Recombinant Full-length Plasmodium falciparum Circumsporozoite Protein-Based Vaccine Adjuvanted With Glucopyranosyl Lipid A-Liposome Quillaja saponaria 21: Results of Phase 1 Testing With Malaria Challenge.

BACKGROUND: Malaria is preventable yet causes >600 000 deaths annually. RTS,S, the first marketed malaria vaccine, has modest efficacy, but improvements are needed for eradication. METHODS: We conducted an open-label, dose escalation phase 1 study of a full-length recombinant circumsporozoite protein vaccine (rCSP) administered with adjuvant glucopyranosyl lipid A-liposome Quillaja saponaria 21 formulation (GLA-LSQ) on days 1, 29, and 85 or 1 and 490 to healthy, malaria-naive adults. The primary end points were safety and reactogenicity. The secondary end points were antibody responses and Plasmodium falciparum parasitemia after homologous controlled human malaria infection. RESULTS: Participants were enrolled into 4 groups receiving rCSP/GLA-LSQ: 10 µg × 3 (n = 20), 30 µg × 3 (n = 10), 60 µg × 3 (n = 10), or 60 µg × 2 (n = 9); 10 participants received 30 µg rCSP alone × 3, and there were 6 infectivity controls. Participants experienced no serious adverse events. Rates of solicited and unsolicited adverse events were similar among groups. All 26 participants who underwent controlled human malaria infection 28 days after final vaccinations developed malaria. Increasing vaccine doses induced higher immunoglobulin G titers but did not achieve previously established RTS,S benchmarks. CONCLUSIONS: rCSP/GLA-LSQ had favorable safety results. However, tested regimens did not induce protective immunity. Further investigation could assess whether adjuvant or schedule adjustments improve efficacy. CLINICAL TRIALS REGISTRATION: NCT03589794.

Saponin and Ribosome-Inactivating Protein Synergistically Trigger Lysosome-Dependent Apoptosis by Inhibiting Lysophagy: Potential to Become a New Antitumor Strategy.

The susceptibility of lysosomal membranes in tumor cells to cationic amphiphilic drugs (CADs) enables CADs to induce lysosomal membrane permeabilization (LMP) and trigger lysosome-dependent cell death (LDCD), suggesting a potential antitumor therapeutic approach. However, the existence of intrinsic lysosomal damage response mechanisms limits the display of the pharmacological activity of CADs. In this study, we report that low concentrations of QS-21, a saponin with cationic amphiphilicity extracted from Quillaja Saponaria tree, can induce LMP but has nontoxicity to tumor cells. QS-21 and MAP30, a type I ribosome-inactivating protein, synergistically induce apoptosis in tumor cells at low concentrations of both. Mechanistically, QS-21-induced LMP helps MAP30 escape from endosomes or lysosomes and subsequently enter the endoplasmic reticulum, where MAP30 downregulates the expression of autophagy-associated LC3 proteins, thereby inhibiting lysophagy. The inhibition of lysophagy results in the impaired clearance of damaged lysosomes, leading to the leakage of massive lysosomal contents such as cathepsins into the cytoplasm, ultimately triggering LDCD. In summary, our study showed that coadministration of QS-21 and MAP30 amplified the lysosomal disruption and can be a new synergistic LDCD-based antitumor therapy.

Structure Elucidation of Triterpenoid Saponins Found in an Immunoadjuvant Preparation of Quillaja brasiliensis Using Mass Spectrometry and 1H and 13C NMR Spectroscopy.

An immunoadjuvant preparation (named Fraction B) was obtained from the aqueous extract of Quillaja brasiliensis leaves, and further fractionated by consecutive separations with silica flash MPLC and reverse phase HPLC. Two compounds were isolated, and their structures elucidated using a combination of NMR spectroscopy and mass spectrometry. One of these compounds is a previously undescribed triterpene saponin (Qb1), which is an isomer of QS-21, the unique adjuvant saponin employed in human vaccines. The other compound is a triterpene saponin previously isolated from Quillaja saponaria bark, known as S13. The structure of Qb1 consists of a quillaic acid residue substituted with a ß-d-Galp-(1→2)-[ß-d-Xylp-(1→3)]-ß-d-GlcpA trisaccharide at C3, and a ß-d-Xylp-(1→4)-α-l-Rhap-(1→2)-[α-l-Arap-(1→3)]-ß-d-Fucp moiety at C28. The oligosaccharide at C28 was further substituted at O4 of the fucosyl residue with an acyl group capped with a ß-d-Xylp residue.

