Evaluation of the AV7909 Anthrax Vaccine Toxicity in Sprague Dawley Rats Following Three Intramuscular Administrations.

AV7909 is a next-generation anthrax vaccine under development for post-exposure prophylaxis following suspected or confirmed Bacillus anthracis exposure, when administered in conjunction with the recommended antibacterial regimen. AV7909 consists of the FDA-approved BioThrax® vaccine (anthrax vaccine adsorbed) and an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909. The purpose of this study was to evaluate the potential systemic and local toxicity of AV7909 when administered via repeat intramuscular injection to the right thigh muscle (biceps femoris) to male and female Sprague Dawley rats. The vaccine was administered on Days 1, 15, and 29 and the animals were assessed for treatment-related effects followed by a 2-week recovery period to evaluate the persistence or reversibility of any toxic effects. The AV7909 vaccine produced no apparent systemic toxicity based on evaluation of clinical observations, body weights, body temperature, clinical pathology, and anatomic pathology. Necrosis and inflammation were observed at the injection sites as well as in regional lymph nodes and adjacent tissues and were consistent with immune stimulation. Antibodies against B. anthracis protective antigen (PA) were detected in rats treated with the AV7909 vaccine, confirming relevance of this animal model for the assessment of systemic toxicity of AV7909. In contrast, sera of rats that received saline or soluble CPG 7909 alone were negative for anti-PA antibodies. Overall, 3 intramuscular immunizations of Sprague Dawley rats with AV7909 were well tolerated, did not induce mortality or any systemic adverse effects, and did not result in any delayed toxicity.

A multi-scale model of the interplay between cell signalling and hormone transport in specifying the root meristem of Arabidopsis thaliana.

The growth of the root of Arabidopsis thaliana is sustained by the meristem, a region of cell proliferation and differentiation which is located in the root apex and generates cells which move shootwards, expanding rapidly to cause root growth. The balance between cell division and differentiation is maintained via a signalling network, primarily coordinated by the hormones auxin, cytokinin and gibberellin. Since these hormones interact at different levels of spatial organisation, we develop a multi-scale computational model which enables us to study the interplay between these signalling networks and cell-cell communication during the specification of the root meristem. We investigate the responses of our model to hormonal perturbations, validating the results of our simulations against experimental data. Our simulations suggest that one or more additional components are needed to explain the observed expression patterns of a regulator of cytokinin signalling, ARR1, in roots not producing gibberellin. By searching for novel network components, we identify two mutant lines that affect significantly both root length and meristem size, one of which also differentially expresses a central component of the interaction network (SHY2). More generally, our study demonstrates how a multi-scale investigation can provide valuable insight into the spatio-temporal dynamics of signalling networks in biological tissues.

Complete Protection in Macaques Conferred by Purified Inactivated Zika Vaccine: Defining a Correlate of Protection.

A critical global health need exists for a Zika vaccine capable of mitigating the effects of future Zika epidemics. In this study we evaluated the antibody responses and efficacy of an aluminum hydroxide adjuvanted purified inactivated Zika vaccine (PIZV) against challenge with Zika virus (ZIKV) strain PRVABC59. Indian rhesus macaques received two doses of PIZV at varying concentrations ranging from 0.016 µg - 10 µg and were subsequently challenged with ZIKV six weeks or one year following the second immunization. PIZV induced a dose-dependent immune response that was boosted by a second immunization. Complete protection against ZIKV infection was achieved with the higher PIZV doses of 0.4 µg, 2 µg, and 10 µg at 6 weeks and  with 10 ug PIZV at  1 year following vaccination. Partial protection was achieved with the lower PIZV doses of 0.016 µg and 0.08 µg. Based on these data, a neutralizing antibody response above 3.02 log10 EC50 was determined as a correlate of protection in macaques. PIZV elicited a dose-dependent neutralizing antibody response which is protective for at least 1 year following vaccination.

Auxin fluxes in the root apex co-regulate gravitropism and lateral root initiation.

