Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination.

This corrects the article DOI: 10.1038/mi.2017.81.

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination.

Mucosal immunity is often required for protection against respiratory pathogens but the underlying cellular and molecular mechanisms of induction remain poorly understood. Here, systems vaccinology was used to identify immune signatures after pulmonary or subcutaneous immunization of mice with pertussis outer membrane vesicles. Pulmonary immunization led to improved protection, exclusively induced mucosal immunoglobulin A (IgA) and T helper type 17 (Th17) responses, and in addition evoked elevated systemic immunoglobulin G (IgG) antibody levels, IgG-producing plasma cells, memory B cells, and Th17 cells. These adaptive responses were preceded by unique local expression of genes of the innate immune response related to Th17 (e.g., Rorc) and IgA responses (e.g., Pigr) in addition to local and systemic secretion of Th1/Th17-promoting cytokines. This comprehensive systems approach identifies the effect of the administration route on the development of mucosal immunity, its importance in protection against Bordetella pertussis, and reveals potential molecular correlates of vaccine immunity to this reemerging pathogen.

Adaptive immune response to whole cell pertussis vaccine reflects vaccine quality: A possible complementation to the Pertussis Serological Potency test.

Whole cell Bordetella pertussis (wP) vaccines are still used in many countries to protect against the respiratory disease pertussis. The potency of whole-cell pertussis vaccine lots is determined by an intracerebral challenge test (the Kendrick test). This test is criticized due to lack of immunological relevance of the read-out after an intracerebral challenge with B. pertussis. The alternative in vivo test, which assesses specific antibody levels in serum after wP vaccination, is the Pertussis Serological Potency test (PSPT). Although the PSPT focuses on a parameter that contributes to protection, the protective immune mechanisms after wP vaccination includes more elements than specific antibody responses only. In this study, additional parameters were investigated, i.e. circulating pro-inflammatory cytokines, antibody specificity and T helper cell responses and it was evaluated whether they can be used as complementary readout parameters in the PSPT to assess wP lot quality. By deliberate manipulation of the vaccine preparation procedure, a panel of high, intermediate and low quality wP vaccines were made. The results revealed that these vaccines induced similar IL-6 and IP10 levels in serum 4h after vaccination (innate responses) and similar antibody levels directed against the entire bacterium. In contrast, the induced antibody specificity to distinct wP antigens differed after vaccination with high, intermediate and low quality wP vaccines. In addition, the magnitude of wP-induced Th cell responses (Th17, Th1 and Th2) was reduced after vaccination with a wP vaccine of low quality. T cell responses and antibody specificity are therefore correlates of qualitative differences in the investigated vaccines, while the current parameter of the PSPT alone was not sensitive enough to distinguish between vaccines of different qualities. This study demonstrates that assessment of the magnitude of Th cell responses and the antigen specificity of antibodies induced by wP vaccination could form valuable complementary parameters to the PSPT.

Trypsin diminishes the rat potency of polio serotype 3.

This study addresses observations made in view of testing in practice the guideline in the European Pharmacopoeia (EP) on omitting the rat potency test for release of polio containing vaccines. In general, use of the guideline is valid and the D-antigen ELISA can indeed be used as an in vitro alternative for the in vivo test. However, the set-up of the ELISA is crucial and should include detection of antigenic site 1 in polio serotype 3 as destruction of that site by trypsin results in a reduced rat potency. Antigenic site 1 in polio serotype 2 may also be modified by trypsin, but the cleavage of viral protein 1 (VP1) did not affect the rat potency. Therefore, any antigenic site, except site 1, can be used for detection of polio serotype 2. It is advised to include testing of the effect of trypsin treatment in the EP-guideline. This allows polio vaccine manufacturers to check whether their in-house ELISA needs improvement.

PDGF and FGF-2 signaling in oligodendrocyte progenitor cells: regulation of proliferation and differentiation by multiple intracellular signaling pathways.

In this paper we address the linking of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (FGF-2) to intracellular signaling molecules in oligodendrocyte progenitors. It is demonstrated that both growth factors activate downstream targets similar to those shown for protein kinase C (PKC) activation. Yet, neither the arrest of terminal oligodendrocyte differentiation nor the proliferation induced by PDGF or FGF-2 can be antagonized by inhibition of PKC. Rather, p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, and pp70 S6 kinase were found to be necessary for the mitogenic activity of PDGF and FGF-2. Paradoxically, these kinases were also necessary for the onset of oligodendrocyte differentiation in control cells. In addition, cAMP-dependent kinase A (PKA) activation inhibited the mitogenic response of oligodendrocyte progenitors to FGF-2. Taken together, the molecular mechanism that controls oligodendrocyte lineage progression is operated by at least two signal pathways, which interfere either with proliferation and/or differentiation of oligodendrocyte progenitors.