Off-line two-dimensional liquid chromatography separation for the quality control of saponins samples from Quillaja Saponaria.

Quil-A is a purified extract of saponins with strong immunoadjuvant activity. While specific molecules have been identified and tested in clinical trials, Quil-A is mostly used as a totum of the Quillaja Saponaria bark extract. Quality control of the extract stability is usually based on the monitoring of specific saponins, whereas the comparison of samples with an initial chromatogram seems more appropriate. A reference fingerprint based on comprehensive two-dimensional liquid chromatography offers a rapid detection of nonconform samples. To fulfill quality control constraints, off-line configuration using basic instrumentation was promoted. Hence, reversed-phase liquid chromatography × reversed-phase liquid chromatography and hydrophilic interaction chromatography × reversed-phase liquid chromatography methods with ultraviolet and single-quadrupole mass spectrometry detection were kinetically optimized. The reversed-phase liquid chromatography × reversed-phase liquid chromatography method used a pH switch between dimensions to maximize orthogonality. Despite diagonalization, it led to a high peak capacity of 831 in 2 h. On the other hand, the combination of hydrophilic interaction chromatography and reversed-phase liquid chromatography offered a larger orthogonality but a lower, yet satisfactory peak capacity of 673. The advantages of both methods were illustrated on degraded samples, where the reversed-phase liquid chromatography × reversed-phase liquid chromatography contour plot highlighted the loss of fatty acid chains, while the hydrophilic interaction chromatography × reversed-phase liquid chromatography method was found useful to evidence enzymatic loss of sugar moieties.

The combination of quillaja and yucca saponins in broilers: effects on performance, nutrient digestibility and ileal morphometrics.

1. Two series of studies were conducted to determine the effects of a combination of ground plant material derived from Quillaja saponaria trees and Yucca schidigera plants (QY) as sources of saponin, on performance, productivity, nutrient digestibility and ileal morphology of growing broilers. In each trial, 480 Cobb male birds were allocated equally to 24 pens at one-day-of-age according to body weight2. The studies consisted of two identical floor pen trials in which performance and nutrient digestibility were assessed and two trials where performance and ileal morphology were determined. In each trial, 0, 250 or 500 ppm QY were included in feed given to the broilers from 1-35 or 1-42 d of age, respectively. Eight (digestibility) or 12 (morphology) randomised replicate pens were used.3. In the digestibility trials, two birds per pen were moved to metabolism cages at d 21. Excreta was collected for a five-day period (d 21 to 25) for the determination of apparent total tract digestibility of dry and organic matter, fat and ash and nitrogen retention. For intestinal morphology, ileal segments were collected from four birds/pen on d 21 to determine villus height and crypt depth. Performance data were collected in each trial series.4. Results showed that feeding graded levels of QY produced significant linear improvements in performance and productivity at d 35, and similar linear effects were observed for N retention and all apparent digestibility measurements. Morphology data showed that birds receiving 250 and 500 ppm QY had significantly increased villus height5. These results indicated that QY exerted a positive influence on the intestinal tract by increasing the absorptive surface and improving nutrient digestibility. These effects were considered to be associated with the performance improvements recorded in both experiments.

Impact of C28 Oligosaccharide on Adjuvant Activity of QS-7 Analogues.

We have synthesized a number of Quillaja saponaria Molina (QS) saponin analogues with a different C28 sugar unit, which features either 3,4-diacetyl groups or a 3,4-cyclic carbonate group at the reducing end fucoside to mimic the naturally occurring saponin adjuvant QS-7. Immunological evaluations of these analogues in BALB/c mice indicate that truncating the C28 oligosaccharide of the natural product to the tetrasaccharide (as in 5d (ß)) could retain the adjuvant's activity in enhancing IgG1 and IgG2a productions, albeit the activity is lower than that of QS-21. Further truncation or changing stereochemistry of glycosidic linkage between the tetrasaccharide and the triterpenoid quillaic acid (QA) core or within the tetrasaccharide eliminated the saponins' adjuvant activity in terms of IgG production. On the other hand, increasing resemblance to QS-7 increased adjuvant activity and led to saponin 3's similar IgG1 and IgG2a activities to QS-21's, indicating that the unique adjuvant activities of QS saponins are determined by their specific structures.