Root architecture plays an important role in water and nutrient acquisition and in the ability of the plant to adapt to the soil. Lateral root development is the main determinant of the shape of the root system and is controlled by external factors such as nutrient concentration. Here it is shown that lateral root initiation and root gravitropism, two processes that are regulated by auxin, are co-regulated in Arabidopsis. A mathematical model was generated that can predict the effects of gravistimulations on lateral root initiation density and suggests that lateral root initiation is controlled by an inhibitory fields mechanism. Moreover, gene transactivation experiments suggest a mechanism involving a single auxin transport route for both responses. Finally, co-regulation may offer a selective advantage by optimizing soil exploration as supported by a simple quantitative analysis.

A Multi-feature Fuzzy Index to Assess Stress Level from Bio-signals.

A mono-feature fuzzy index that evaluates the stress level from one feature extracted from ECG or GSR is presented. It is build using several measures of the feature recorded when the subject is at rest. The mono-feature fuzzy index can be merged in a multi-feature stress index without any tuning. It can be used to select relevant features and to detect stress. The performance of the stress index is analyzed on a data set made of 160 time periods of time when 20 subjects had to perform stressful tasks and corresponding control tasks. The stress was induced by 4 different tasks. The performances reached are 72% of correctly classified time periods in stress and no stress situations. Interesting conclusions could also be made on the tasks ability to induce stress.

Free and membrane-bound ribosomes. VI. Distribution of free sulphydryl groups in rabbit reticulocyte ribosomes.

When free and membrane-bound ribosomes were titrated with N-ethyl[14C]-maleimide or 5,5'-dithio-bis(2-nitrobenzoic acid), 25 free SH groups were detected in polysomes, 10 in the 60 S subunit and 14 in the 40 S subunits. When individual ribosomal proteins were analysed, one large subunit protein L14 was found to be strongly labelled compared to the other ribosomal proteins, in polysomes, monosomes and subunits. However, two additional protein spots from the small subunit were more radioactive in monosomes and subunits than inpolysomes. In all these studies, it was found that the distribution of free SH groups in free and membrane-bound ribosomes was identical except for the few proteins which were reported before to be specific for free or membrane-bound ribosomes (Fehlmann, ., Bellemare, G. and Godin, C. (1975) Biochim. Biophys. Acta 378, 119-124 and Fehlmann, M., Bellemare, G. and Godin, C. (1975) FEBS Lett. 59, 8-12).

Free and membrane-bound ribosomes. II. Two-dimensional gel electrophoresis of proteins from free and membrane-bound rabbit reticulocyte ribosomes.

Free and membrane-bound polysomes were isolated from rabbit reticulocytes. The membrane-bound polysomes were liberated form the membrane with deoxycholate. Monosomes were prepared from the two types of polysomes by incubation with puromycin. The ribosomal proteins were extracted and analyzed by two-dimensional gel electrophoresis. Two proteins of the large subunit, L11 and L17 present in the free monosomes were not found in the membrane-bound monosomes. On the other hand, four additional spots were found in the protein pattern of the membrane-bound monosomes.

Androgen formation and metabolism in the pulmonary epithelial cell line A549: expression of 17beta-hydroxysteroid dehydrogenase type 5 and 3alpha-hydroxysteroid dehydrogenase type 3.

Surfactant synthesis within developing fetal lung type II cells is affected by testosterone and 5alpha-dihydrotestosterone (5alpha-DHT). The pulmonary epithelial cell line A549, isolated from a human lung carcinoma, like normal lung type II cell, produces disaturated phosphatidylcholines and has been widely used for studying the regulation of surfactant production. Androgen receptor has been detected in A549 cells; however, the capacity of these cells for androgen synthesis and metabolism has not been investigated at molecular level. This study was undertaken to identify the steroidogenic enzymes involved in the formation and metabolism of androgens from adrenal C19 steroid precursors in A549 cells. When cultured in the presence of normal FCS, A549 intact cells converted DHEA to androstenediol, androstenedione principally to testosterone, and 5alpha-DHT to 5alpha-androstane 3alpha,17beta-diol. High levels of 17beta-hydroxysteroid dehydrogenase (HSD) and 3alpha-HSD activities were detected in both cytosol and microsomes isolated from homogenates. Analysis of A549 RNA indicated the presence of 17beta-HSD type 4 and type 5, and of 3alpha-HSD type 3 messenger RNAs. Very low levels of 3beta-HSD type 1 and 5alpha-reductase type 1 messenger RNAs and activities were detected. With regard to active androgen formation, there was little or no capacity for the conversion of DHEA to 5alpha-DHT. In contrast, androstenedione was rapidly transformed to testosterone. The pattern of steroid metabolism was not affected by the use of charcoal-stripped FCS or by the synthetic glucocorticoid dexamethasone. Together, our findings show that A549 cells express a pattern of steroid metabolism in which 17beta-HSD type 5 and 3alpha-HSD type 3 are the predominant enzymes. The level of androgens is regulated at the level of catalysis in intact cells such that the intracellular level of testosterone is stabilized, whereas 5alpha-DHT is rapidly inactivated by reduction to 3alpha,17beta-diol. This pattern of androgen metabolism has implications for the relative importance of testosterone and 5alpha-DHT in normal lung development and surfactant production.

