RESUMO
There is a high prevalence of aeroallergen sensitivity in asthmatic populations, and seroreactivity to aeroallergens early in infancy is associated with increased risk of developing asthma later in life. In addition to allergen sensitivity, asthma development has been associated with differential microbial exposure and infection in early life. We have previously shown that cord blood mononuclear cells respond to common aeroallergens (i.e., house dust mite [Der f1] and cockroach [Bla g2]) as assayed by lymphoproliferation and cytokine (IL-13 and IFN-γ) production. We hypothesized that there is a relationship between perinatal microbial exposure and response to specific aeroallergens. To test this hypothesis, we isolated DNA from cord blood serum samples with known lymphoproliferative and cytokine responses to Bla g2 and Der f1. Bacterial 16S ribosomal DNA amplicon libraries were generated and analyzed using high throughput sequencing of cord blood serum samples. In our analysis, we identified major compositional differences, including diversity and abundance of specific taxa, between groups whose IL-13 response to Der f1 and Bla g2 differed. We demonstrate a strong association between the ratio of Acinetobacter to Proteobacteria and IL-13 production and the probability of IL-13 production after allergen exposure. IL-13 concentrations in serum were also significantly correlated with the diversity of bacterial DNA. Together, these results underscore the relationship between immune responses to allergens and bacterial exposure during perinatal development.
Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Ácido Aspártico Endopeptidases/imunologia , Asma/imunologia , Infecções Bacterianas/imunologia , Cisteína Endopeptidases/imunologia , Exposição Ambiental/efeitos adversos , Interleucina-13/imunologia , Acinetobacter/imunologia , Asma/epidemiologia , Asma/microbiologia , Infecções Bacterianas/epidemiologia , DNA Bacteriano/imunologia , DNA Ribossômico/imunologia , Feminino , Humanos , Recém-Nascido , Interferon gama/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Masculino , RNA Ribossômico 16S/imunologiaRESUMO
Common variable immunodeficiency (CVID) is characterized by profound primary antibody defects and frequent infections, yet autoimmune/inflammatory complications of unclear origin occur in 50% of individuals and lead to increased mortality. Here, we show that circulating bacterial 16S rDNA belonging to gut commensals was significantly increased in CVID serum (P < 0.0001), especially in patients with inflammatory manifestations (P = 0.0007). Levels of serum bacterial DNA were associated with parameters of systemic immune activation, increased serum IFN-γ, and the lowest numbers of isotype-switched memory B cells. Bacterial DNA was bioactive in vitro and induced robust host IFN-γ responses, especially among patients with CVID with inflammatory manifestations. Patients with X-linked agammaglobulinemia (Bruton tyrosine kinase [BTK] deficiency) also had increased circulating bacterial 16S rDNA but did not exhibit prominent immune activation, suggesting that BTK may be a host modifier, dampening immune responses to microbial translocation. These data reveal a mechanism for chronic immune activation in CVID and potential therapeutic strategies to modify the clinical outcomes of this disease.
Assuntos
Agamaglobulinemia/sangue , Imunodeficiência de Variável Comum/sangue , DNA Bacteriano/sangue , DNA Ribossômico/sangue , Microbioma Gastrointestinal/genética , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Inflamação/sangue , Adolescente , Adulto , Agamaglobulinemia/imunologia , Idoso , Anemia Hemolítica Autoimune/sangue , Anemia Hemolítica Autoimune/complicações , Anemia Hemolítica Autoimune/imunologia , Linfócitos B/imunologia , Translocação Bacteriana , Criança , Pré-Escolar , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/imunologia , DNA Bacteriano/imunologia , DNA Ribossômico/imunologia , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Granuloma/sangue , Granuloma/complicações , Granuloma/imunologia , Humanos , Switching de Imunoglobulina , Memória Imunológica/imunologia , Inflamação/imunologia , Interferon gama/sangue , Doenças Pulmonares Intersticiais/sangue , Doenças Pulmonares Intersticiais/complicações , Doenças Pulmonares Intersticiais/imunologia , Masculino , Pessoa de Meia-Idade , Poliendocrinopatias Autoimunes/sangue , Poliendocrinopatias Autoimunes/complicações , Poliendocrinopatias Autoimunes/imunologia , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/complicações , Púrpura Trombocitopênica Idiopática/imunologia , Esplenomegalia/sangue , Esplenomegalia/complicações , Esplenomegalia/imunologia , Adulto JovemRESUMO
Monoclonal antibodies were raised to a DNA.RNA heteropolymer duplex prepared by transcription of phi X174 single-stranded DNA with DNA-dependent RNA polymerase. A monoclonal antibody with the highest affinity and specificity was selected. This antibody bound the DNA.RNA heteropolymer and poly(I).poly(dC) equally but 100-fold higher levels of poly(A).poly(dT) were required to achieve a similar degree of binding in competitive binding assays using DNA.[3H]RNA. Single-stranded DNA, double-stranded DNA and RNA, and ribosomal RNA were not bound by the antibody. The observed association constant for the antibody and DNA.[3H]RNA, determined by Scatchard analysis, was 8.5 X 10(10) l/mol assuming independent antibody binding sites. The antibody and an alkaline phosphatase-labeled second antibody were used in an immunodetection method for measurement of hybrids formed between immobilized DNA probes of various lengths and 23 S ribosomal RNA. The colorimetric response of this assay increased linearly with the amount of hybrid formed.
