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Immunological adjuvants such as the saponin natural product QS-21 help stimulate the immune response to co-administered antigens and have become increasingly important in the development of prophylactic and therapeutic vaccines. However, clinical use of QS-21 is encumbered by chemical instability, dose-limiting toxicity, and low-yielding purification from the natural source. Previous studies of structure-activity relationships in the four structural domains of QS-21 have led to simplified, chemically stable variants that retain potent adjuvant activity and low toxicity in mouse vaccination models. However, modification of the central glycosyl ester linkage has not yet been explored. Herein, we describe the design, synthesis, immunologic evaluation, and molecular dynamics analysis of a series of novel QS-21 variants with different linker lengths, stereochemistry, and flexibility to investigate the role of this linkage in saponin adjuvant activity and conformation. Despite relatively conservative structural modifications, these variants exhibit striking differences in in vivo adjuvant activity that correlate with specific conformational preferences. These results highlight the junction of the triterpene and linear oligosaccharide domains as playing a critical role in the immunoadjuvant activity of the Quillaja saponins and also suggest a mechanism of action involving interaction with a discrete macromolecular target, in contrast to the non-specific mechanisms of emulsion-based adjuvants. © The Royal Society of Chemistry 2016.

Polysialic acid (polySA) is a polymer side chain bound to the neural cell adhesion molecule that is extensively expressed on the surface of small cell lung cancer (SCLC) cells. In our previous study, a robust antibody response was noted in patients with SCLC after vaccination with 30 μg of keyhole limpet hemocyanin (KLH)-conjugated N-propionylated (NP-) polySA, but peripheral neuropathy and ataxia were detected in several vaccinated patients. The objectives of the current trial were to establish the lowest optimal dose and to confirm the safety of the induction of antibodies against polySA with the NP-polySA vaccine.Patients with SCLC who completed initial treatment and had no evidence of disease progression were injected with either 10 or 3 μg of NP-polySA conjugated to KLH and mixed with 100 μg of immunologic adjuvant (QS-21) at weeks 1, 2, 3, 4, 8, and 16.Nine patients were enrolled at each of the two dose levels. Prior to vaccination, one patient in each group had low-titer antibodies against polysialic acid. All patients at the 10 μg vaccine dose level responded to vaccination with IgM antibody titers against polysialic acid (median titer 1/1,280 by ELISA), and all but one patient made IgM and IgG antibodies against the artificial vaccine immunogen, NP-polysialic acid (median titer 1/10,240). The antibody responses at the 3 μg vaccine dose level were lower; six of nine patients developed antibodies against polysialic acid (median titer 1/160). Post-vaccination sera from 6/9 and 3/9 patients in the 10 and 3 μg groups reacted strongly with human SCLC cells by fluorescent-activated cell sorting (FACS). Sera from all patients in the 10 μg dose group also had bactericidal activity against group B meningococci with rabbit complement. Self-limited grade 3 ataxia of unclear etiology was seen in 1 of 18 patients.Vaccination with NP-polySA-KLH resulted in consistent high-titer antibody responses, with the 10 μg dose significantly more immunogenic than the 3 μg dose. This study establishes the lowest optimally immunogenic dose of NP-polysialic acid in this NP-polysialic acid-KLH conjugate vaccine to be at least 10 μg, and it establishes the vaccine's safety. We plan to incorporate NP-polySA into a polyvalent vaccine against SCLC with four glycolipid antigens also widely expressed in SCLC-GD2, GD3, fucosylated GM1, and globo H.

The 95 % ethanol extract of Astragalus has been demonstrated to have potent activity as an immunological adjuvant when administered with vaccines of various types. We endeavor here to identify the components of this extract that are responsible for this adjuvant activity. Mice were immunized with KLH conjugated to cancer carbohydrate antigens globo H and GD3 and cancer peptide antigen MUC1 combined with different Astragalus fractions or with commercially available Astragalus saponins and flavonoids. The antibody responses against cancer antigens and KLH were quantitated in ELISA assays, and toxicity was calculated by weight loss. Astragalosides II and IV were the most active components, but the toxicity of these two differed dramatically. Astragaloside II was the most toxic Astragalus component with 5-10 % weight loss at a dose of 500 µg while astragaloside IV showed no weight loss at all at this dose, suggesting that astragaloside IV might be utilized as an immunological adjuvant in future studies. Several flavonoids also had significant adjuvant activity. However, when the activities of these known immunologically active components of Astragalus (and of endotoxin) are calculated based on the extent of their presence in the 95 % ethanol extract, they provide only a small proportion of the immunological activity. This raises the possibility that additional uniquely active components of Astragalus may contribute to adjuvant activity, or that the adjuvant activity of Astragalus is greater than the activity of the sum of its parts.