A liquid chromatography coupled to quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS) method for identification analysis of saponins from Quillaja saponaria bark extracts in foot-and-mouth disease vaccines: Development, validation and applicability.

Saponins from Quillaja saponaria have been commonly used as immunomodulatory adjuvants in foot-and-mouth disease vaccines (FMDVs). However, due to the lack of consensus over the possible exacerbation of local inflammatory responses in cattle and its economic impacts, their use has been discouraged by Brazilian authorities. A qualitative method intended to determine the presence of saponins from Q. saponaria bark extracts in FMDVs was developed and validated. Instrumental analysis was performed using an liquid chromatography (LC) coupled to a quadrupole-time-of-flight-mass spectrometry (TOF-MS) system. The method was validated according to the International Conference on Harmonization Harmonized Tripartite Guideline Q2 (R1) and Brazilian Ministry of Agriculture, Livestock and Food Supply Analytical Quality Assurance Guidelines. Validation parameters were determined and considered suitable to the established criteria. The validated method has been applied in routine analysis in the National Agricultural Laboratory at Rio Grande do Sul (LANAGRO-RS). All results obtained were in agreement with the vaccine's composition described by the manufacturer. The method is easy and adequate for analysis in routine laboratories. To the best of the authors' knowledge, this is the first report of a method which intends to investigate the presence of saponins from Q. saponaria bark extracts in veterinary vaccines.

Formulation and Optimization of Nanoemulsions Using the Natural Surfactant Saponin from Quillaja Bark.

Replacing synthetic surfactants by natural alternatives when formulating nanoemulsions has gained attention as a sustainable approach. In this context, nanoemulsions based on sweet almond oil and stabilized by saponin from Quillaja bark with glycerol as cosurfactant were prepared by the high-pressure homogenization method. The effects of oil/water (O/W) ratio, total surfactant amount, and saponin/glycerol ratio on their stability were analyzed. The formation and stabilization of the oil-in-water nanoemulsions were analyzed through the evaluation of stability over time, pH, zeta potential, and particle size distribution analysis. Moreover, a design of experiments was performed to assess the most suitable composition based on particle size and stability parameters. The prepared nanoemulsions are, in general, highly stable over time, showing zeta potential values lower than -40 mV, a slight acid behavior due to the character of the components, and particle size (in volume) in the range of 1.1 to 4.3 µm. Response surface methodology revealed that formulations using an O/W ratio of 10/90 and 1.5 wt% surfactant resulted in lower particle sizes and zeta potential, presenting higher stability. The use of glycerol did not positively affect the formulations, which reinforces the suitability of preparing highly stable nanoemulsions based on natural surfactants such as saponins.

Combining Monophosphoryl Lipid A (MPL), CpG Oligodeoxynucleotide (ODN), and QS-21 Adjuvants Induces Strong and Persistent Functional Antibodies and T Cell Responses against Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) of Plasmodium falciparum in BALB/c Mice.

Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate that has a crucial role in the traversal of the malaria parasite in both mosquito and mammalian hosts. As recombinant purified proteins are normally poor immunogens, they require to be admixed with an adjuvant(s); therefore, the objective of the present study was to evaluate the capacity of different vaccine adjuvants, monophosphoryl lipid A (MPL), CpG, and Quillaja saponaria Molina fraction 21 (QS-21), alone or in combination (MCQ [MPL/CpG/QS-21]), to enhance the immunogenicity of Escherichia coli-expressed PfCelTOS in BALB/c mice. This goal was achieved by the assessment of anti-PfCelTOS IgG antibodies (level, titer, IgG isotype profile, avidity, and persistence) and extracellular Th1 cytokines using an enzyme-linked immunosorbent assay (ELISA) on postimmunized BALB/c mouse sera and PfCelTOS-stimulated splenocytes, respectively. Also, an assessment of the transmission-reducing activity (TRA) of anti-PfCelTOS obtained from different vaccine groups was carried out in female Anopheles stephensi mosquitoes by using a standard membrane feeding assay (SMFA). In comparison to PfCelTOS alone, administration of PfCelTOS with three distinct potent Th1 adjuvants in vaccine mouse groups showed enhancement and improvement of PfCelTOS immunogenicity that generated more bias toward a Th1 response with significantly enhanced titers and avidity of the anti-PfCelTOS responses that could impair ookinete development in A. stephensi However, immunization of mice with PfCelTOS with MCQ mixture adjuvants resulted in the highest levels of induction of antibody titers, avidity, and inhibitory antibodies in oocyst development (88%/26.7% reductions in intensity/prevalence) in A. stephensi It could be suggested that adjuvant combinations with different mechanisms stimulate better functional antibody responses than adjuvants individually against challenging diseases such as malaria.

