismo , Difenilexatrieno , Ácidos Graxos/metabolismo , Ácidos Graxos Monoinsaturados/análise , Polarização de Fluorescência , Metabolismo dos Lipídeos , Lipídeos/análise , Fluidez de Membrana , Fosfatidilcolinas/análise , Fosfatidiletanolaminas/análise , Fosfatidilinositóis/análise , Fosfatidilserinas/análise , Fosfolipídeos/metabolismo , Espectrometria de Fluorescência , Esfingomielinas/análise , Medula Espinal/metabolismoRESUMO
The relationship between lipid composition, the physical properties of microsomal phospholipids and the kinetics of liver UDP-glucuronyltransferase was studied in microsomes from guinea pigs supplied with a normal or a fat-free diet for 28 days. Fatty acid deficiency did not modify either the cholesterol/phospholipid molar ratio or the polar head group composition, but exclusively redistributed the unsaturated fatty acid pattern, by partially exchanging oleic for linoleic acid. This phenomenon accounts for the decrease of both rotational and translational mobilities of the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and pyrene respectively. When the thermotropic behaviour of the different systems was assessed, no transition temperature (gel-liquid-crystalline) between 10 and 40 degrees C was seen as a consequence of the lower degree of unsaturation, either in the microsomal membranes or in the total lipid or total phospholipid extracts from the treated animals. In spite of this, the polarization ratio of trans-parinaric acid and the fluorescence intensity of merocyanine 540 revealed that a significant lateral phase separation occurred at 20-22 degrees C in the extracted phospholipids, which was smoother in the total lipid fractions and in the native microsomal membranes. Fatty acid deficiency caused an upward shift of the midpoint temperature of the lateral phase separation. Furthermore, the phosphatidylcholine extracted from the 'normal' microsomes showed a lateral phase separation centred at a lower temperature than that extracted from 'fat-deficient' microsomes. In contrast, the Arrhenius plot of UDP-glucuronyltransferase from 'normal' microsomes exhibited a change in slope at a higher temperature than that from treated microsomes. These results would suggest that fatty acid deficiency in guinea-pig liver microsomes, while rigidizing the bulk lipids, would segregate the most unsaturated phosphatidylcholine molecules towards the UDP-glucuronyltransferase microenvironment, in accordance with our previous results with cholesterol incorporation [Castuma & Brenner (1986) Biochemistry 25, 4733-4738].