Cloning of the gene encoding a Schistosoma mansoni antigen homologous to human Ro/SS-A autoantigen.

A cDNA library was constructed from the mRNA of adult worms of Schistomsoma mansoni in the expression vector lambda gt11 and screened with a rabbit antiserum raised against a 60-65-kDa electroeluted adult worm fraction. Two overlapping clones were selected and a partial nucleotide sequence was deduced (1172 bp). The full-length sequence was obtained by the amplification of the 5' end of first strand cDNA using PCR. The overall mRNA size was 1335 nt including a 25 nt 5' non-coding region and a 131 nt untranslated region with the poly(A) tail. The predicted amino acid sequence of 393 aa (45 kDa) has 52% identity with the human Ro/SS-A autoantigen, which is considered to be the human calreticulin. As for the human Ro/SS-A, the protein encoded by the cDNA described here contains a hydrophobic leader sequence and a carboxyl terminal sequence, HDEL consensus signal sequence for retention in the ER. An antiserum raised against the fusion protein of one clone recognized a 58-kDa antigen in homogenates of cercariae and of adult worms. The expression of the protein in the pGEX-2T fusion system allowed us to show the presence of specific antibodies in S. mansoni infected patients' sera and in the sera of patients with systemic lupus erythematosus, reflecting a cross-immunoreactivity between the S. mansoni protein and the human calreticulin autoantigen.

Inter-species variation of schistosome 28-kDa glutathione S-transferases.

The 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) is a candidate vaccine antigen. To evaluate the antigenic and phylogenetic variations between the 28-kDa GSTs from 4 species of schistosome, we have cloned and sequenced the 28-kDa GSTs from Schistosoma haematobium (Sh28GST) and Schistosoma bovis (Sb28GST). Sb28GST and Sh28GST are more similar to each other (97%) than to Sm28GST (90%) and particularly to the 28-kDa GST from Schistosoma japonicum (Sj28GST, 77%). Antisera directed against the major Sm28GST epitopes revealed differences in the recognition of the 28-kDa GSTs from the other schistosome species suggesting that these regions have been subjected to evolutionary pressure. The consequences of such species-specific epitopes on the development of a multi-species anti-schistosome vaccine are discussed.

Bradykinin stimulates DNA synthesis in competent Balb/c 3T3 cells and enhances inositol phosphate formation induced by platelet-derived growth factor.

Both platelet-derived growth factor (PDGF) and bradykinin were found to induce a growth response in Balb/c 3T3 cells. However, whereas PDGF brought about a five-fold increase in the incorporation of [3H]thymidine into DNA, the response to bradykinin was never more than 50%. When bradykinin was present simultaneously with sub-optimal concentrations of PDGF the response was about 15% greater than with PDGF alone. In contrast, if the cells were made competent by a 5 hr preincubation with PDGF which was then washed away, subsequent addition of bradykinin induced a more than two-fold increase in incorporation of [3H]thymidine into DNA compared with competent cells subsequently incubated with serum-free medium alone. Bradykinin also acted synergistically with insulin when the two agents were added simultaneously to competent cells. PDGF induced marked increases in the concentration of inositol phosphates at 30 min after stimulation, but by this time point any effect of bradykinin had disappeared. However, the simultaneous presence of PDGF and bradykinin induced increases at 30 min that were 50-100% greater than with PDGF alone. It is concluded that the pathways by which PDGF and bradykinin initiate a growth response in BALB/c 3T3 cells only partly overlap. Their actions on the synthesis of inositol phosphates exhibit distinctive temporal characteristics, but can be co-operative at 30 min and at earlier time intervals. This effect was found to be time-dependent, and developed over the first 5 min.