Assuntos
Anticorpos Monoclonais/imunologia , DNA/imunologia , Hibridização de Ácido Nucleico , RNA/imunologia , Animais , Especificidade de Anticorpos , DNA Ribossômico/imunologia , Escherichia coli , Imunoensaio/métodos , Técnicas de Imunoadsorção , Camundongos , RNA Ribossômico/imunologiaRESUMO
In an attempt to study Giardia at the DNA sequence level, the rRNA genes of three species, Giardia duodenalis, Giardia ardeae and Giardia muris were cloned and restriction enzyme maps were constructed. The rDNA repeats of these Giardia show completely different restriction enzyme recognition patterns. The size of the rDNA repeat ranges from approximately 5.6 kb in G. duodenalis to 7.6 kb in both G. muris and G. ardeae. These size differences are mainly attributable to the variation in length of the spacer. Minor differences exist among these Giardia in the sizes of their small subunit rRNA and the internal transcribed spacer between small and large subunit rRNA. The genetic maps were constructed by sequence analysis of the DNA around the 5' and 3' ends of the mature rRNA genes and between the rRNA covering the 5.8S rRNA gene and internal transcribed spacer. Comparison of the 5.8S rDNA and 3' end of large subunit rDNA from these three Giardia species showed considerable sequence variation, but the rDNA sequences of G. duodenalis and G. ardeae appear more closely related to each other than to G. muris.
Assuntos
DNA Ribossômico/genética , DNA Ribossômico/imunologia , Giardia/genética , RNA Ribossômico/genética , Mapeamento por Restrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular/métodos , DNA de Protozoário/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA de Protozoário/genética , Homologia de Sequência do Ácido NucleicoRESUMO
A collaborative study was initiated by the European Directorate for the Quality of Medicines (EDQM), to assign a potency value for candidate batch 2 of European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for Hepatitis B (rDNA) antigen in vitro assays, for both method A and method B by calibrating them against the Ph. Eur. BRPs, batch 1 for methods A and B respectively. The study was prompted by the observation that the first batch of BRP for method B appeared to have lost potency over time. BRP 1 for method A showed no loss in potency, however stocks of the material were nearing depletion. Eleven laboratories participated in the study and all reported results. Participants performed 3 independent assays using both method A and method B. Method A was used to assess BRPs for method A and method B was used to assess BRPs for method B. Since BRP 1B was suspected to have lost potency, an additional sample was included in the method B test in an attempt to clarify the situation. BRP 1B was also assayed in method A against BRP 1A in the hope of also attaining further information by comparing the results from this study to those obtained in the original study to establish the first batch of BRP [1]. Although it was not the primary aim of this study to correlate in vitro potency with the immunogenicity assay in mice, a number of interested parties also performed the mouse in vivo assay to obtain data on the behaviour of the candidate BRPs in this assay. For method A, potency estimates were satisfactory in terms of repeatability and reproducibility. The candidate material was therefore assigned a value of 16.6 micrograms/ml. For method B, it appeared that the observation of reduced in vitro potency of BRP1 was confirmed. Despite the attempt to clarify the situation with additional studies, it was not possible to assign a potency value with the results obtained. A small-scale collaborative study will be organised to determine an appropriate value for the candidate BRP for method B. The results from the in vivo study while highly variable showed no evidence of a shift in the in vivo potency for either BRP 1A or BRP 1B. It should be noted that the in vitro method for determination of hepatitis B vaccine potency is under revision due to the discontinuation of the Auszyme kit from Abbott, which is required to perform the current assays. Once an alternative assay has been established, the suitability of the reference preparations or establishment of new reference preparations will be required. The candidate material for method A BRP was adopted by the European Pharmacopoeia Commission at its session in November 2003, as the European Pharmacopoeia Hepatitis B vaccine (rDNA) method A, batch 2.
