RESUMO
BACKGROUND: As a result of effective combination antiretroviral therapy (cART) and advanced supportive healthcare, a growing number of human immunodeficiency virus (HIV)-infected children survive into adulthood. The period of transition to adult care is often associated with impaired adherence to treatment and discontinuity of care. We aimed to evaluate virological and social outcomes of HIV-infected adolescents and young adults (AYAs) before and after transition, and explore which factors are associated with virological failure. METHODS: We included 59 HIV-infected AYAs from the Netherlands who had entered into pediatric care and transitioned from pediatric to adult healthcare. We used HIV RNA load and cART data from the Dutch Stichting HIV Monitoring database (1996-2014), and collected social and treatment data from patients' medical records from all Dutch pediatric HIV treatment centers and 14 Dutch adult treatment centers involved. We evaluated risk factors for virological failure (VF) in a logistic regression model adjusted for repeated measurements. RESULTS: HIV VF occurred frequently during the study period (14%-36%). During the transition period (from 18 to 19 years of age) there was a significant increase in VF compared with the reference group of children aged 12-13 years (odds ratio, 4.26 [95% confidence interval, 1.12-16.28]; P = .03). Characteristics significantly associated with VF were low educational attainment and lack of autonomy regarding medication adherence at transition. CONCLUSIONS: HIV-infected AYAs are vulnerable to VF, especially during the transition period. Identification of HIV-infected adolescents at high risk for VF might help to improve treatment success in this group.
Assuntos
Infecções por HIV/epidemiologia , Transição para Assistência do Adulto , Adolescente , Fatores Etários , Terapia Antirretroviral de Alta Atividade , Criança , Pré-Escolar , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/transmissão , Infecções por HIV/virologia , Humanos , Perda de Seguimento , Masculino , Países Baixos/epidemiologia , Razão de Chances , Fatores de Risco , Fatores Socioeconômicos , Falha de Tratamento , Resultado do Tratamento , Adulto JovemRESUMO
ß-Propiolactone is often applied for inactivation of viruses and preparation of viral vaccines. However, the exact nature of the reactions of ß-propiolactone with viral components is largely unknown. The purpose of the current study was to elucidate the chemical modifications occurring on nucleotides and amino acid residues caused by ß-propiolactone. Therefore, a set of nucleobase analogues was treated with ß-propiolactone, and reaction products were identified and quantified. NMR revealed at least one modification in either deoxyguanosine, deoxyadenosine, or cytidine after treatment with ß-propiolactone. However, no reaction products were found from thymidine and uracil. The most reactive sides of the nucleobase analogues and nucleosides were identified by NMR. Furthermore, a series of synthetic peptides was used to determine the conversion of reactive amino acid residues by liquid chromatography-mass spectrometry. ß-Propiolactone was shown to react with nine different amino acid residues. The most reactive residues are cysteine, methionine, and histidine and, to a lesser degree, aspartic acid, glutamic acid, tyrosine, lysine, serine, and threonine. Remarkably, cystine residues (disulfide groups) do not react with ß-propiolactone. In addition, no reaction was observed for ß-propiolactone with asparagine, glutamine, and tryptophan residues. ß-Propiolactone modifies proteins to a larger extent than expected from current literature. In conclusion, the study determined the reactivity of ß-propiolactone with nucleobase analogues, nucleosides, and amino acid residues and elucidated the chemical structures of the reaction products. The study provides detailed knowledge on the chemistry of ß-propiolactone inactivation of viruses.
Assuntos
Desinfetantes/química , Nucleosídeos/química , Peptídeos/química , Propiolactona/química , Proteínas Virais/química , Inativação de Vírus , Vírus/químicaRESUMO
Acridinium esters traditionally are triggered using basic hydrogen peroxide. By serendipity, we have found that acridinium esters can also be triggered with emission of chemiluminescence by reductive triggering, e.g., by zinc metal or reduced forms of ferric and cupric salts. Furthermore, organic reducing compounds like dithiothreitol, tricarboxyethylphosphine or glutathione could be used in combination with organic oxidants like quinones or inorganic ferric or cupric salts. Mechanisms are proposed which involve the intermediacy of superoxide. Two forms of reactive oxygen species (i.e., hydrogen peroxide and superoxide) could be discriminated based on differences in kinetics. Some applications (improved detection of acridinium ester, use of acridinium ester as redox probes) are discussed.
