Ovalbumin Solution Research Articles

Effects of MnTnHex-2-PyP on lung antioxidant defence system in asthma mice model.

We aimed to study the MnTnHex-2-PyP effect on some markers of lung antioxidant defence system in mice asthma model.The study was carried out on 28 C57B1/6 mice divided into four treatment groups: group 1 - controls; group 2 - injected and inhaled with ovalbumin; group 3 - treated with MnTnHex-2-PyP and inhaled with phosphate buffered saline; group 4 - injected with ovalbumin and MnTnHex-2-PyP but also inhaled with ovalbumin. On days 24, 25 and 26, mice from groups 1 and 2 were inhaled with PBS for 30 min, and those from groups 2 and 4 were given a 1% ovalbumin solution. One hour before inhalation, and 12 hours later the animals from groups 1 and 2 were injected i.p. with 100 μl PBS, and those from groups 3 and 4 received a 100 μl MnTnHex-2-PyP solution in PBS, сontaining 0,05mg/kg. The animals were killed by exsanguination 48 hours after the last inhalation for obtaining a lung homogenate. The activities of superoxide dismutase, catalase, glutathione peroxidase and the non-protein sulphhydryl group content in the lung homogenate were investigated. Ovalbumin decreased the activities of superoxide dismutase (p=0.01), catalase (p=0.002), glutathione peroxidase and non-protein sulphhydryl groups content (p<0.001) in comparison to controls. In group 4 (ovalbumin and MnTnHex-2-PyP) the activities of superoxide dismutase (p=0.044), catalase (p=0.045), glutathione peroxidase (p=0.002), and the non-protein sulphhydryl groups content (p<0.001) were significantly increased compared to ovalbumin (group 2).MnTnHex-2-PyP restored the activities of basic enzymes in the lung antioxidant defence system in ovalbumin-induced asthma mice model, 48 hours after the last nebulization.

Effect of protein release rates from tablet formulations on the immune response after sublingual immunization

Dry vaccine formulations for sublingual administration would provide great advantages for public health use, especially in developing countries, since they are easy to administer and might also have improved stability properties. This study investigates the influence of protein release rate from mucoadhesive two-layer tablets on the elicited antibody responses after sublingual immunization. Two fast release tablets, one based on a mixture of lactose and microcrystalline cellulose (MCC) and one protein coated ethylcellulose (EC) tablet, and three hydrophilic matrix tablets with extended release (ER) properties based on HPMC 90 SH 100000 or Carbopol® 974-P NF were tested. The in vitro release profiles of the model protein ovalbumin (OVA) from these tablets were characterized and correlated to the in vivo potential of the tablets to induce an immune response after sublingual immunization in BALB/c mice. It could be concluded that a tablet with fast protein release elicits antibody titres not significantly different from titres obtained with OVA in solution, whereas low immune responses were observed with a slow release of OVA from the ER formulations. Thus, an ER tablet seems not favorable for vaccine delivery to the sublingual mucosa. Thus, we can present a fast releasing tablet formulation with attractive features for sublingual immunization, whereas the use of ER formulations for sublingual vaccination has to be investigated more in detail.

IgE-binding and in vitro gastrointestinal digestibility of egg allergens in the presence of polysaccharides

This work studies the IgE-binding and in vitro gastrointestinal digestibility of the main egg allergens, ovalbumin (OVA) and ovomucoid (OM), in the presence of pectin (P), gum arabic (G) and xylan (X), functional biopolymers commonly used in the food industry. To this aim, solutions of OVA or OM and P, G or X were digested by using a model that mimics physiological conditions. Gastric and duodenal digests were analysed by SDS-PAGE, RP-HPLC and SEC and the specific human-IgE binding capacity was assessed by immunoblotting and ELISA using sera from egg-sensitized patients. The reactivity towards human IgE of OVA and OM was considerably increased in the presence of the polysaccharides and their susceptibility to digestion was diminished when compared with the isolated proteins. As a result, the duodenal digests obtained in the presence of polysaccharides retained more IgE-binding than the isolated protein digests. Overall, the present results underline the importance of the food matrix in the digestibility of food allergens and in their potential to trigger an immune response.