Assuntos
DNA Ribossômico/análise , Vacinas contra Hepatite B/normas , Animais , DNA Ribossômico/imunologia , Europa (Continente) , Vacinas contra Hepatite B/análise , Cooperação Internacional , Camundongos , Farmacopeias como Assunto/normas , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Enzyme immunoassay showed that blood serum from healthy donors contains specific high-affinity antibodies (apparent association constant > or = 5 x 10(9) M(-1)) against a fragment of transcribed region of ribosomal DNA repeat of human serum, which are present in a free form or are bound to extracellular DNA. Preheating of the serum at 55 degrees C and high ionic strength (1.5 M NaCl) had no effect on the interaction of antibodies with this fragment. Competitive binding assay showed that these antibodies recognize DNA epitopes, which differ from the epitopes recognized by most anti-DNA antibodies in systemic lupus erythematosus.
Assuntos
Anticorpos Antinucleares/sangue , DNA Ribossômico/imunologia , Soro/imunologia , Adulto , Animais , Feminino , HumanosRESUMO
We previously hypothesized that the sequence of transcribed region of human ribosomal repeats is selectively accumulated in circulating extracellular DNA due to its increased resistance to double-strand breaks caused by accumulation of single-chain breaks produced by nucleases. The contents of rDNA in blood serum DNA and in DNA from leukocytic nuclei both in healthy donors and in patients with rheumatoid arthritis were compared using dot hybridization method. By the content of non-methylated CpG-repeats, transcribed region of rDNA is identical to bacterial DNA, which is characterized by potent immunostimulatory effect. The transcribed region of rDNA (13.3 kb) contains more than 200 CpG-motifs capable of interacting with TLR9 receptors, which are the mediators of the cell immune response to the action of CpG-rich DNA fragments. The data suggest that DNA from dead cells circulating in the peripheral blood is enriched with sequences possessing potent immunostimulatory properties.
Assuntos
Artrite Reumatoide/imunologia , Ilhas de CpG/imunologia , DNA Ribossômico/imunologia , Proteínas de Ligação a DNA/imunologia , DNA/sangue , Artrite Reumatoide/genética , Estudos de Casos e Controles , Humanos , Receptor Toll-Like 9/imunologiaRESUMO
Intranucleolar DNA, including ribosomal DNA (rDNA), was localized in situ in proliferating onion cells under the electron microscope using an anti-DNA monoclonal antibody and a postembedding indirect immunogold procedure. In the interphase nucleolus of this species, characterized by a very high amount of rRNA genes, we found DNA concentrated mostly in fibrillar centres (FCs) and in the region of the dense fibrillar component (DFC) immediately surrounding them. Clusters of gold particles were frequently seen covering both of these structural components of the nucleolus at the same time. Moreover, the same technique, applied to transcriptionally arrested quiescent onion cells, showed the nucleolar DFC devoid of DNA. Also, in mitotic cells at telophase, the prenucleolar material, which has the same morphological and cytochemical features as the DFC, does not contain DNA. These data suggest the existence of at least two subcomponents of the DFC in the onion cell nucleolus, one associated with pre-rRNA synthesis, and the other, with further processing of transcripts, already released from the rDNA template. We conclude that the first subcomponent forms part of the "transition between FC and DFC", which is the in situ structural counterpart of pre-rRNA synthesis. This transition is morphologicaly sizeable in onion cells, because of their high number of rRNA genes and the large size of the DFC mass; however, it would be largely detectable in situ in other cell systems, where the whole DFC comprises only a thin layer and the amount of rDNA is considerably reduced.