Formation of quaternary amines by N-methylation of azaheterocycles with homogeneous amine N-methyltransferases.

Catalytic activities of two amine N-methyltransferases were documented for the following azaheterocycles: isomeric phenyl- and bispyridyls; 2-, 3- and 4-mono-substituted pyridines; and a miscellaneous group of azaheterocycles that included mono- and diazabenzenes and mono- and diazanaphthalenes. The broad substrate specificities of the two amine N-methyltransferases for primary and secondary amines are here extended to a large number of aromatic azaheterocycles in which N-methylation results in the formation of quaternary ammonium metabolites. Pyridine was the best substrate for both enzymes. Substitution in the ring at the 2-position sterically hindered methylation of the pyridyl nitrogen; 2-phenylpyridine and 2,2'-bispyridyl were not substrates.

Intrathecal administration of dynorphin A and its fragments increase heart rate and arterial pressure in the urethane anesthetized rat: mediation by a nonopioid mechanism.

Intrathecal administration of 6.50 nmol of dynorphin A (dyn A) (1-13) and (1-17) to the ninth thoracic (T9) spinal segment provoked a transient (5-10 min) increased in heart rate (40-60 beats per minute (bpm] and arterial pressure (20-25 mmHg). Intravenous administration and administration to the second thoracic (T2) segment failed to mimic the effect of T9 administration, suggesting that the cardiovascular effects of T9 administration did not occur via diffusion to the periphery or to the brainstem. The cardioacceleratory and hypertensive responses to T9 dyn A (1-13) administration were prevented by pretreatment with the nicotinic ganglion blocker hexamethonium (10 mg/kg), but were unaffected by bilateral adrenalectomy. These results suggest that the cardiovascular effects of dyn A were mediated predominantly via a sympathetic pathway that does not innervate the adrenal glands. The effects were not antagonized by pretreatment with the opiate receptor antagonist naloxone or by the specific kappa opiate receptor antagonist nor-binaltorphimine, suggesting that they were not mediated via activation of kappa opiate receptors. Further support for this conclusion was provided by experiments demonstrating that dyn A (3-13) (30 nmol), a dynorphin fragment which is devoid of kappa activity, mimicked the effect of dyn A (1-13), whereas administration of the synthetic kappa agonist U50, 488H (100 nmol), failed to elicit effects similar to those provoked by dyn A (1-13). It is concluded that the cardiovascular effects of intrathecal dyn A administration are mediated via a nonopioid mechanism.

A multiscale model of plant topological structures

In applications dealing with plant growth modeling, increasing attention is being devoted to the topological structure of plants. Different models, based on tree-graphs, have been introduced to represent plants. These models assume that the scale of description is fixed. However, this hypothesis is too restrictive for new modeling applications that aim to tackle analysis or simulation of plant growth at different time and space scales. In order to make such multiscale descriptions available to computer applications, we have defined a general methodology for measuring and representing multiscale plant topological structures. This paper discusses the design of a model of plant topological structures and sketches out its general formal properties. The model supports multiscale, attributed and time-varying descriptions of plants. It is intended to be used for plant analysis methodologies and plant growth simulations.Copyright 1998 Academic Press Limited

Ivermectin-based control of onchocerciasis in northern Cameroon: individual factors influencing participation in community treatment.

A study aimed at determining individual factors associated with participation in community treatment with ivermectin was conducted in a village hyperendemic for onchocerciasis in northern Cameroon. The respective influences of sex, age, place of residence, distance between the compound and the dosing point, compound size, and participation in treatment by authoritative individuals in the compound was evaluated using univariate and multivariate analysis. Participation in treatment was closely associated with the attitude of the compound heads. Participation of compound heads in treatment increased as the household size increased, and as the distance to the distribution point diminished. This may be explained by the fact that getting information on health programmes is easier in large households whose members are involved in various social activities, and in compounds located near the village centre. Staff involved in health education should take this issue into account, and try to ensure circulation of information particularly to those living in small or remote compounds.