알레르기 비염 유발 생쥐에 대한 형개연교탕(荊芥連翹湯)의 iNOS 생성 억제 효과

Background and Objectives : Allergic rhinitis is one of the most common diseases in the otorhinolaryngology area. in oriental clinic, Hyunggaeyungyo-tang(HYT) has been used as a primary prescription to treat allergic rhinitis. However, there have been no studies so far performed on the effect of this HYT use. The purpose of this study was find out therapeutic effects of its exclusive use on the rat with allergic rhinitis. Material and Methods : Thirty BALB/c mice were divided into three group : normal group(NOR), control group(CON) inoculated with allergic rhinitis and sample group(SAM) treated with the HYT extract after it was treated the same as the control group. Rats were sensitized intraperitoneally with ovalbumin solution 4times at intervals of 2 days. After that time, rats in SAM were administered by HYT to treat the inflammation. Results : 1. The number of eosinophil in SAM noticeably decreased than CON and this decrease had probability. The inhibition of eosinophil distribution. The infiltration of eosinophil in SAM noticeably decreased than CON. 2. The damaged mucosa as disruption of cilia in respiratory cell, vacant mucose secreting cell and infiltration of inflammation intricate cells in CON were increased than NOR, but SAM same as normal configuration. Decrease of icthing and sneezing intricate neurotransmitter (substance P). Decrease of angiogenesis intricate cytokine(MIP-2). 3. Transcription factor(NF-<TEX>${\kappa}B$</TEX> p65) was decreased. 4. Transcription factor inhibitor(p-<TEX>$I{\kappa}B$</TEX>) was decreased. 5. Inflammation cytokine(iNOS) was decreased. Conclusion : The results suggest that HYT is significantly effective in the treatment of inflammation caused by allergic rhinitis through the suppression of NF-<TEX>${\kappa}B$</TEX> activation and iNOS production.

Two-and Three-Layered Dissolving Microneedles for Transcutaneous Delivery of Model Vaccine Antigen in Rats

Purpose: Comparison of transcutaneous immunization of ovalbumin (OA) between two-and three-layered dissolving microneedles (MN) in rats. Methods: We prepared 500 μm long two-layered and three-layered dissolving microneedle (2-MN and 3-MN, respectively) arrays from chondroitin sulfate as the base, and OA as the model antigen. The 2-MN containing OA at the acral portion and 3-MN with OA at the second portion were administered to rat skin transcutaneously. As a positive control, OA solution was injected subcutaneously (sc). The OA delivery and diffusion in the rat skin were studied using confocal microscopy with fluorescein-conjugated OA (FL-OA). Results: The formulated positions of OA were 0-155 ± 5 μm for 2-MN and 175 ± 4 – 225 ± 5 μm for 3-MN. The administered doses of OA were 2.2 ± 0.1 μg, 12.0 ± 0.2 μg and 22.0 ± 0.2 μg for 2-MN, 1.8 ± 0.2 μg, 12.6 ± 0.7 μg, and 20.4 ± 0.3 μg for 3-MN, 10 μg, 100 μg and 1000 μg for sc injection. At 4 weeks after the first administration, 3-MN showed about 2.5-7.0 fold and 5.4 fold higher total Ig (G + A + M) antibody than 2-MN and sc injection of the OA solution. Conclusions: The 3-MN, which delivered OA to the epidermis, is a useful drug delivery system for transcutaneous antigen delivery.

Ffects Of Mntnhex-2-Pyp On Markers Of Inflammation And Lipid Peroxidation In Asthma Mice Model

Background and objective: Iinvestigation of the effects of MnTnHex-2-PyP on some markers of inflammation and lipid peroxidation in an asthma mice model.Methods: The experiment was carried out on 24 female mice C57Bl/6, divided into four groups: group 1, controls; group 2, injected with ovalbumin (OVA); group 3, treated with MnTnHex-2-PyP and group 4, treated with OVA and MnTnHex-2-PyP. The animals from groups 1 and 3 were injected i.p. on days 0 and 14 with a 100 μl phosphate-buffered saline (PBS), and those from groups 2 and 4 were injected with a 100 μl ovalbumin solution, containing 20 μg OVA. On days 24, 25 and 26 the mice from groups 1 and 2 were inhaled with PBS for 30 min, and those from groups 2 and 4 were given a 1% ovalbumin solution. One hour before inhalation, and 12 hours later the animals from groups 1 and 2 were injected i.p. with 100 μl PBS, and those from groups 3 and 4 received a 100 μl MnTnHex-2-Pyp solution in PBS сontaining 0.05mg/kg.Results: Ovalbumin alone (group 2) increased the total cell number, total protein content, the levels of IL-4, IL-5 and 8-isoprostane in bronchoalveolar lavage. Elevations were observed in IgE level in serum, and the malone dialdehyde (MDA) content in the lung homogenate. These markers were decreased significantly in group 4 as compared to the OVA group.Conclusions: MnTnHex-2-Pyp reduces the inflammation and lipid peroxidation in Ovalbumin-induced mice asthma model.