The saponin fraction QS-21 from Quillaja saponaria has been demonstrated to be a potent immunological adjuvant when mixed with keyhole limpet hemocyanin conjugate vaccines, as well as with other classes of subunit antigen vaccines. QS-21 adjuvant is composed of two isomers that include the apiose and xylose forms in a ratio of 65:35, respectively. The chemical syntheses of these two isomers in pure form have recently been disclosed. Herein we describe detailed in vivo immunological evaluations of these synthetic QS-21 isomeric constituents, employing the GD3-KLH melanoma antigen. With this vaccine construct, high antibody titers against GD3 ganglioside and KLH were elicited when GD3-KLH was co-administered with adjuvant, either as the individual separate synthetic QS-21 isomers (SQS-21-Api or SQS-21-Xyl), or as its reconstituted 65:35 isomeric mixture (SQS-21). These antibody titer levels were comparable to that elicited by vaccinations employing naturally derived QS-21 (PQS-21). Moreover, toxicities of the synthetic saponin adjuvants were also found to be comparable to that of naturally derived PQS-21. These findings demonstrate unequivocally that the adjuvant activity of QS-21 resides in these two principal isomeric forms, and not in trace contaminants within the natural extracts. This lays the foundation for future exploration of structure-function correlations to enable the discovery of novel saponins with increased potency, enhanced stability, and attenuated toxicity.

Sodium-selective amiloride-sensitive epithelial channel (ENaC) located in the apical membrane is involved in the reabsorption of sodium in tight epithelia. The soluble N-ethylmaleimide-sensitive attachment receptors (SNAREs) mediate vesicle trafficking in a variety of cell systems. Syntaxin (a t-SNARE) has been shown to interact with and functionally regulate a number of ion channels including ENaC. In this study, we investigated the role of SNAP-23, another SNARE protein, on ENaC activity in the HT-29 colonic epithelial cell system and Xenopus oocytes. Recording of amiloride-sensitive currents in both systems suggest that SNAP-23 modulates channel function, though a much higher concentration is required to inhibit ENaC in Xenopus oocytes. The introduction of Botulinum toxin A (a neurotoxin which cleaves SNAP-23), but not Botulinum toxin B or heat-inactivated Botulinum toxin A, reversed the inhibitory effect of SNAP-23 on amiloride-sensitive currents. However, syntaxin 1A and SNAP-23 combined portray a complex scenario that suggests that this channel interacts within a quaternary complex. Synaptotagmin expression neither interacts with, nor showed any effect on amiloride-sensitive currents when co-expressed with ENaC. Pull down assays suggest mild interaction between ENaC and SNAP-23, which gets stronger in the presence of syntaxin 1A. Data further suggest that SNAP-23 possibly interacts with the N-terminal alphaENaC. These functional and biochemical approaches provide evidence for a complex relationship between ENaC and the exocytotic machinery. Our data suggest that SNARE protein interplay defines the fine regulation of sodium channel function.

Cystic fibrosis (CF), an autosomal recessive disorder, is caused by the disruption of biosynthesis or the function of a membrane cAMP-activated chloride channel, CFTR. CFTR regulatory mechanisms include recruitment of channel proteins to the cell surface from intracellular pools and by protein–protein interactions. Rab proteins are small GTPases involved in regulated trafficking controlling vesicle docking and fusion. Rab4 controls recycling events from endosome to the plasma membrane, fusion, and degradation. The colorectal cell line HT-29 natively expresses CFTR and responds to cAMP stimulation with an increase in CFTR-mediated currents. Rab4 over-expression in HT-29 cells inhibits both basal and cAMP-stimulated CFTR-mediated currents. GTPase-deficient Rab4Q67L and GDP locked Rab4S22N both inhibit channel activity, which appears characteristically different. Active status of Rab4 was confirmed by GTP overlay assay, while its expression was verified by Western blotting. The pull-down and immunoprecipitation experiments suggest that Rab4 physically interacts with CFTR through protein–protein interaction. Biotinylation with cell impermeant NHS-Sulfo-SS-Biotin implies that Rab4 impairs CFTR expression at cell surface. The enhanced cytosolic CFTR indicates that Rab4 expression restrains CFTR appearance at the cell membrane. The study suggests that Rab4 regulates the channel through multiple mechanisms that include protein-protein interaction, GTP/GDP exchange, and channel protein trafficking. We propose that Rab4 is a dynamic molecule with a significant role in CFTR function.