Assuntos
Acridinas/química , Imunoensaio , Indicadores e Reagentes/química , Hibridização de Ácido Nucleico/métodos , Coloração e Rotulagem/métodos , Acridinas/análise , Ésteres/análise , Ésteres/química , Glutationa/química , Glutationa/metabolismo , Peróxido de Hidrogênio , Cinética , Luminescência , Metais Pesados/química , Oxirredução , Fosfinas/química , Fosfinas/metabolismo , Quinonas/química , Quinonas/metabolismo , Superóxidos/químicaRESUMO
Neisseria meningitidis serogroup B is a pathogen that can infect diverse sites within the human host. According to the N. meningitidis genomic information and experimental observations, glucose can be completely catabolized through the Entner-Doudoroff pathway and the pentose phosphate pathway. The Embden-Meyerhof-Parnas pathway is not functional, because the gene for phosphofructokinase (PFK) is not present. The phylogenetic distribution of PFK indicates that in most obligate aerobic organisms, PFK is lacking. We conclude that this is because of the limited contribution of PFK to the energy supply in aerobically grown organisms in comparison with the energy generated through oxidative phosphorylation. Under anaerobic or microaerobic conditions, the available energy is limiting, and PFK provides an advantage, which explains the presence of PFK in many (facultatively) anaerobic organisms. In accordance with this, in silico flux balance analysis predicted an increase of biomass yield as a result of PFK expression. However, analysis of a genetically engineered N. meningitidis strain that expressed a heterologous PFK showed that the yield of biomass on substrate decreased in comparison with a pfkA-deficient control strain, which was associated mainly with an increase in CO(2) production, whereas production of by-products was similar in the two strains. This might explain why the pfkA gene has not been obtained by horizontal gene transfer, since it is initially unfavourable for biomass yield. No large effects related to heterologous expression of pfkA were observed in the transcriptome. Although our results suggest that introduction of PFK does not contribute to a more efficient strain in terms of biomass yield, achievement of a robust, optimal metabolic network that enables a higher growth rate or a higher biomass yield might be possible after adaptive evolution of the strain, which remains to be investigated.
Assuntos
Neisseria meningitidis Sorogrupo B/enzimologia , Fosfofrutoquinases/biossíntese , Biomassa , Clonagem Molecular , Escherichia coli/genética , Perfilação da Expressão Gênica , Redes e Vias Metabólicas , Neisseria meningitidis Sorogrupo B/classificação , Neisseria meningitidis Sorogrupo B/genética , Fosfofrutoquinases/genética , Filogenia , RNA Bacteriano/genéticaRESUMO
BACKGROUND: Despite nearly complete vaccine coverage, a small number of fully vaccinated children in the Netherlands have experienced invasive disease caused by Haemophilus influenzae serotype b (Hib). This increase started in 2002, nine years after the introduction of nationwide vaccination in the Netherlands. The capsular polysaccharide of Hib is used as a conjugate vaccine to protect against Hib disease. To evaluate the possible rise of escape variants, explaining the increased number of vaccine failures we analyzed the composition of the capsular genes and the expressed polysaccharide of Dutch Hib strains collected before and after the introduction of Hib vaccination. RESULTS: The DNA sequences of the complete capsular gene clusters of 9 Dutch Hib strains were assessed and two variants, designated type I and type II were found. The two variants displayed considerable sequence divergence in the hcsA and hcsB genes, involved in transport of capsular polysaccharide to the cell surface. Application of hcsA type specific PCRs on 670 Hib strains collected from Dutch patients with invasive Hib disease showed that 5% of the strains collected before 1996 were type II. No endogenous type II Hib strains were isolated after 1995 and all type II strains were isolated from 0-4 year old, non-vaccinated children only. Analysis of a worldwide collection of Hib strains from the pre-vaccination era revealed considerable geographic differences in the distribution of the type I and type II strains with up to 73% of type II strains in the USA. NMR analysis of type I and type II capsule polysaccharides did not reveal structural differences. However, type I strains were shown to produce twice as much surface bound capsular polysaccharide. CONCLUSION: Type II strains were only isolated during the pre-vaccination era from young, non-vaccinated individuals and displayed a lower expression of capsular polysaccharide than type I strains. The higher polysaccharide expression may have provided a selective advantage for type I strains resulting in the rapid elimination of type II from the Dutch Hib population after introduction of nationwide Hib vaccination. However, this phenomenon does not explain the increase in the number of Hib vaccine failures in the Netherlands.