Randomized comparison of direct thrombin inhibition versus heparin in conjunction with fibrinolytic therapy for acute myocardial infarction: results from the GUSTO-IIb Trial. Global Use of Strategies to Open Occluded Coronary Arteries in Acute Coronary Syndromes (GUSTO-IIb) Investigators.

OBJECTIVES: We sought to show that hirudin might interact differently with streptokinase (SK) and tissue-type plasminogen activator (t-PA), which could reduce the incidence of death or reinfarction at 30 days. BACKGROUND: In a large-scale trial of patients with acute coronary syndromes, hirudin provided modest benefit compared with heparin. However, the interaction with thrombolytic agents was not specifically assessed. METHODS: Patients with symptoms of acute myocardial infarction and electrocardiographic ST segment elevation were treated with thrombolytic therapy and randomly assigned to receive hirudin or heparin. RESULTS: A total of 2,274 patients received t-PA, and 1,015 received SK. Baseline characteristics were balanced by antithrombin assignment. Among SK-treated patients, death or reinfarction at 30 days occurred more often in those treated with adjunctive heparin (14.4%) rather than hirudin (8.6%, odds ratio [OR] 1.78, 95% confidence interval [CI] 1.20 to 2.66, p = 0.004). Among t-PA-treated patients, the rates were 10.9% with heparin and 10.3% with hirudin (OR 1.06, 95% CI 0.81 to 1.38, p = 0.68; for treatment heterogeneity: chi-square 4.20, degrees of freedom [df] 1, p = 0.04). After adjustment for baseline differences between thrombolytic groups, the rates were 9.1% for SK with hirudin, 10.3% for t-PA with hirudin, 10.5% for t-PA with heparin and 14.9% for SK with heparin (for treatment heterogeneity: chi-square 4.5, df 1, p = 0.03), suggesting that the beneficial treatment effect of hirudin was limited to the SK-treated patients. CONCLUSIONS: Hirudin interacts favorably with SK but not t-PA, highlighting the importance of thrombin activity after SK therapy and the potential for simulating the effects of a more potent fibrinolytic agent through direct antithrombin therapy.

Heparin as an adjuvant to thrombolytic therapy in acute myocardial infarction.

For the treatment of acute myocardial infarction, heparin has been a topic of continuing debate for the past four decades. After review of the available data, the American College of Cardiology/American Heart Association Guidelines for the Early Management of Patients with Acute Myocardial Infarction, published in 1990, recommended intravenous heparin administration together or immediately after thrombolytic therapy to maintain the activated partial thromboplastin time approximately 1.5 to 2.0 times the control value for 24 to 72 hours. Over the past five years, with the proven benefits or thrombolytic therapy and antiplatelet therapy, investigators have been in search of the ideal thrombolytic agent as well as the best adjunctive antithrombotic strategy. We review a number of angiographic patency trials as well as the major thrombolytic mortality reduction trials in which adjunctive heparin therapy was directly assessed. These trials established the need for intravenous heparin administration with tissue plasminogen activator, but, on the other hand, do not substantiate the need for either subcutaneous or intravenous heparin use with streptokinase. New data from a large scale trial emphasizes the importance of maintaining the aPTT in the 55-70 second range to prevent bleeding complications and optimize clinical outcomes.

Ergonomics and international standards: history, organizational structure and method of development.

Egonomics international standards were formally considered in 1973 by a symposium of the International Ergonomics Association (IEA) held at Loughborough University in the UK. Recommendations led to the establishment of ISO TC 159 'Ergonomics', with Germany (DIN) holding the secretariat. Six subcommittees (SC) were established and have worked towards standards. These have been rationalized to the present four subcommittees: SC1, Ergonomics guiding principles; SC3, Anthropometry and biomechanics; SC4, Ergonomics and human system interaction; and SC5, Ergonomics of the physical environment. The subcommittees have over 15 working groups (WG) covering over 50 work items that will lead to ergonomics ISO standards. Over 20 ISO standards have already been produced and a number have been reconfirmed at automatic five-yearly reviews. The organizational structure is presented as well as methods of standards production from proposed work item to international standard. European standards (EN) are produced under CEN TC 122 'Ergonomics', which has 11 working groups. How European Standards are produced and organizational links with ISO TC 159 are described.

