Pharmaceutical and preclinical evaluation of Advax adjuvant as a dose-sparing strategy for ant venom immunotherapy.

A major challenge in broader clinical application of Jack Jumper ant venom immunotherapy (JJA VIT) is the scarcity of ant venom which needs to be manually harvested from wild ants. Adjuvants are commonly used for antigen sparing in other vaccines, and thereby could potentially have major benefits to extend JJA supplies if they were to similarly enhance JJA VIT immunogenicity. The purpose of this study was to evaluate the physicochemical and microbiological stability and murine immunogenicity of low-dose JJA VIT formulated with a novel polysaccharide adjuvant referred to as delta inulin or Advax™. Jack Jumper ant venom (JJAV) protein stability was assessed by UPLC-UV, SDS-PAGE, SDS-PAGE immunoblot, and ELISA inhibition. Diffraction light scattering was used to assess particle size distribution of Advax; pH and benzyl alcohol quantification by UPLC-UV were used to assess the physicochemical stability of JJAV diluent, and endotoxin content and preservative efficacy test was used to investigate the microbiological properties of the adjuvanted VIT formulation. To assess the effect of adjuvant on JJA venom immunogenicity, mice were immunised four times with JJAV alone or formulated with Advax adjuvant. JJA VIT formulated with Advax was found to be physicochemically and microbiologically stable for at least 2 days when stored at 4 and 25 °C with a trend for an increase in allergenic potency observed beyond 2 days of storage. Low-dose JJAV formulated with Advax adjuvant induced significantly higher JJAV-specific IgG than a 5-fold higher dose of JJAV alone, consistent with a powerful allergen-sparing effect. The pharmaceutical data provides important guidance on the formulation, storage and use of JJA VIT formulated with Advax adjuvant, with the murine immunogenicity studies providing a strong rationale for a planned clinical trial to test the ability of Advax adjuvant to achieve 4-fold JJAV dose sparing in JJA-allergic human patients.

Preparation of inulin-type fructooligosaccharides using fast protein liquid chromatography coupled with refractive index detection.

A fast protein liquid chromatography coupled with refractive index detection (FPLC-RID) method was firstly developed for preparation and purification of fructooligosaccharides with different degree of polymerization from burdock, Arctium lappa. After extraction with 60% ethanol and decolorization with MCI gel CHP20P, total fructooligosaccharides were purified on Bio-Gel P-2 column eluted with water at the flow rate of 0.3 ml/min, which was the optimized conditions. The obtained fructooligosaccharides with degree of polymerization of 3-9 were identified based on their methylation analysis, MS and NMR data. This method has the advantages of high automation, good recovery and easy performance, which could be used for preparation of FOS from other sources, as well as other targeted compounds without UV absorbance.

[Relationship between the anticoagulant activity of sulfated plant polysaccharides and the area of their precipitation with polycations during biospecific electrophoresis].

Polyanions (in an amount within 1.5 - 6.0 mg), including cellulose sulfates (excreted from Gossipium hirsutum L., molecular weight 22.0 kDa, degree of sulfation within 0.8 - 1.8), inulin sulfates (excreted from Helianthus tuberosus, molecular weight 8.0 kDa, degree of sulfation within 0.6 - 1.6), pectin sulfates (excreted from Abies sibirica L., molecular weight 24.0 kDa, degree of sulfation within 0.8 - 1.1), give rise to peaks of precipitation with polycations of protamine sulfate. Only cellulose sulfates (in amount within 0.38 - 6.00 mg) give the peaks of precipitation with chitosan polycations (molecular weight 10 kDa, degree of deacetylation 85%) during horizontal biospecific electrophoresis. The height of the peak of precipitation with protamin sulfate was found to grow with increasing antithrombin activity of cellulose sulfates and pectin sulfate (for polyanions in an amount within 1.5 - 6 mg). The size of the area of precipitation with chitosan was found to decrease with increasing antithrombin activity of cellulose sulfates.

[Dependence of the anticoagulant activity of starch and inulin on their degree of sulfonation].

