Phase I study of safety and immunogenicity of an Escherichia coli-derived recombinant protective antigen (rPA) vaccine to prevent anthrax in adults

Background

The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA).

Methodology/Principal Findings

A total of 73 healthy adults ages 18–40 were enrolled and 67 received 2 injections separated by 4 weeks of either buffered saline placebo, or rPA formulated with or without 704 µg/ml Alhydrogel® adjuvant in increasing doses (5, 25, 50, 100 µg) of rPA. Participants were followed for one year and safety and immunologic data were assessed. Tenderness and warmth were the most common post-injection site reactions. No serious adverse events related to the vaccine were observed. The most robust humoral immune responses were observed in subjects receiving 50 µg of rPA formulated with Alhydrogel® with a geometric mean concentration of anti-rPA IgG antibodies of 283 µg/ml and a toxin neutralizing geometric 50% reciprocal geometric mean titer of 1061. The highest lymphoproliferative peak cellular response (median Lymphocyte Stimulation Index of 29) was observed in the group receiving 25 µg Alhydrogel®-formulated rPA.

Conclusions/Significance

The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00057525","term_id":"NCT00057525"}}NCT00057525     

A Mutational Analysis of the Endophilin-A N-BAR Domain Performed in Living Flies

Background

Endophilin is a cytoplasmic protein with an important function in clathrin-dependent endocytosis at synapses and elsewhere. Endophilin has a BAR (Bin/Amphiphysin/Rvs-homology) domain, which is implicated in the sensing and induction of membrane curvature. Previous structure-function studies of the endophilin-A BAR domain have almost exclusively been made in reduced systems, either in vitro or ex vivo in cultured cells. To extend and complement this work, we have analyzed the role played by the structural features of the endophilin-A BAR domain in Drosophila in vivo.

Methodology/Principal Findings

The study is based on genetic rescue of endophilin-A (endoA) null mutants with wild type or mutated endoA transgenes. We evaluated the viability of the rescuants, the locomotor behavior in adult flies and the neurotransmission at the larval neuromuscular junction. Whereas mutating the endophilin BAR domain clearly affected adult flies, larval endophilin function was surprisingly resistant to mutagenesis. Previous reports have stressed the importance of a central appendage on the convex BAR surface, which forms a hydrophobic ridge able to directly insert into the lipid bilayer. We found that the charge-negative substitution A66D, which targets the hydrophobic ridge and was reported to completely disrupt the ability of endophilin-BAR to tubulate liposomes in vitro, rescued viability and neurotransmission with the same efficiency as wild type endoA transgenes, even in adults. A similar discrepancy was found for the hydrophilic substitutions A63S/A66S and A63S/A66S/M70Q. The A66W mutation, which introduces a bulky hydrophobic side chain and induces massive vesiculation of liposomes in vitro, strongly impeded eye development, even in presence of the endogenous endoA gene. Substantial residual function was observed in larvae rescued with the EndoA(Arf) transgene, which encodes a form of endophilin-A that completely lacks the central appendage. Whereas a mutation (D151P) designed to increase the BAR curvature was functional, another mutation (P143A, ΔLEN) designed to decrease the curvature was not.

Conclusions/Significance

Our results provide novel insight into the structure/function relationship of the endophilin-A BAR domain in vivo, especially with relation to synaptic function.     

Mechanisms ensuring optimal conditions of implantation and embryo development in the pig

The paper summarizes results of a series of studies concerning luteolysis and early pregnancy in pigs. The involvement of the oxytocin (OT)/OT receptor system in the mechanism of corpus luteum (CL) protection during early pregnancy as well as the implication of luteinizing hormone (LH) in the endometrial prostaglandin (PG) release and synthesis are described. In addition, the role of leptin in the regulation of ovarian steroidogenesis and the expression of leptin and its receptor (OB-Rb) genes in hypothalamus, pituitary and reproductive tissues are reported. Moreover, a strong emphasis was placed on the mechanism of PGE2 participation in the local endocrine regulations of reproductive processes occurring in the utero-ovarian area as well as on the vascular endothelial growth factor (VEGF) ligand-receptor system in the ovary and uterus.     