Interaction of a rhizobial DNA-binding protein with the promoter region of a plant leghemoglobin gene.

A nucleotide sequence was identified approximately 650 bp upstream of the Sesbania rostrata leghemoglobin gene Srglb3 start codon, which interacts specifically with a proteinaceous DNA-binding factor found in nodule extracts but not in extracts from leaves or roots. The binding site for this factor was delimited using footprinting techniques. The DNA-binding activity of this factor was found to be heat stable, dependent on divalent cations, and derived from the (infecting) Azorhizobium caulinodans bacteria or bacteroids (A. caulinodans bacterial binding factor 1, AcBBF1). A 9- to 10-kD protein was isolated from a free-living culture of A. caulinodans that co-purifies with the DNA-binding activity (A. caulinodans bacterial binding protein 1, AcBBP1) and interacts specifically with its target (S. rostrata bacterial binding site 1, SrBBS1). The amino acid sequence of the N-terminal 27 residues of AcBBP1 was determined and was found to share significant similarity (46% identity; 68% similarity) with a domain of the herpes simplex virus major DNA-binding protein infected cell protein 8 (ICP8). An insertion mutation in the SrBBS1 was found to result in a substantial reduction of the expression of a Srglb3-gus reporter gene fusion in nodules of transgenic Lotus corniculatus plants, suggesting a role for this element in Srglb3 promoter activity. Based on these results, we propose that (a) bacterial transacting factor(s) may play a role in infected cell-specific expression of the symbiotically induced plant lb genes.

Endogenous expression of E1A in human cells enhances the effect of adenovirus E3 on class I major histocompatibility complex antigen expression.

Group C human adenovirus (Ad) serotypes (e.g., Ad type 2 [Ad2] and Ad5) cause persistent infections in humans. One explanation for Ad persistence is an ineffective cytotoxic T-lymphocyte response due to diminished cell surface expression of class I major histocompatibility antigen (MHC Ag) on Ad-infected cells, an effect mediated by the Ad E3 19-kDa glycoprotein (E3 effect). However, we previously reported that, except for the Ad5 E1-transformed human cell line 293, a variety of human lymphoid, epithelial, and fibroblastic cells are resistant to the E3 effect during Ad5 infection (J. M. Routes and J. L. Cook, J. Immunol. 144:2763-2770, 1990). The present study tested the hypothesis that endogenous expression of E1A proteins in 293 cells sensitizes cells to this E3 effect, resulting in an enhanced downregulation of surface class I MHC Ag expression following Ad5 infection. Human epithelial and fibroblastic cells expressing E1A gene products for at least 72 h exhibited an enhanced E3 effect following Ad5 infection that was independent of baseline levels of surface class I MHC Ag expression and of E1A induction of E3 19-kDa glycoprotein expression. There was a direct correlation between the level of endogenous E1A expressed and the magnitude of the E3 effect. We postulate that the in vivo existence of cells stably expressing either E1A proteins or E1A-like activities in the microenvironment of Ad5 infection provides a reservoir of Ad-infected cells that is relatively protected from the virus-specific cytotoxic T-lymphocyte response, thereby favoring Ad persistence in humans.

[Gamma-1 heavy chain disease with the demonstration of Bence-Jones proteins]. / Gamma-1-Schwerkettenkrankheit mit Nachweis von Bence-Jones-Proteinen.

We describe a 61-year-old patient suffering from gamma-1-heavy-chain disease (gamma 1-HCD) associated with Bence-Jones-lambda proteinemia and proteinuria. The analysis of the patients gamma 1-HCD protein (WIN) shows a deletion of the complete Fd fragment. The N-terminal seven amino-acid residue does not resemble any of the known immunoglobulin-heavy-chain variable regions. Unexpectedly, in PBL-DNA and in DNA from EBV-immortalized cells we found in addition to the expected predominantly rearranged Ig-lambda-light-chain gene a predominant rearrangement of an Ig-kappa gene. These findings show that the gamma-1-heavy-chain disease of the patient involves a defective regulation of Ig-light-chain-gene activation as well.

Regulation of plant genes specifically induced in nitrogen-fixing nodules: role of cis-acting elements and trans-acting factors in leghemoglobin gene expression.