Biodistribution Study Using Egg Protein ELISA Kit after Administration of FITC-labeled Ovalbumin Solution and Its Double Liposomes in the In Situ Loop Method, and Its Implication in Oral Immunization

Ovalbumin (OVA) is often used as a model antigen, and its biodistribution is important for the induction of immunization, especially oral immunization. In this study, an allergic substance-detecting kit, Egg Protein ELISA kit, was applied to the investigation of the biodistribution of fluorescein isothiocyanate-labeled ovalbumin (FITC-OVA). After FITC-OVA solution and its double liposomes were administered into the intestinal loop with one Peyer's patch, the biodistribution of FITC-OVA was examined with the Egg Protein ELISA kit. Each calibration was performed by fitting a quadratic curve to the observed ELISA response points. The ELISA response was almost the same between OVA and FITC-OVA. Similar ELISA response curves were obtained in Peyer's patch (PP) homogenate, spleen (SP) homogenate and plasma (PL). The concentration of FITC-OVA could be determined at 4 - 64 ng/ml for aqueous solution and SP homogenate and at 1 - 64 ng/ml for PP homogenate and PL. Thus, it was suggested that the ELISA kit should be useful for measurement of OVA biodistribution in an oral immunization study. After the administration of FITC-OVA solution and its double liposomes into the intestinal loop, the biodistribution of OVA-FITC in PP, SP and PL was investigated. The distributed amount was the greatest in PP. At the early time, the distributed amount in PP, SP and PL tended to be greater with FITC-OVA solution than the double liposomes. FITC-OVA was retained longer in PP with the double liposomes than FITC-OVA solution. The present results indicated that OVA could transfer well to PP and systemic circulation even with the solution dosage form in the loop method, probably because it was not exposed to harsh conditions such as a gastric fluid. Namely, it implied that the protection from gastric pH and enzyme by the double liposomes, which had been reported before, would be importantly associated with the promotion of immune induction. In addition, the double liposomes could retain OVA longer in PP, which might cause the enhancement of oral immunization.

Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode

The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20–80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.

Antigen−Adjuvant Nanoconjugates for Nasal Vaccination: An Improvement over the Use of Nanoparticles?

Entrapment of antigens in mucoadhesive nanoparticles prepared from N-trimethyl chitosan (TMC) has been shown to increase their immunogenicity. However, because of their large size compared to soluble antigens, particles poorly diffuse through the nasal epithelium. The aim of this work was to study whether nasal vaccination with a much smaller TMC-antigen nanoconjugate would result in higher antibody responses as compared to TMC nanoparticles. TMC was covalently linked to a model antigen, ovalbumin (OVA), using thiol chemistry. For comparison, TMC/OVA nanoparticles and solutions of OVA and a physical mixture of TMC and OVA were made. As shown previously for TMC/OVA nanoparticles, TMC-OVA conjugate prolonged the nasal residence time of the antigen. TMC-OVA conjugate diffused significantly better through a monolayer of lung carcinoma (Calu-3) cells than TMC/OVA nanoparticles did. Moreover, nasal immunization of mice with the conjugate resulted in significantly more OVA positive DCs in the cervical lymph nodes as compared to TMC/OVA nanoparticles. Mice nasally immunized with TMC-OVA conjugate produced high levels of secretory IgA in nasal washes and higher titers of OVA-specific IgG than mice immunized with TMC/OVA nanoparticles after a priming dose. Moreover, as compared to TMC/OVA nanoparticles, TMC-OVA conjugate induced a more balanced IgG1/IgG2a response. In conclusion, the TMC-antigen nanoconjugate improves nasal delivery and immunogenicity of the antigen. This suggests that efficient codelivery of antigen and adjuvant to DCs, rather than a particulate form of the antigen/adjuvant combination, is decisive for the immunogenicity of the antigen.