Assuntos
Cápsulas Bacterianas/genética , Genes Bacterianos , Infecções por Haemophilus/microbiologia , Haemophilus influenzae tipo b/genética , Haemophilus influenzae tipo b/metabolismo , Polissacarídeos Bacterianos/metabolismo , Idoso , Cápsulas Bacterianas/classificação , Criança , Pré-Escolar , Variação Genética , Infecções por Haemophilus/prevenção & controle , Vacinas Anti-Haemophilus/administração & dosagem , Haemophilus influenzae tipo b/classificação , Humanos , Lactente , Proteínas de Membrana Transportadoras/genética , Países Baixos/epidemiologia , Reação em Cadeia da Polimerase , Polissacarídeos Bacterianos/genética , Sorotipagem , VacinaçãoRESUMO
Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. At the Netherlands Vaccine Institute (NVI) a vaccine against serogroup B organisms is currently being developed. This study describes the influence of the growth rate of N. meningitidis on its macro-molecular composition and its metabolic activity and was determined in chemostat cultures. In the applied range of growth rates, no significant changes in RNA content and protein content with growth rate were observed in N. meningitidis. The DNA content in N. meningitidis was somewhat higher at the highest applied growth rate. The phospholipid and lipopolysaccharide content in N. meningitidis changed with growth rate but no specific trends were observed. The cellular fatty acid composition and the amino acid composition did not change significantly with growth rate. Additionally, it was found that the PorA content in outer membrane vesicles was significantly lower at the highest growth rate. The metabolic fluxes at various growth rates were calculated using flux balance analysis. Errors in fluxes were calculated using Monte Carlo Simulation and the reliability of the calculated flux distribution could be indicated, which has not been reported for this type of analysis. The yield of biomass on substrate (Y(x/s)) and the maintenance coefficient (m(s)) were determined as 0.44 (+/-0.04) g g(-1) and 0.04 (+/-0.02) g g(-1) h(-1), respectively. The growth associated energy requirement (Y(x/ATP)) and the non-growth associated ATP requirement for maintenance (m(ATP)) were estimated as 0.13 (+/-0.04) mol mol(-1) and 0.43 (+/-0.14) mol mol(-1) h(-1), respectively. It was found that the split ratio between the Entner-Doudoroff and the pentose phosphate pathway, the sole glucose utilizing pathways in N. meningitidis, had a minor effect on ATP formation rate but a major effect on the fluxes going through for instance the citric-acid cycle. For this reason, we presented flux ranges for underdetermined parts of metabolic network rather than presenting single flux values, which is more commonly done in literature.
Assuntos
Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Neisseria meningitidis Sorogrupo B/crescimento & desenvolvimento , Neisseria meningitidis Sorogrupo B/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoácidos/análise , DNA Bacteriano/análise , Metabolismo Energético/fisiologia , Ácidos Graxos/análise , Glucose/metabolismo , Cinética , Lipopolissacarídeos/análise , Método de Monte Carlo , Neisseria meningitidis Sorogrupo B/genética , Via de Pentose Fosfato/fisiologia , Fosfolipídeos/análise , Porinas/análise , RNA Bacteriano/análise , Reprodutibilidade dos TestesRESUMO
The use of detergent-extracted outer membrane vesicles (OMVs) is an established approach for development of a multivalent PorA vaccine against N. meningitidis serogroup B. Selective removal of lipopolysaccharide (LPS) decreases toxicity, but promotes aggregation and narrows the immune response. Detergent-free OMV vaccines retain all LPS, which preserves the native vesicle structure, but result in high toxicity and lower yield. The present study assessed the effects of gene mutations that attenuated LPS toxicity (lpxL1) or improved OMV yield (rmpM) in combination with the available OMV purification processes. The results substantiate that OMVs from a strain with both mutations, produced with a detergent-free process provide better vaccine characteristics than the traditional detergent-based approach. With comparable toxicity and yield, no aggregation and cross-protection against other PorA subtypes, these OMV vaccines are potentially safe and effective for parenteral use in humans.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Infecções Meningocócicas/imunologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/genética , Neisseria meningitidis Sorogrupo B/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Engenharia Genética , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/toxicidade , Vacinas Meningocócicas/química , Vacinas Meningocócicas/toxicidade , Camundongos , MutaçãoAssuntos
Terapia Antirretroviral de Alta Atividade/efeitos adversos , Glicemia/metabolismo , Fluordesoxiglucose F18 , Infecções por HIV/tratamento farmacológico , Síndrome de Lipodistrofia Associada ao HIV/diagnóstico por imagem , Compostos Radiofarmacêuticos , Adulto , Fluordesoxiglucose F18/farmacocinética , Síndrome de Lipodistrofia Associada ao HIV/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons/métodos , Estudos Prospectivos , Compostos Radiofarmacêuticos/farmacocinéticaRESUMO