Structural characterisation of new immunoadjuvant saponins from leaves and the first study of saponins from the bark of Quillaja brasiliensis by liquid chromatography electrospray ionisation ion trap mass spectrometry.

INTRODUCTION: Quillaja brasiliensis (St. A. -Hil. & Tul) Mart (Quillajaceae) is a species native to South America, which is rich in saponins. Saponins are used in different industries, so there is a constant demand for this type of compound. Based on the wide range of applications for the saponins found in this species, notably as immunoadjuvants, we conducted a comprehensive study of this tree and its saponins. OBJECTIVE: The purpose of this work is to complete the characterisation of the immunoadjuvant saponin fraction from Q. brasiliensis leaves and further study the saponin fraction obtained from Q. brasiliensis bark. METHODOLOGY: Saponin fractions were studied using mass spectrometry in combination with classical methods of monosaccharide and methylation analysis. We performed direct infusion and liquid chromatography/electrospray ionisation ion trap multiple-stage mass spectrometry (DI-ESI-IT-MSn and LC-ESI-IT-MS2 ). RESULTS: Seventy-five saponins, 21 from leaves and 54 from bark, were tentatively identified according to their molecular mass, fragmentation pattern and chromatographic behaviour. This work represents the first investigation of saponins from the bark of Q. brasiliensis and some of them presented new structural motifs not previously reported in the genus Quillaja. CONCLUSION: The efficiency and selectivity of the data dependent LC-MS2 method allowed the rapid profiling of saponins from Q. brasiliensis. The results of the monosaccharide and methylation analysis performed in saponins from Q. brasiliensis fractions and Q. saponaria Molina (Quillajaceae) fraction gives further support to the structures proposed according to the mass spectral data, validating the strategy used in the present work.

Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities.

Quillaja saponaria Molina represents the main source of saponins for industrial applications. Q. saponaria triterpenoids have been studied for more than four decades and their relevance is due to their biological activities, especially as a vaccine adjuvant and immunostimulant, which have led to important research in the field of vaccine development. These saponins, alone or incorporated into immunostimulating complexes (ISCOMs), are able to modulate immunity by increasing antigen uptake, stimulating cytotoxic T lymphocyte production (Th1) and cytokines (Th2) in response to different antigens. Furthermore, antiviral, antifungal, antibacterial, antiparasitic, and antitumor activities are also reported as important biological properties of Quillaja triterpenoids. Recently, other saponins from Q. brasiliensis (A. St.-Hill. & Tul.) Mart. were successfully tested and showed similar chemical and biological properties to those of Q. saponaria barks. The aim of this manuscript is to summarize the current advances in phytochemical and pharmacological knowledge of saponins from Quillaja plants, including the particular chemical characteristics of these triterpenoids. The potential applications of Quillaja saponins to stimulate further drug discovery research will be provided.

Saponin Adsorption at the Air-Water Interface-Neutron Reflectivity and Surface Tension Study.

Saponins are a large group of glycosides present in many plant species. They exhibit high surface activity, which arises from a hydrophobic scaffold of triterpenoid or steroid groups and attached hydrophilic saccharide chains. The diversity of molecular structures, present in various plants, gives rise to a rich variety of physicochemical properties and biological activity and results in a wide range of applications in foods, cosmetics, medicine, and several other industrial sectors. Saponin surface activity is a key property in such applications and here the adsorption of three triterpenoid saponins, escin, tea saponins, and Quillaja saponin, is studied at the air-water interface by neutron reflectivity and surface tension. All these saponins form adsorption layers with very high surface visco-elasticity. The structure of the adsorbed layers has been determined from the neutron reflectivity data and is related to the molecular structure of the saponins. The results indicate that the structure of the saturated adsorption layers is governed by densely packed hydrophilic saccharide groups. The tight molecular packing and the strong hydrogen bonds between the neighboring saccharide groups are the main reasons for the unusual rheological properties of the saponin adsorption layers.

Quillaja saponaria bark saponin protects Wistar rats against ferrous sulphate-induced oxidative and inflammatory liver damage.