Assuntos
Allium/genética , Nucléolo Celular/análise , DNA Ribossômico/análise , Anticorpos Monoclonais/genética , DNA Ribossômico/imunologia , Interfase , Microscopia Eletrônica , Precursores de RNA/biossínteseRESUMO
The distribution of nucleolar RNA polymerase in the nucleolus of onion root meristematic cells has been studied by means of an antibody originally raised against Drosophila RNA polymerase II. This antibody recognizes the homologous domains of the large subunit of the enzyme, which are highly conserved throughout evolution in the three classes of eucaryotic RNA polymerases. Given that RNA polymerase I is confined to the nucleolus, and that the onion cell nucleolus lacks digitations of extranucleolar chromatin, we conclude that the nucleolar enzyme localized is RNA polymerase I. A quantitative approach, independent of the existence of borderlines between nucleolar fibrillar centres and the dense fibrillar component, allowed us to show that the enzyme is localized in fibrillar centres and in the transition area between them and the dense fibrillar component, in parallel with the nucleolar DNA. These results, together with previous autoradiographic, cytochemical and immunocytochemical results, in this and other species, lead us to conclude that the activation of rDNA for transcription occurs in the fibrillar centres and pre-rRNA synthesis is expressed at the transition area between fibrillar centres and the dense fibrillar component. Fibrillar centres are connected to each other by extended RNA polymerase-bound DNA fibres, presumably active in transcription. This work provides evidence of the high evolutionary conservation of some domains of the large subunit of RNA polymerases and of the existence of fibrillar centres in the nucleolus of plant cells, totally homologous to those described in mammalian cells.
Assuntos
Evolução Biológica , Nucléolo Celular/química , RNA Polimerase I/análise , Allium/química , Allium/genética , Animais , Anticorpos Monoclonais , Nucléolo Celular/imunologia , Reações Cruzadas , DNA Ribossômico/genética , DNA Ribossômico/imunologia , Drosophila/química , Drosophila/genética , Microscopia Imunoeletrônica , RNA Polimerase I/imunologiaRESUMO
The immunogenicities of hepatitis B virus vaccines containing S and pre-S2 regions were compared using two different schedules of immunization (A: 0-1-2-12 months and B: 0-1-6 months). Two hundred males and females aged 17-22 years were vaccinated with 20 micrograms per dose. The follow-up period was extended up to 13 months. One month after the booster dose anti-HBs were detected in 98.9% of those vaccinated with schedule A and 100% of those vaccinated with schedule B. Geometric mean titres (GMT) of anti-HBs were significantly higher with schedule A than schedule B, reaching GMT of 16269.7 mIU ml-1 and 4372.4 mIU ml-1, respectively, one month after the booster dose. Seroconversion rates for the anti-pre-S2 antibodies one month after the booster dose were 89.4% for schedule A and 76.6% for schedule B. GMT were 157.8 mIU ml-1 and 67.5 mIU ml-1, respectively. We conclude that both vaccines elicit high titres of anti-HBs and anti-pre-S2 antibodies. Immunity lasts longer in schedule A than in schedule B.
Assuntos
DNA Ribossômico/imunologia , Vacinas contra Hepatite B/imunologia , Vacinas Sintéticas/imunologia , Adolescente , Adulto , Esquema de Medicação , Estudos de Avaliação como Assunto , Feminino , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/análise , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/administração & dosagem , Vacinas contra Hepatite B/efeitos adversos , Vacinas contra Hepatite B/química , Vacinas contra Hepatite B/farmacologia , Humanos , Imunização , Masculino , Precursores de Proteínas/análise , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/farmacologiaRESUMO
This investigation was designed to investigate mechanisms underlying oral immunization in humans after ingestion of the ribosomal vaccine D53. Immunofluorescence and ELISA (Enzyme-Linked ImmunoSorbent Assay) spot techniques were used for peripheral blood studies. The first part of the investigation was a double-blind placebo-controlled study of 12 healthy volunteers; counts of cells containing immunoglobulins and cells producing specific antibodies were higher in the individuals given the oral ribosomal vaccine than in the placebo-treated controls. In the second part of the investigation, analysis of the kinetics of apparition of the immunoglobulin-containing and specific antibody-producing cells suggested prompt stimulation of Peyer patch B lymphocytes following ingestion of the vaccine. Lastly, a study of 5 children given the vaccine on a long-term basis demonstrated increased counts of both above-described cell types after one month treatment.