Immunoadjuvants are used to potentiate the activity of modern subunit vaccines that are based on molecular antigens. An emerging approach involves the combination of multiple adjuvants in a single formulation to achieve optimal vaccine efficacy. Herein, to investigate such potential synergies, we synthesized novel adjuvant conjugates based on the saponin natural product QS-21 and the aldehyde tucaresol via chemoselective acylation of an amine at the terminus of the acyl chain domain in QS saponin variants. In a preclinical mouse vaccination model, these QS saponin–tucaresol conjugates induced antibody responses similar to or slightly higher than those generated with related QS saponin variants lacking the tucaresol motif. The conjugates retained potent adjuvant activity, low toxicity, and improved activity–toxicity profiles relative to QS-21 itself and induced IgG subclass profiles similar to those of QS-21, indicative of both Th1 cellular and Th2 humoral immune responses. This study opens the door to installation of other substituents at the terminus of the acyl chain domain to develop additional QS saponin conjugates with desirable immunologic properties.

Adjuvants are materials added to vaccines to enhance the immunological response to an antigen. QS-21 is a natural product adjuvant under investigation in numerous vaccine clinical trials, but its use is constrained by scarcity, toxicity, instability and an enigmatic molecular mechanism of action. Herein we describe the development of a minimal QS-21 analogue that decouples adjuvant activity from toxicity and provides a powerful platform for mechanistic investigations. We found that the entire branched trisaccharide domain of QS-21 is dispensable for adjuvant activity and that the C4-aldehyde substituent, previously proposed to bind covalently to an unknown cellular target, is also not required. Biodistribution studies revealed that active adjuvants were retained preferentially at the injection site and the nearest draining lymph nodes compared with the attenuated variants. Overall, these studies have yielded critical insights into saponin structure-function relationships, provided practical synthetic access to non-toxic adjuvants, and established a platform for detailed mechanistic studies.

There is a great deal of importance associated with the problem of location analysis, but there is an appreciable lack of adequate methods that can be used by an independent business having limited financial means and inadequate training. Because of this condition, a study was undertaken to determine if any patterns existed which could be used to test future locations. A secondary purpose of the study was to provide limited information on other competitive aspects indirectly related to location. One such example would be an increase in price awareness and the extent to which this has changed customer buying habits in regard to the convenience of a store location. The methodology employed to gain the information was to interview customers as they left six neighborhood independent retail pharmacies in Salt Lake County and determine their place of residence as well as some information about buying attitudes. The information was then punched on Key-Sort cards for ease of tabulation and the addresses were plotted on maps. The area around each store on the map was then divided into quadrants and half-mile increments. The results demonstrated some very definite customer patterns. The quadrant farthest away from the central business district had the largest number of customers (forty-five per cent). Nearly four-fifths (seventy-nine per cent) of the customers live within the first one and one-half miles of the stores. More customers live on the outboard side (sixty-five per cent), the area beyond the store site away from the central business district, than on the inboard side (thirty-five) per cent, the area between the store site and the central business district. Other results obtained from the interviews showed that sixty-seven per cent of the people were at the closest store to their home when they were interviewed, ninety-five per cent drove to the store, sixty-eight per cent did the majority of their purchasing at the store at which they were interviewed, and only sixteen per cent of the people shopped at a store that was not nearest to their home. The average purchase size was |2.52, the average age was 36.3 years, and more men (fifty-two and three-tenths per cent) were interviewed than women (forty-seven and seven-tenths per cent). This latter result is assumed to be due to the time of the day in which the interviews were performed. Definite customer patterns were noted for all the stores surveyed. One should be able to determine the feasibility of a given site with the application of three pattern tests derived. If the tests are affirmative, it is recommended that further evaluation of the site be done before a definite decision to locate is made. If the tests are negative, the costs of further testing can be avoided, Since the average purchase size was relatively small and most of the store's customers lived within a limited area, it is assumed that the convenience-goods concept is still in existence and that large chain drug stores have not greatly influenced a considerable segment of the buying public.