Transgenic alfalfa plants harboring a gene fusion between the soybean leghemoglobin (lbc3) promoter region and the chloramphenicol acetyl transferase (cat) gene were used to determine the influence of rhizobial mutants on lb gene expression in nodules. The promoter region of the Sesbania rostrata glb3 (Srglb3) leghemoglobin gene was examined for the presence of conserved motifs homologous to binding site 1 and 2 of the soybean lbc3 promoter region, found to interact with a trans-acting factor present in soybean nodule nuclear extracts (Jensen EO, Marcker KA, Schell J, de Bruijn FJ, EMBO J 7:1265-1271, 1988). Subfragments of the S. rostrata glb3 (Srglb3) promoter region were examined for binding to trans-acting factors from nodule nuclear extracts. In addition to the binding sites previously identified (Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ, Mol Gen Genet 214: 181-191), several other sites were found to interact with trans-acting factors. In most cases the same trans-acting factor(s) were shown to be involved. One fragment (202) was found to bind specifically to a different factor (protein) which was extremely heat-resistant (100 degrees C). The appearance of this factor was shown to be developmentally regulated since the expected protein-DNA complexes were first observed around 12 days after infection, concomitant with the production of leghemoglobin proteins. Fragments of the Srglb3 5' upstream region were fused to the beta-glucuronidase reporter gene with its own CAAT and TATA box region or those of the cauliflower mosaic virus 35S and nopaline synthase (nos) promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure and promoter analysis of leghemoglobin genes of the stem-nodulated tropical legume Sesbania rostrata: conserved coding sequences, cis-elements and trans-acting factors.

The primary structure of a leghemoglobin (lb) gene from the stem-nodulated, tropical legume Sesbania rostrata and two lb gene promoter regions was analysed. The S. rostrata lb gene structure and Lb amino acid composition were found to be highly conserved with previously described lb genes and Lb proteins. Distinct DNA elements were identified in the S. rostrata lb promoter regions, which share a high degree of homology with cis-active regulatory elements found in the soybean (Glycine max) lbc3 promoter. One conserved DNA element was found to interact specifically with an apparently universal, trans-acting factor present in nuclear extracts of nodules. These results suggest a conserved mechanism for nodule specific induction of lb genes in leguminous plants.

[Detection of antibodies to extractable nuclear antigens with counterimmunoelectrophoresis and the Western blot technic]. / Nachweis von Antikörpern gegen extrahierbare nukleäre Antigene (ENA) mit Uberwanderungselektrophorese (CIE) und Western-Blot.

Detection of antibodies against extractable nuclear antigens (ENA) by counter immunoelectrophoresis (CIE) is currently used to screen sera from patients with collagen vascular disease. Although fast and reliable, the method has its limitations with regard to the differentiation of various antigen-antibody systems. In this study, results of the anti-ENA analysis by CIE and by Western-Blot technique are compared. Except for a higher frequency of Sm antibodies by Western-blot, the results of CIE were confirmed. The source of antigen and the higher sensitivity of the Western-Blot technique are responsible for the different results obtained. With respect to the relationship of antibody profile and illness, both methods demonstrated a high frequency of antibodies against U1-sn-RNP in mixed connective tissue disease and of anti-SS-B antibodies in Sjögren's syndrome.

Developmentally regulated transcription of Dictyostelium discoideum plasmid Ddp1.

The presence of high copy number plasmid DNA has been described for several independent wild isolates of the lower eukaryote Dictyostelium discoideum, a model organism in developmental biology. The function of these plasmids has remained unclear so far. To understand the possible functions of the plasmids we have analyzed the transcription of one plasmid, Ddp1, in the wild strains NC4 and V12. The plasmid is transcribed in an identical fashion in both strains. Ddp1 codes for three RNA species in growth-phase cells, at least two of which are present in the poly(A) fraction. RNA isolated from cells of different developmental stages revealed the existence of five more transcripts, the majority of them being found in the poly(A) fraction. These transcripts are present at distinct time points during development. Both growth-phase and development-specific transcripts were found exclusively in Ddp1-containing strains. Mapping of the transcripts to the plasmid showed that some of them originated from the same area. These results indicated an overlapping of their coding regions.

Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum.

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.

Presence of nuclear associated plasmids in the lower eukaryote Dictyostelium discoideum.

High copy number plasmids have been identified in six out of 25 wild-type strains of the cellular slime mould Dictyostelium discoideum, a model organism in developmental biology (Loomis, 1982). The characterization of three plasmids, from the NC4 (Ddp1), WS380B (Ddp2) and OHIO (Ddp3) wild isolates, is presented here. We show that they are nuclear associated and non-homologous to the mitochondrial DNA and extrachromosomal ribosomal DNA.

Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes.

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.