Assuntos
Vacinas Bacterianas/imunologia , Células Sanguíneas/fisiologia , DNA Ribossômico/imunologia , Infecções por Haemophilus/prevenção & controle , Infecções Estreptocócicas/prevenção & controle , Administração Oral , Adulto , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/uso terapêutico , Criança , Feminino , Haemophilus influenzae/genética , Haemophilus influenzae/imunologia , Humanos , Imunoglobulina A/imunologia , Imunoglobulina M/imunologia , Infecções por Klebsiella/prevenção & controle , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/imunologia , Masculino , Streptococcus/genética , Streptococcus/imunologiaRESUMO
Nucleic acid vaccines provide an exciting approach for antigen presentation to the immune system. As a test of this new methodology, the immune response to the in vivo-expressed Brucella abortus ribosomal L7/12 gene in the muscle cells of mice was examined. To accomplish this goal the eukaryotic expression systems pcDNA3 and p6 were used. Single intramuscular injection of the L7/L12 gene driven by the human cytomegalovirus (CMV) promoter (pcDNA3) or bovine MHC 1 promoter (p6) resulted in intracellular expression of the B. abortus L7/L12 immunodominant protein encoded by this gene. This application facilitated directed antigen presentation to the immune system and established specific antibody and T-cell responses compared with vector only (pcDNA3) negative controls and B. abortus S19 injected positive controls. Although pcDNA3-encoded L7/L12 gene-inoculated mice possessed significant protection, p6-L7/L12 did not engender significant protection against B. abortus S2308 infection compared to positive control mice. These data suggest a promising antigen-specific response, and L7/L12 nucleic acid vaccination may be an initial step in the development of genetically engineered candidate vaccines against brucellosis. This study for the first time focuses on DNA immunization of a gene from B. abortus.
Assuntos
Anticorpos Antibacterianos/biossíntese , Vacina contra Brucelose/imunologia , Vacina contra Brucelose/uso terapêutico , Brucella abortus/genética , Brucella abortus/imunologia , Proteínas Ribossômicas/genética , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico , Animais , Formação de Anticorpos/imunologia , Especificidade de Anticorpos , Vacina contra Brucelose/genética , Brucelose/prevenção & controle , Bovinos , DNA Ribossômico/administração & dosagem , DNA Ribossômico/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Genes Bacterianos , Vetores Genéticos , Humanos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia , Vacinas de DNA/genéticaRESUMO
The development of additional methods for detecting and identifuing Babesia and Plasmodium infections may be useful in disease monitoring, management and control efforts. To preliminarily evaluate sunthetic peptide-based serodiagnosis, a hydrophilic sequence (DDESEFDKEK)was selected from published BabR gene of B. bovis. Immunization of rabbits and cattle with the hemocyanin-conjugated peptide elicited antibody responses that specifically detected both P. falciparum and B. bovis antigens by immunofluorescence and Western blots. Using a dot-ELISA with this peptide, antisera from immunized and naturally-infected cattle, and immunized rodents, were specifically detected. Reactivity was weak and correlated with peptide immunization or infection. DNA-based detection using repetitive DNA was species-specific in dot-blot formats for B. bovis DNA, and in both dot-blot and in situ formats for P. falciparum; a streamlined enzymelinked synthetic DNA assay for P. falciparum detected 30 parasites/mm(cúbicos) from patient blood using either colorimetric (2-15 h color development) or chemiluminescent detection (0.5-6-min. exposures). Serodiagnostic and DNA hybridization methods may be complementary in the respective detection of both chronic and acute infections. However, recent improvements in the polymerase chain reaction (PCR) make feasible a more sensitive and uniform approach to the diagnosis of these and other infectious disease complexes, with appropriate primers and processing methods. An analysis of ribosomal DNA genes of Plasmodium and Toxoplasma identified Apicomplexa-conserved sequence regions. Specific and distinctive PCR profiles were obtained for primers spanning the internal transcribed spacer locus for each of several Plasmodium and Babesia species