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.

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.

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

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

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.

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.

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.

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.

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.

Chromosome rearrangements in Dictyostelium discoideum.

A tandem duplication (D350(III,III] of the whiB to radB interval of linkage group III has been characterized. The gene order on the duplication-bearing chromosome is: centromere, whiB500, radB+, whiB+, radB24, bsgA5, acrC4. Slow-growing, duplication-bearing strains (yellow-spored, radiation-resistant) produced four classes of faster growing sectors involving the whiB and radB loci: white-spored, radiation-sensitive (whiB500, radB24); white-spored, radiation-resistant (whiB500, radB+); yellow-spored, radiation-sensitive (whiB+, radB24); and yellow-spored, radiation-resistant. The first three classes can be explained as the products of single recombination events in which one copy of the whiB to radB interval was lost. The yellow-spored, radiation-resistant sectors probably arose by mutation elsewhere in the genome, but alternatively may represent multiple recombination events or deletion of part of one copy of the duplicated region. Loss of the duplicated segment was enhanced by irradiation with ultraviolet light (254 nm). Heterozygosity for a DNA repair mutation at the radB locus may have been involved in the formation of the duplication. It is proposed that translocations are a major cause of nonrandom segregation patterns such as the cosegregation of unlinked markers in Dictyostelium discoideum. Translocations involving all known linkage groups are tabulated and DNA damage by N-methyl-N'-nitro-N-nitrosoguanidine is implicated in the formation of translocations in D. discoideum.

[Electron paramagnetic resonance study of the interactions between steroid hormones and binding proteins]. / Etude en résonance paramagnétique électronique (RPE) des interactions stéroïdes hormonaux-protéines de liaison.

Interaction of a spin labeled corticosteroid (desoxycorticosterone nitroxyde: DOC -NO) with three purified proteins (albumin, transcortin, progesterone binding protein: PBG) was studied by electron spin resonance (ESR) spectroscopy. DOC-NO was competitive with natural corticosteroids and therefore bound at the same site to specific binding proteins. ESR spectra in the presence of each of the proteins showed an immobilized (bound) form of the spin labeled steroid and allowed the calculation of the corresponding association constant (Ka) at equilibrium. The three binding proteins could be characterized by the ESR parameters of the DOC-NO bound form. The thermodynamic parameters (deltaH, deltaS) of the steroid-protein interactions were calculated from the ESR data obtained within a wide temperature range (3--40 degrees C). The ESR spectra width (2T) was used to evaluate the polarity of the spin label environment within the steroid binding site: a hydrophobic character was observed for transcortin whereas PBG exhibited a more hydrophilic steroid binding sits. The rotational correlation time of the three protein DOC-NO complexes at equilibrium were calculated from ESR data; the results were correlated with the protein molecular size and suggested a non spherical shape for the binding macromolecule in solution. Spin labelling of biologically active steroids thus provides a novel approach for the study of the interaction of these hormones with their binding protein. Providing a suitable spin label, the ESR parameters may allow the characterization of several types of binding sites of different biological significance for the same hormone, in biological fluids as well as in target tissues.

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.

Penetration of gentamicin into heart valves, subcutaneous and muscular tissue of patients undergoing open heart surgery.

Concentrations of gentamicin in plasma, heart valves, subcutaneous tissue and muscle were determined in 38 patients undergoing open heart surgery. Gentamicin reached peak levels in plasma and tissue within 60 min after a 5 min intravenous bolus injection of 1.5 mg/kg body weight. Subcutaneous and muscle concentrations varied between 0.51 microgram/g and 2.1 microgram/g. Gentamicin peak concentrations in cardiac valvar tissue wre 3.6 mug/g between 2 and 5 hours after administration; gentamicin heart valve concentrations varied between 1.2 microgram/g and 1.59 microgram/g. Gentamicin tissue concentrations during open heart surgery are high enough to inhibit most Klebsiella/Enterobacter and Staphylococcus aureus and epidermidis strains. However Gentamicin heart valve concentrations do not exceed 1.5 microgram/g for more than 1 h, which may explain treatment failures of patients with endocarditis.

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.

Hypercapnia and acetylcholine release from the cerebral cortex and medulla.

1. The cerebral cortex and medulla of fifty-eight anaesthetized dogs released ACh spontaneously through push-pull cannulae after perfusion with the anticholinesterase, sarin. Hypercapnia (12% CO(2)) evoked a significant release of ACh above the basic spontaneous level, from the medullary and cortical areas. Hypercapnia + hypoxia (12% CO(2) + 8% O(2)), in combination, produced an ACh release comparable to hypercapnia; hypoxia (8% O(2)) had no effect in any region.2. Areas in the medullary reticular formation responsive to injections of CO(2)-bicarbonate solutions (;respiratory responsive areas') produced a significant increase of ACh after exposure to hypercapnia or hypercapnia + hypoxia, over that obtained from either the ;non-respiratory responsive areas' of the medulla or the cerebral cortex.3. The evidence supports the concept that ACh may participate as a neurotransmitter within the cerebral cortex and medulla. Also the results would suggest but do not prove, that a cholinergic factor may be a component in respiratory control under certain circumstances, such as exposure to hypercapnia.

[Gas exchanges on effort in diffuse pulmonary emphysema (author's transl)]. / Les échanges gazeux à l'effort dans l'emphysème pulmonaire diffus.

With the aim of determining the causes of hypoxia which develops on effort in certain emphysema sufferers, gas exchange at rest and effort were studied in 28 patients with diffuse pulmonary emphysema (including one third with large bullous form), with the exclusion of any clinical evidence of bronchitis or asthma. These patients, selected on the basis of the similarity of their clinical, radiological, mechano-spirographic and diffusion criteria, behaved very differently in terms of their gas exchanges during a moderate effort (mean VO2 = 750 ml/mn). The first group of 12 subjects showed a significant fall in pO2 (- 10.5 mmHg on average). The second group of 16 subjects improved by 4 mmHg hypoxaemia present at rest (though more marked than in the first group). The various parameters studied of ventilation, blood gases, and diffusion appeared to indicate a preponderant role played by distributive problems, and in particular the Qs/Qt ratio (calculated on the Fenn and Rahn pO2 and pCO2 diagram) which worsened on effort in the first group whilst it improved in the second, as well as the greater degree of desaturation of mixed venous blood on effort in the first group. The difficulties of a valid physiopathological interpretation are emphasized.

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.