CONTEXT: Saponins from different sources are historically reported in Chinese medicine to possess many beneficial effects. However, insufficient experimental data are available regarding the hepatoprotective potential of Quillaja bark saponin. OBJECTIVE: The protective effect of Quillaja saponaria Molina (Quillajaceae) bark triterpenoid saponin against iron-induced hepatotoxicity is compared to the standard N-acetylcysteine in adult male Wistar rats. MATERIALS AND METHODS: Animals were divided into (six) groups, namely a normal control, an N-acetylcysteine control (300 mg/kg/day, p.o., 10 days), a saponin control (100 mg/kg/day, p.o., for 10 days), a hepatotoxicity control (two doses of ferrous sulphate, 30 mg/kg/day each, i.p., on 9th and 10th day), an N-acetylcysteine plus ferrous sulphate (standard treatment) and a saponin plus ferrous sulphate (test treatment) group. Hepatocyte integrity loss markers (serum ALT, AST, ALP, GGT and LDH), oxidative stress markers (hepatic MDA, GSH and NOx), dyslipidaemic markers (serum TC and TG) and hepatocyte functioning markers (serum bilirubin and albumin) were assessed. RESULTS: Quillaja bark saponin decreased iron-induced elevation of ALT (reaching 57% of hepatotoxicity control), AST (66%), ALP (76%), GGT (60%), LDH (54%), MDA (65%), NOx (77%), TC (70%), TG (54%), and total (54%), direct (54%) and indirect (54%) bilirubin, coupled with increased GSH (219%) and albumin (159%) levels. Histopathological study strongly supported biochemical estimations, while immunohistochemical study showed marked effect on eNOS and iNOS expression. CONCLUSIONS: Quillaja bark saponin has a good hepatoprotective effect. Amelioration of oxidative stress and suppression of NOS expression, with resultant maintenance of hepatocyte integrity and functioning, may explain this beneficial effect.

Saponin Interactions with Model Membrane Systems - Langmuir Monolayer Studies, Hemolysis and Formation of ISCOMs.

Saponins are used in medicine due to their pharmacological and immunological effects. To better understand interactions of saponins with model membranes and natural membranes of, for example, erythrocytes, Langmuir film balance experiments are well established. For most saponins, a strong interaction with cholesterol was demonstrated in dependence of both the aglycone part and the sugar moieties and is suggested to be correlated with a strong hemolytic activity, high toxicity, and high surface activity, as was demonstrated for the steroid saponin digitonin. In general, changes in the sugar chain or in substituents of the aglycone result in a modification of the saponin properties. A promising saponin with regard to fairly low hemolytic activity and high adjuvant effect is α-tomatine, which still shows a high affinity for cholesterol. An interaction with cholesterol and lipids has also been proven for the Quillaja saponin from the bark of Quillaja saponaria Molina. This triterpene saponin was approved in marketed vaccines as an adjuvant due to the formation of immunostimulating complexes. Immunostimulating complexes consist of a Quillaja saponin, cholesterol, phospholipids, and a corresponding antigen. Recently, another saponin from Quillaja brasiliensis was successfully tested in immunostimulating complexes, too. Based on the results of interaction studies, the formation of drug delivery systems such as immunostimulating complexes or similar self-assembled colloids is postulated for a variety of saponins.

Adjuvant Effect of Quillaja saponaria Saponin (QSS) on Protective Efficacy and IgM Generation in Turbot (Scophthalmus maximus) upon Immersion Vaccination.

The adjuvant effect of Quillaja saponaria saponin (QSS) on protection of turbot fry was investigated with immersion vaccination of formalin-killed Vibrio anguillarum O1 and various concentrations of QSS (5, 25, 45 and 65 mg/L). Fish were challenged at days 7, 14 and 28 post-vaccination. Significantly high relative percent of survival (RPS) ((59.1 ± 13.6)%, (81.7 ± 8.2)%, (77.8 ± 9.6)%) were recorded in the fish that received bacterins immersion with QSS at 45 mg/L, which is comparable to the positive control group vaccinated by intraperitoneal injection (IP). Moreover, a remarkably higher serum antibody titer was also demonstrated after 28 days in the vaccinated fish with QSS (45 mg/L) than those vaccinated fish without QSS (p < 0.05), but lower than the IP immunized fish (p < 0.05). Significant upregulation of IgM gene expression has also been identified in the tissues of skin, gill, spleen and kidney from the immunized fish in comparison to the control fish. Taken together, the present study indicated that QSS was able to dramatically evoke systemic and mucosal immune responses in immunized fish. Therefore, QSS might be a promising adjuvant candidate for fish vaccination via an immersion administering route.