We have studied a relationship between the degree of sulfonation and anticoagulant activity of starch from Solanum tuberosum (molecular weight, 25000-30000 Da; sulfonation degree, 0.4-2.5) and inulin from Helianthus tuberosus (molecular weight, 7000-8000 Da; sulfonation degree, 0.6-1.6). Starch and inulin sulfates (i) increased the time of appearance of fibrin clots in plasma in coagulometric tests and (ii) reduced (via antithrombin) the rate of thrombin-induced hydrolysis of a chromogen substrate. The antithrombin (aIIa) activity of starch sulfates reached 16.8-70.0 IU/mg and the activity against factor Xa (aXa activity) was 2.3-16.6 IU/mg. The antithrombin activity of inulin sulfates was within 5.5-11.4 IU/mg and the activity against factor Xa (aXa activity) was within 0-1.4 IU/mg. An increase in the degree of sulfonation led to a growth in the anticoagulant activity of starch sulfates. The anticoagulant activity of starch sulfates and inulin sulfate with sulfonation degree 1.0 is mediated by antithrombin, which is the plasma inhibitor of serine proteases.

Inulin-type prebiotics: a review. (Part 2).

This is part 2 of a two-part review of inulin-type prebiotics. This article discusses the clinical research on inulin-type prebiotics, including effects on infant nutrition, gastrointestinal health, colon cancer prevention, blood sugar and lipid metabolism, bone mineralization, fatty liver disease, obesity, and immunity. Gastrointestinal side effects and dosage recommendations are also considered.

Inulin-type prebiotics--a review: part 1.

This article is part 1 of a two-part review of inulin-type prebiotics. Prebiotics are a category of nutritional compounds grouped together by the ability to promote the growth of specific beneficial (probiotic) gut bacteria. Inulin-type prebiotics contain fructans of the inulin-type. Fructans are a category of nutritional compounds that encompasses naturally occurring plant oligo- and polysaccharides in which one or more fructosyl-fructose linkages comprise the majority of glycosidic bonds. To be inulin-type a fructan must have beta (2(1) fructosyl-fructose glycosidic bonds, which gives inulin its unique structural and physiological properties, allowing it to resist enzymatic hydrolysis by human salivary and small intestinal digestive enzymes. Inulin-type prebiotics include fructooligosaccharides (FOS), oligofructose, and inulin - terms that have been used inconsistently in both the scientific literature and in food applications. Commercially available inulin-type prebiotics can be extracted from food (typically chicory root) or synthesized from a more fundamental molecule (typically sucrose). Depending on the starting source and degree of processing, inulin-type prebiotics can be produced with very different chemical compositions. Some inulin-type prebiotics are relatively high in free sugars (the monosaccharides fructose and glucose and the disaccharide sucrose), while others have most or all free sugars removed. Processing can also result in mixes consisting exclusively of inulin-type oligosaccharides, polysaccharides, or both. Because inulin, oligofructose, and FOS resist enzymatic digestion in the upper gastrointestinal tract, they reach the colon virtually intact where they undergo bacterial fermentation. All inulin-type prebiotics are bifidogenic - stimulating the growth of Bifidobacteria species. The effects they have on other gut organisms are less consistent. A minimal dose of inulin-type prebiotic appears to be needed to produce a bifidogenic effect. However, intra-individual response to an identical dose of the same inulin-type prebiotic, in terms of stimulation of total number of Bifidobacteria and individual Bifidobacteria species, can be variable. Research on therapeutic uses of inulin-type prebiotics will be covered in part 2 of this review.

Polymeric surfactants based on inulin, a polysaccharide extracted from chicory. 1. Synthesis and interfacial properties.

Inulin, the polydisperse reserve polysaccharide from chicory, has been modified by carbamoylation in organic solvents. The reaction of inulin with a range of alkyl isocyanates resulted, after crystalization, in a variety of carbamoylated inulins from which the interfacial properties were determined. The medium and long chain carbamoylated inulins showed a good to very good reduction of the interfacial tension which makes these biopolymers interesting in the field of biodegradable surface active agents.