The protective effect of fenretinide against allergic asthma

Objective/purposeFenretinide [N-4-hydroxyphenyl, 4HPR] is a syntheticretinoid derived from vitamin A with pro-apoptotic andanti-inflammatory properties. Studies have shown thatat high concentrations fenretinide is an effective anti-neoplastic agent, regulating cell growth and differentia-tion. We have recently demonstrated that fenretinide, atdoses 5-10× lower than for cancer treatment, has pro-tective effect against bacterial infection[1] and osteo-porosis[2] in Cftr-knockout mice. The effect offenretinide as a potential treatment for allergic asthmainduced inflammation has not been evaluated in asth-matics. The goal of this study was to determine if, bynormalizing inflammatory mediators, fenretinide wouldbe able to alleviate the symptoms associated with aller-gic asthma.MethodsHyperresponsive to methacholine, mice of A/J strainwere sensitized weekly using ovalbumin (OVA) forthree consecutive weeks, then age-matched and sepa-rated into two study groups and two control groups.For a period of 4 weeks, the study groups were orallytreated with fenretinide while the controls were treatedwith the drug vehicle. During the last week of treat-ment, the control and study groups were split into twoadditional groups and underwent allergen challengesfor three consecutive days, with either an OVA solu-tion or PBS. Forty-eight hours after the third challenge,resistance of the respiratory system of the mice inresponse to methacholine was measured using a Buxcoplethysmograph. Total IgE in plasma was measured byELISA. Lung histopathology was observed using H&Eand PAS stains.FindingsVehicle treated OVA challenged mice exhibited highvalues of airway resistance, plasma IgE concentration,and immune cell infiltration into the airways comparedto PBS challenged animals. Interestingly, fenretinidetreated OVA challenged mice had a statistically lowerrespiratory resistance. In addition, fenretinide treatmentabrogated the recruitment of eosinophils to the regionsurrounding the blood vessels and airways. Similarly,after drug treatment a decrease in goblet cell hyperplasiawas also observed through histopathology. However, nodifference was observed in the level of plasma IgEbetween the control and study groups.DeliverablesFenretinide has been demonstrated to have great poten-tial as a therapeutic agent. Our findings provide a novelapproach to treat allergen-induced asthma and will helpus in pursuing these studies towards the use of thisdrug for patients suffering from allergic asthma.RelevanceThe data presented herein will facilitate the develop-ment of fenretinide as a therapeutic compound in thetreatment of allergic asthma and provide the foundationfor the identification of drug targets for the developmentof effective future drugs.

Preparation of macroporous hydroxyapatite by foaming and gelcasting using ovalbumin and its optimisation by Taguchi method

Macroporous hydroxyapatite is a widely researched implant material due to its biocompatibility and bone bonding ability. The process of preparing macroporous HAp foam by foaming and gelcasting using ovalbumin involved four variables, namely ovalbumin amount, pH of ovalbumin solution, method of foaming and hydroxyapatite (HAp) amount, each varied in two levels. The properties for optimisation in this process were density and porosity. Pore size of the foam was considered as an additional criterion. The normal trial and error method of optimising the foam properties do not infer the effect of each factor on the properties. Design of experiment based on Taguchi method reduced the number of trials to eight and also identified the effect of each factor on the properties using the objective function calculated for each trial. The factor effect analysis indicated that HAp amount had the maximum influence and ovalbumin amount had least influence on density, and pH amount had the maximum influence and ovalbumin amount and foaming method had least influence on porosity. It also enabled the calculation of the objective functions for untried combinations which assisted in identifying the combinations that could offer optimum properties. The optimum properties were identified to be produced by one tried combination and two untried combinations. The identified untried combinations were then tried and the properties were calculated experimentally. The optimum combination with higher porosity and higher density with pore size >150 μm was identified using the objective functions and SEM images. This analysis indicated that the macroporous HAp prepared by the addition of 16 vol.-% HAp to 70 wt-% ovalbumin foam prepared at pH 4·7 by agitator had appropriate properties.