Unusual penetration of phospholipid mono- and bilayers by Quillaja bark saponin biosurfactant.

The interactions between a model phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and a biosurfactant Quillaja Bark Saponin (QBS) obtained from the bark of Quillaja saponaria Molina were studied using simple models of biological membranes. QBS is known to interact strongly with the latter, exerting a number of haemolytic, cytotoxic and anti-microbial actions. The interaction of QBS dissolved in the subphase with DPPC monolayers and silicon-supported bilayers was studied above the cmc (10(-3)M). Surface pressure relaxation and surface dilatational rheology combined with quartz crystal microbalance (QCM) and neutron reflectivity (NR) were employed for this purpose. The DPPC-penetrating abilities of QBS are compared with those of typical synthetic surfactants (SDS, CTAB and Triton X-100). We show that the penetration studies using high surface activity (bio)surfactants should be performed by a subphase exchange, not by spreading onto the surfactant solution. In contrast to the synthetic surfactants of similar surface activity, QBS does not collapse DPPC mono- and bilayers, but penetrates them, improving their surface dilatational elastic properties even in the highly compressed solid state. The dilatational viscoelasticity modulus increases from 204 mN/m for pure DPPC up to 310 mN/m for the QBS-penetrated layers, while it drops to near zero values in the case of the synthetic surfactants. The estimated maximum insertion pressure of QBS into DPPC monolayers exceeds the maximum surface pressure achievable in our setup, in agreement with the surface rheological response of the penetrated layers.

Effects of garlic oil, nitrate, saponin and their combinations supplemented to different substrates on in vitro fermentation, ruminal methanogenesis, and abundance and diversity of microbial populations.

AIMS: To investigate the effect of garlic oil (G), nitrate (N), saponin (S) and their combinations supplemented to different forage to concentrate substrates on methanogenesis, fermentation, diversity and abundances of bacteria and Archaea in vitro. METHODS AND RESULTS: The study was conducted in an 8 × 2 factorial design with eight treatments and two substrates using mixed ruminal batch cultures obtained. Quillaja S (0·6 g l(-1) ), N (5 mmol l(-1) ) and G (0·27 g l(-1) ) were used separately or in binary and tertiary combinations. The two substrates contained grass hay and a dairy concentrate mixture at a 70 : 30 (high-forage substrate) ratio or a 30 : 70 (high-concentrate substrate) ratio. Ruminal fermentation and cellulolytic bacterial populations were affected by interaction between substrate and anti-methanogenic compounds. The inhibitor combinations decreased the methane production additively regardless of substrate. For the high-concentrate substrate, S decreased methane production to a greater extent, so did G and N individually for the high-forage substrate. Feed degradability and total volatile fatty acid (VFA) concentrations were not decreased by any of the treatments. Fibre degradability was actually improved by N+S for the high-forage substrate. VFA concentrations and profiles were affected differently by different anti-methanogenic inhibitors and their combinations. All treatments inhibited the growth of Archaea, but the effect on Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens varied. CONCLUSIONS: The results suggest that substrate influences the efficacy of these inhibitors when they are used separately, but in combinations, they can lower methanogenesis additively without much influence from the substrate. SIGNIFICANCE AND IMPACT OF THE STUDY: The presented research provided evidence that binary and tertiary combination of garlic oil, nitrate and saponin can lower the methane production additively without adversely impacting rumen fermentation and degradability, and forage to concentrate ratio does not change the above effects. These anti-methanogenic inhibitors in combination may have practical application to mitigate methane emission from ruminants.

Inclusion of phytogenic blends in different nutrient density diets of meat-type ducks.