Effects of oxytocin alone and in combination with selected hypothalamic hormones on ACTH, beta-endorphin, LH and PRL secretion by anterior pituitary cells of cyclic pigs

Oxytocin (OT) is involved in the stimulation of secretion of anterior pituitary hormones in females during the periovulatory and periparturient periods. In the present study we examined the role of OT in control of ACTH, beta-endorphin, LH and PRL secretion in vitro from dispersed anterior pituitary cells collected from gilts during the luteal (Days 10-12; n=6) and follicular (Days 18-20; n=5) phases of the estrous cycle. Isolated anterior pituitary cells (1 x 10(6)/ml) were transferred into 24-well plates, separately for each animal, and were pre-incubated for three days at 37 degrees C in atmosphere of 5% CO(2) and 95% air. The cells which attached to the dishes were incubated (3.5 h, 37 degrees C) in McCoy's medium in the absence (control) or in the presence of the following factors: CRH alone (10(-10), 10(-9), 10(-8), 10(-7) M), OT alone (10(-8), 10(-7), 10(-6) M), LVP alone (10(-7) M), OT (10(-7) M) plus CRH (10(-9) M) and LVP (10(-7) M) plus CRH (10(-9) M) for studying ACTH and beta-endorphin secretion; OT alone (10(-8), 10(-7), 10(-6) M), GnRH alone (100 ng/ml), CRH alone (10(-9) M), OT (10(-7) M) plus GnRH (100 ng/ml) and OT (10(-7) M) plus CRH (10(-9) M) for studying LH and PRL secretion. Concentrations of the studied hormones in media were analyzed by RIA. Oxytocin alone increased ACTH (at doses 10(-7), 10(-6) M), beta-endorphin (at dose 10(-8) M), LH (at dose 10(-8) M) and PRL (at doses 10(-7), 10(-6) M) secretion by pituitary cells isolated only from luteal-phase gilts. None of the studied hormone concentrations in the medium was increased in response to OT when pituitary cells of follicular-phase gilts were examined. Oxytocin in combination with CRH exerted an additive effect on beta-endorphin secretion during the luteal phase. Summarizing, in the present study the stimulatory effect of oxytocin on ACTH, beta-endorphin, LH and PRL secretion by pituitary cells isolated from gilts during the luteal phase was demonstrated. However, the cells collected from follicular-phase gilts appeared to be unresponsive to OT. Moreover, interaction between OT and CRH in affecting beta-endorphin secretion was shown. These results suggest that OT may be transiently involved in the modulation of anterior pituitary hormone secretion in cyclic pigs.     

65Zn2+ Transport by lobster hepatopancreatic lysosomal membrane vesicles

In crustaceans, the hepatopancreas is the major organ system responsible for heavy metal detoxification, and within this structure the lysosomes and the endoplasmic reticulum are two organelles that regulate cytoplasmic metal concentrations by selective sequestration processes. This study characterized the transport processes responsible for zinc uptake into hepatopancreatic lysosomal membrane vesicles (LMV) and the interactions between the transport of this metal and those of calcium, copper, and cadmium in the same preparation. Standard centrifugation methods were used to prepare purified hepatopancreatic LMV and a rapid filtration procedure, to quantify 65Zn2+ transfer across this organellar membrane. LMV were osmotically reactive and exhibited a time course of uptake that was linear for 15-30 sec and approached equilibrium by 300 sec. 65Zn2+ influx was a hyperbolic function of external zinc concentration and followed Michaelis-Menten kinetics for carrier transport (Km = 32.3 +/- 10.8 microM; Jmax = 20.7 +/- 2.6 pmol/mg protein x sec). This carrier transport was stimulated by the addition of 1 mM ATP (Km = 35.89 +/- 10.58 microM; Jmax = 31.94+/-3.72 pmol/mg protein/sec) and replaced by an apparent slow diffusional process by the simultaneous presence of 1 mM ATP+250 microM vanadate. Thapsigargin (10 microM) was also a significant inhibitor of zinc influx (Km = 72.87 +/- 42.75 microM; Jmax =22.86 +/- 4.03 pmol/mg protein/sec), but not as effective in this regard as was vanadate. Using Dixon analysis, cadmium and copper were shown to be competitive inhibitors of lysosomal membrane vesicle 65Zn2+ influx by the ATP-dependent transport process (cadmium Ki = 68.1 +/- 3.2 microM; copper Ki = 32.7 +/- 1.9 microM). In the absence of ATP, an outwardly directed H+ gradient stimulated 65Zn2+ uptake, while a proton gradient in the opposite direction inhibited metal influx. The present investigation showed that 65Zn2+ was transported by hepatopancreatic lysosomal vesicles by ATP-dependent, vanadate-, thapsigargin-, and divalent cation-inhibited, carrier processes that illustrated Michaelis-Menten influx kinetics and was stimulated by an outwardly directed proton gradient. These transport properties as a whole suggest that this transporter may be a lysosomal isoform of the ER Sarco-Endoplasmic Reticulum Calcium ATPase.     