In recent years, spars have become a preferred industry solution for certain offshore deepwater developments. Perdido is the first spar platform to be operated by Shell. The Perdido spar has been recently installed in the ultradeepwater Gulf of Mexico Alaminos Canyon and is scheduled for first oil in 2010. This Direct Vertical Access (DVA) spar will operate at a water depth of 7,825′ and will be the deepest spar production and drilling facility in the world. Numerical predictions of the spar global motions in waves, wind and current are presented in this paper. Motivation for this study comes from two facts: 1. Each spar platform design is unique in terms of its size, number and geometry of heave plates, riser system and mooring system. 2. Metocean design criteria have been increased in view of the recent hurricanes. Model tests of the Perdido spar were conducted at MARIN at a scale of 1:59.94. In these experiments, several Gulf of Mexico (GOM) wave, current and wind environments were considered. The six-degree-of-freedom motions, deck accelerations, air gap, as well as the loads on the heave plates, mooring lines and risers were measured. In this paper, global motion predictions of the Perdido spar are given using Shell’s in-house COSMOS/ WAMIT suite of programs. Extensive comparisons between the numerical predictions and the experimental results were undertaken. In all cases, the comparisons are very good. In order to include heave viscous loads and damping, special line members were included at the bottom of the hard tank, the bottom of the soft tank and each heave plate, in addition to standard line members used to describe the truss. These special members contribute heave viscous loads with drag coefficients selected from the Perdido experiments. Several heave plate configurations were considered to systematically study the impact of heave plates on the spar global motions. The influence of the heave plate geometry on the heave added mass and on the global motions was derived using WAMIT. The strakes’ actual geometry was also included in the WAMIT diffraction analysis. Most of the moonpool area at the bottom of the Perdido hard tank is closed. As a result, the pumping mode was not excited during the experiments. However, numerical simulations with WAMIT showed a sharp peak at the “pumping mode” resonant frequency. This peak was suppressed by introducing a second floating body that capped the moonpool at the water surface. Based on these learnings, recommendations for global motion modeling are presented in this paper.Copyright © 2010 by ASME

Carbohydrate and peptide-based antitumor vaccine constructs featuring clusters of both tumor associated carbohydrate antigens and mucin-like peptide epitopes have been designed, synthesized, and studied. The mucin-based epitopes are included to act, potentially, as T-cell epitopes in order to provoke a strong immune response. Hopefully the vaccine will simulate cell surface architecture, thereby provoking levels of immunity against cancer cell types displaying such characteristics. With this central idea in mind, we designed a new vaccine type against ovarian cancer. Following advances in glycohistology, our design is based on clusters of Gb(3) antigen and also incorporates a MUC5AC peptide epitope. The vaccine is among the most complex targeted constructs to be assembled by chemical synthesis to date. The strategy for the synthesis employed a Gb(3)-MUC5AC thioester cassette as a key building block. Syntheses of both nonconjugate and KLH-conjugated vaccines constructs have been accomplished.

Tanzania is endowed with vast quantities and types of resources whose extraction has been central to the country¡¯s economic growth. However, the challenge has been translating growth in extraction activities into inclusive and sustainable socio-economic development and transformation. Instead of being of benefit to all, mineral resource extraction has only served to put Tanzania into the map of foreign capital flows but with limited benefits to the local citizens. Drawing on academic literature, government policy and legal documents as well as other secondary materials, this paper examines challenges of translating resource extraction into inclusive and sustainable socio-economic development in Tanzania. It focuses on large scale gold mining and local content provisions. The paper argues that having in place a good policy and legal framework is nothing if it is not implemented to the benefit of local communities and the entire nation. There is, therefore, a need to move from papers to implementation if mineral resource extraction is to be of any significance to socio-economic transformation. This is very significant especially now that Tanzania has a new mining sector legal and policy framework in place and is looking forward to making the most out of its recently discovered natural gas.