A total of 160 1-day-old ducklings (average initial body weight of 53 g), were used in a 42-d feeding trial to evaluate the effects of reducing nutrient density of diets, and supplementing the diets with a phytogenic blend (quillaja, anise, and thyme) on their growth, carcass quality, and nutrient digestibility. After checking body weight on d 1, the birds were sorted into pens with 5 birds/pen and 8 pens/treatment. The treatments were: T1, Basal diet; T2, T1+150 ppm phytogenic blend; T3, T1-(1% CP, 0.04% Lys, 0.05% Met+Cys, 0.02% Ca, and 0.02% P, and 50 kcal ME); T4, T3+150 ppm phytogenic blend. The results indicated that reducing nutrient density of the diets had an adverse effect (P<0.05) on body weight gain (BWG) on d 1 to 21, d 21 to 42, and the overall experimental period. Supplementing the diets with the phytogenic blend improved (P<0.05) BWG and feed conversion ratio (FCR) on d 21 to 42 and the overall experimental period. Feed intake was not affected by treatments. Low nutrient density diets increased (P<0.05) the cooking loss percentage of breast meat. Supplementing the diets with the phytogenic blend decreased (P<0.05) the lightness of breast meat. The percentage of drip loss was influenced (P<0.05) by nutrient density and the phytogenic blend on d 1 and d 7. The relative weights of breast meat, abdominal fat, gizzard, liver, spleen, and bursa of Fabricius, pH, and TBARS values were not affected by the treatments. The digestibility of dry matter, energy, nitrogen, ADF, and NDF was decreased (P<0.05) by reducing nutrients density of the diets, but addition of the phytogenic blend alleviated (P<0.05) the negative effects of lowering the nutrient density. The results indicated that the ducks fed high nutrient density diets supplemented with the phytogenic blend showed higher BWG and nutrient digestibility and lower FCR, cooking loss, drip loss, and TBARS value, without any negative effect on meat quality and relative organs weights.

[PREPARATION OF HUMAN TISSUE PROTEIN EXTRACTS ENRICHED WITH THE SPHINGOMYELIN SYNTHASE 1].

Sphingomyelin synthase 1 (SMS 1) catalyzes sphingomyelin biosynthesis in eukaryotic cells. We previously studied the structure of the human SGMS1 gene, which encodes the enzyme and its numerous transcripts. The tissue-specific expression of the transcripts was also described. Analysis of the SMS1 protein expression in human tissues using immunoblotting of tissue extracts prepared in the RIPA (Radio Immuno-Precipitation Assay) buffer revealed a weak signal in renal cortex, testis, lung, and no signal in placenta and lymphatic node. In this work, a new method of preparation of the tissue protein extracts enriched with SMS1 was suggested. The method based on the consecutive extraction with a buffer containing 0.05 and 1 mg/ml of the Quillaja saponaria saponin allowed SMS1 to be detected in all tissues tested. The SMS1 content in the saponin extract of kidney cortex is about 12-fold higher compared to the RIPA extraction procedure.

The predominant Quillaja Saponaria fraction, QS-18, is safe and effective when formulated in a liposomal murine cancer peptide vaccine.

Extracts of the Chilean soapbark tree, Quillaja Saponaria (QS) are the source of potent immune-stimulatory saponin compounds. This study compared the adjuvanticity and toxicity of QS-18 and QS-21, assessing the potential to substitute QS-18 in place of QS-21 for vaccine development. QS-18, the most abundant QS saponin fraction, has been largely overlooked due to safety concerns. We found that QS-18 spontaneously inserted into liposomes, thereby neutralizing hemolytic activity, and following administration did not induce local reactogenicity in a footpad swelling test in mice. With high-dose intramuscular administration, transient weight loss was minor, and QS-18 did not induce significantly more weight loss compared to a liposome vaccine adjuvant system lacking it. Two days after administration, no elevation of inflammatory cytokines was detected in murine serum. In a formulation including cobalt-porphyrin-phospholipid (CoPoP) for short peptide sequestration, QS-18 did not impact the formation of peptide nanoparticles. With immunization, QS-18 peptide particles induced higher levels of cancer neoepitope-specific and tumor-associated antigen-specific CD8+ T cells compared to QS-21 particles, without indication of greater toxicity based on mouse body weight. T cell receptor sequencing of antigen-specific CD8+ T cells showed that QS-18 induced significantly more T cell transcripts. In two murine cancer models, vaccination with QS-18 peptide particles induced a similar therapeutic effect as QS-21 particles, without indication of increased toxicity. Antigen-specific CD8+ T cells in the tumor microenvironment were found to express the exhaustion marker PD-1, pointing to the rationale for exploring combination therapy. Taken together, these data demonstrate that QS-18, when formulated in liposomes, can be a safe and effective adjuvant to induce tumor-inhibiting cellular responses in murine models with potential to facilitate or diminish costs of production for vaccine adjuvant systems. Further studies are warranted to assess liposomal QS-18 immunogic, reactogenic and toxicological profiles in mice and other animal species.