Mapping data elements to terminological resources for integrating biomedical data sources

Background

Data integration is a crucial task in the biomedical domain and integrating data sources is one approach to integrating data. Data elements (DEs) in particular play an important role in data integration. We combine schema- and instance-based approaches to mapping DEs to terminological resources in order to facilitate data sources integration.

Methods

We extracted DEs from eleven disparate biomedical sources. We compared these DEs to concepts and/or terms in biomedical controlled vocabularies and to reference DEs. We also exploited DE values to disambiguate underspecified DEs and to identify additional mappings.

Results

82.5% of the 474 DEs studied are mapped to entries of a terminological resource and 74.7% of the whole set can be associated with reference DEs. Only 6.6% of the DEs had values that could be semantically typed.

Conclusion

Our study suggests that the integration of biomedical sources can be achieved automatically with limited precision and largely facilitated by mapping DEs to terminological resources.

     

Evidence of self-sustained motoneuron firing in young and older adults.

Motoneurons demonstrate a type of self-sustained firing behavior that seems to be produced by a prolonged period of depolarization caused by intrinsic long-term changes in the motoneuron. Such self-sustained firing behavior has previously been reported in human motor units. The purpose of the present study was to investigate the occurrence of self-sustained firing behavior in older adults. Eight young (mean age 24 yrs) and eight older (mean age 73 yrs) individuals participated in the investigation. While subjects produced light dorsiflexion contractions, a brief vibration stimulus was applied to the tibialis anterior muscle. Motor unit recordings were also obtained from the tibialis anterior muscle. Self-sustained firing behavior was evidenced by the appearance of new motor unit recruitment following vibration, even as the motor units that fired before the vibratory stimulus maintained a steady firing rate. The proportion of motor units exhibiting self-sustained firing activity was similar in both young and older adults (approx. 23% of trials). We conclude that self-sustained firing behavior is a ubiquitous phenomenon that does not seem to be affected by the aging process.     

Characteristics of rabbit muscle adenylate kinase inhibition by ascorbate

Earlier studies [1-3] showed that of the glycolytic enzymes, the muscle isozymes PFK-1, LDH, and AK were inhibited by ascorbic acid. These studies on the characteristics of the inhibition of RMAK by ascorbate are part of a hypothesis [3] that ascorbate facilitates the storage of skeletal muscle glycogen by inhibiting glycolysis when the muscle is at rest. These studies examine conditions for RMAK inhibition, prevention of inhibition, and reversal of ascorbate inhibition. We found that the concentration of RMAK was an important condition for inhibition. Above 200 nM RMAK, inhibition by ascorbate could not be demonstrated and below that concentration RMAK became increasingly sensitive to ascorbate inhibition. Associated with increased sensitivity to inhibition by ascorbate is a deviation from a linear to a concave relationship between low RMAK concentrations and enzyme activity. At low RMAK concentrations, the concave relationship becomes convex in the presence of muscle aldolase. In addition, aldolase reverses inhibitions by ascorbate. A comparison of inhibition of RMAK byascorbate and inhibition of LDH-m4 [3] is discussed. Other proteins prevent RMAK inhibition but do not reverse inhibition by ascorbate. The role of RMAK as a factor in the control of the rate of glycolysis is presented as is the role of compartmentalization with respect to the proposed role for ascorbate inhibition.