BACKGROUND: Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years. RESULTS: Using the genomic database of N. meningitidis serogroup B together with biochemical and physiological information in the literature we constructed a genome-scale flux model for the primary metabolism of N. meningitidis. The validity of a simplified metabolic network derived from the genome-scale metabolic network was checked using flux-balance analysis in chemostat cultures. Several useful predictions were obtained from in silico experiments, including substrate preference. A minimal medium for growth of N. meningitidis was designed and tested successfully in batch and chemostat cultures. CONCLUSION: The verified metabolic model describes the primary metabolism of N. meningitidis in a chemostat in steady state. The genome-scale model is valuable because it offers a framework to study N. meningitidis metabolism as a whole, or certain aspects of it, and it can also be used for the purpose of vaccine process development (for example, the design of growth media). The flux distribution of the main metabolic pathways (that is, the pentose phosphate pathway and the Entner-Douderoff pathway) indicates that the major part of pyruvate (69%) is synthesized through the ED-cleavage, a finding that is in good agreement with literature.
Assuntos
Biologia Computacional , Genoma Bacteriano , Modelos Biológicos , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Redes e Vias Metabólicas/genéticaRESUMO
The production of acellular pertussis in comparison with whole cell pertussis vaccines demands 5-25 times the amount of Bordetella pertussis' virulence factors, such as Pertussis Toxin (PT), to produce the same number of vaccine doses. An increase in the volumetric productivity by employing fed-batch rather than the currently used batch cultivations of B. pertussis could reduce the cost price of acellular pertussis vaccines. This study defined the conditions that enable fed-batch cultivations at high specific PT production. A solution containing lactate and glutamate was fed to the cultures at various rates. The feed rate and whether or not the fed substrates were completely consumed, significantly influenced cellular metabolism. If lactate was detectable in the culture broth while glutamate was not, poly-hydroxy-butyrate (PHB) was formed. Any PHB present was metabolized when glutamate became detectable again in the culture liquid. At higher lactate and glutamate concentrations, free fatty acids were produced. Though toxic, free fatty acids were not the reason the cultures stopped growing. By choosing appropriate conditions, a cell density of 6.5 g/L dry weight was reached, i.e. a 7-fold increase compared to batch culture. The metabolic mechanisms behind the formation of PHB and fatty acids are discussed, as well as how to increase the cell density further. The PT production stopped at 12 mg/L, well before growth stopped, indicating that regulatory mechanisms of PT production may be involved.
Assuntos
Bordetella pertussis/crescimento & desenvolvimento , Bordetella pertussis/metabolismo , Toxina Pertussis/biossíntese , Técnicas Bacteriológicas , Modelos Biológicos , OxirreduçãoRESUMO
BACKGROUND: Adherence to protease inhibitor-containing antiretroviral therapy is crucial, but difficult to measure. OBJECTIVE: To compare and combine various methods of measuring adherence to the strict protease inhibitor-containing regimens. METHODS: The following methods were used: medication event monitoring system (MEMS) caps (electronic monitoring), therapeutic drug monitoring, pill count, pharmacy refill data, questionnaires, diaries (for registration of food patterns and special events related to the use of MEMS), adherence assessment by the physician and clinical nurse specialist, and in-depth interviews. In addition, ultrasensitive viral load and resistance testing was performed. RESULTS: Twenty-eight patients were included; data could be evaluated in 26. According to MEMS data, 25% of the patients took fewer than 95% of all doses, and two thirds of the patients took fewer than 95% of the doses on time. Only 43% of the patients showed good adherence with food restrictions. Methods that showed significant correlations with MEMS results were patients' self-reported adherence; therapeutic drug monitoring, indicating plasma levels outside predefined ranges; and estimation of adherence by a clinical nurse specialist, especially by in-depth interview. CONCLUSION: Diary-corrected MEMS data gave a detailed insight into patients' adherence patterns. Patients' self-report and therapeutic drug monitoring were significantly correlated with the MEMS data, and the clinical nurse specialist may also play a role in identifying patients who are imperfectly adherent.