Molecular cloning and sequencing of the rat interleukin-12 p40 gene.

The nucleotide sequences containing the rat interleukin-12 p40 gene was determined. Sequencing revealed the presence of six exons and five introns. Analysis of the 5' non-coding region showed the presence of several possible sites involved in cytokine gene regulation at the transcriptional level. Comparison of the deduced amino acid sequence of rat IL-12 p40 with that of the mouse and of human p40, showed 92% and 65% identity respectively.

Separation by thin-layer chromatography of the most common androgen-derived C19 steroids formed by mammalian cells.

Several methods have been developed in the past for the separation and identification of closely related steroid hormones. Although these methods are effective, most of them use HPLC-derived systems and are expensive, laborious, or time-consuming. In the course of our studies of the metabolism of dehydroepiandrosterone and androstenedione in tissues, we have modified a previously published technique in such a way that in one TLC step we can separate most of the androgen C19 steroid derivatives produced by mammalian cells. We have used this modified technique for the past 2 years with considerable success and reproducible results, and we find it to be rapid and relatively inexpensive.

In vivo depletion of S-adenosyl-L-homocysteine and S-adenosyl-L-methionine in guinea pig lung after chronic S-(-)-nicotine administration.

Guinea pig lung tissue is dramatically depleted of S-adenosyl-L-homocysteine (SAH) after chronic exposure (600 micrograms/h s.c. for 21 days) of animals to either R-(+)- or S-(-)-nicotine enantiomers; S-(-)-nicotine decreased lung SAH levels by 60-fold, while R-(+)-nicotine caused an 11-fold reduction of SAH, relative to control values. Lung S-adenosyl-L-methionine (SAM) levels were also reduced (15-fold and 9-fold reductions with R-(+)- and S-(-)-isomers, respectively) in nicotine-treated animals, compared to controls. These depletions are more pronounced with the S-(-)-enantiomer. Liver tissue levels of SAH and SAM are less affected, in fact, a 4-fold increase in liver SAH levels was observed after exposure of animals to R-(+)-nicotine. These results indicate that chronic exposure to nicotine may perturb important endogenous methyltransferase reactions, which could be a contributory factor in the toxicological effects produced by cigarette smoking.

Effect of continuous administration of nicotine on urinary histamine and N tau-methylhistamine levels in the guinea pig.

The effect of continuous subcutaneous administration of S-(-)- and R-(+)-nicotines on urinary excretion levels of histamine and N tau-methylhistamine in guinea pigs, over a 23-day period, has been studied. Urinary levels of these endogenous compounds were measured utilizing paired-ion reversed-phase high performance liquid chromatography with flow-through electrochemical detection. Urinary histamine levels of animals that had been administered either of these nicotine isomers were not significantly different from control values. Initial levels of urinary N tau-methylhistamine (days 2-3) in R-(+)- and S-(-)-nicotine-treated animals were, 2-fold and 8-fold higher, respectively than control levels but in both cases these levels returned to control values over the remainder of the time course examined (days 6-23). These results suggest that exposure to S-(-)-nicotine results in initial histamine release and/or inhibition of histamine uptake. However, longer term exposure to S-(-)-nicotine may not result in significantly altered levels of circulating histamine.

Lack of detectable metabolism for solubilized 2,3,4-trimethylpentane by rat kidney proximal tubules.

Primary proximal tubule suspension cultures exposed to solubilized 2,3,4-trimethylpentane (2,3,4-TMP) resulted in a linear dose response, as determined by cellular lactate dehydrogenase leakage. The EC50 for 2,3,4-TMP was 16.3 mM. Metabolite analysis by gas chromatography/mass spectrometry of supernate and cell extracts from cultures exposed to 2,3,4-TMP (12.0 mM) failed to detect the presence of metabolites. Electron-microscopic examination of proximal tubules exposed to 2,3,4-TMP indicated ultrastructural changes that included increased mitochondrial swelling, increased vesiculation, decreased microvilli and pyknotic nuclei. This study indicates that kidney proximal tubules do not appear to metabolize 2,3,4-TMP.