Chitin Particles Are Multifaceted Immune Adjuvants

Chitin is a ubiquitous polysaccharide in fungi, insects, allergens, and parasites that is released at sites of infection. Its role in the generation of tissue inflammation, however, is not fully understood. We hypothesized that chitin is an important adjuvant for adaptive immunity. Mice were injected with a solution of ovalbumin and chitin. We used in vivo and ex vivo/in vitro approaches to characterize the ability of chitin fragments to foster adaptive immune responses against ovalbumin and compared these responses to those induced by aluminum hydroxide (alum). In vivo, ovalbumin challenge caused an eosinophil-rich pulmonary inflammatory response, Th2 cytokine elaboration, IgE induction, and mucus metaplasia in mice that had been sensitized with ovalbumin plus chitin or ovalbumin plus alum. Toll-like receptor-2, MyD88, and IL-17A played critical roles in the chitin-induced responses, and MyD88 and IL-17A played critical roles in the alum-induced responses. In vitro, CD4(+) T cells from mice sensitized with ovalbumin plus chitin were incubated with ovalbumin-stimulated bone marrow-derived dendritic cells. In these experiments, CD4(+) T-cell proliferation, IL-5, IL-13, IFN-γ, and IL-17A production were appreciated. Toll-like receptor-2, MyD88, and IL-17A played critical roles in these in vitro adjuvant properties of chitin. TLR-2 was required for cell proliferation, whereas IL-17 and TLR-2 were required for cytokine elaboration. IL-17A also inhibited the generation of adaptive Th1 responses. These studies demonstrate that chitin is a potent multifaceted adjuvant that induces adaptive Th2, Th1, and Th17 immune responses. They also demonstrate that the adjuvant properties of chitin are mediated by a pathway(s) that involves and is regulated by TLR-2, MyD88, and IL-17A.

Phosphorylation of ovalbumin by dry-heating in the presence of pyrophosphate: Effect of carbohydrate chain on the phosphorylation level and heat stability

Ovalbumin (OVA) derived from egg white (N-OVA) and recombinant OVA (Re-OVA), which was essentially carbohydrate-free, expressed by Escherichia coli, were phosphorylated by dry-heating for 1 day in the presence of pyrophosphate (PP-N- and PP-Re-OVA). The phosphorus contents of N- and Re-OVA were 0.79% and 0.46%, respectively, after phosphorylation. The secondary structural change of both OVAs was small, and the midpoint temperature of both OVAs determined from the thermal unfolding curve was decreased by phosphorylation. In addition, the differential scanning calorimetry thermograms of both OVAs showed a lowering of denaturation temperature by phosphorylation. The stability of both OVA solutions after heating at pH 7.0 was improved, and the degree of stabilization was higher for PP-N-OVA than for PP-Re-OVA. This suggests that the phosphate groups introduced to the carbohydrate chain also play an important role in the heat stability of OVA.

Protein−Protein Interactions in Ovalbumin Solutions Studied by Small-Angle Scattering: Effect of Ionic Strength and the Chemical Nature of Cations

The influence of ionic strength and of the chemical nature of cations on the protein-protein interactions in ovalbumin solution was studied using small-angle X-ray and neutron scattering (SAXS/SANS). The globular protein ovalbumin is found in dimeric form in solutions as suggested by SANS/SAXS experiments. Due to the negative charge of the proteins at neutral pH, the protein-protein interactions without any salt addition are dominated by electrostatic repulsion. A structure factor related to screened Coulombic interactions together with an ellipsoid form factor was used to fit the scattering intensity. A monovalent salt (NaCl) and a trivalent salt (YCl(3)) were used to study the effect of the chemical nature of cations on the interaction in protein solutions. Upon addition of NaCl, with ionic strength below that of physiological conditions (150 mM), the effective interactions are still dominated by the surface charge of the proteins and the scattering data can be understood using the same model. When yttrium chloride was used, a reentrant condensation behavior, i.e., aggregation and subsequent redissolution of proteins with increasing salt concentration, was observed. SAXS measurements reveal a transition from effective repulsion to attraction with increasing salt concentration. The solutions in the reentrant regime become unstable after long times (several days). The results are discussed and compared with those from bovine serum albumin (BSA) in solutions.

Accumulation of xylem transported protein at pit membranes and associated reductions in hydraulic conductance

Proteins and traces of polysaccharide are the only polymeric colloids consistently transported in the xylem sap of plants. The hypothesis that such proteins could have physical inhibitory effects on xylem water transport was investigated. Ovalbumin, with a molecular weight of 45 kDa and a molecular diameter of 5.4 nm, is an inert, water-soluble protein that is midway along the size range of endogenous xylem sap proteins. Solutions of ovalbumin conjugated to a fluorescent marker and supplied to transpiring shoot explants of tobacco (Nicotiana tabacum L.) and olive (Olea europaea L.) were shown by confocal laser scanning microscopy to accumulate specifically at wall-based pit membranes that connect neighbouring xylem conduits. In addition, pressure-induced perfusion of micro-filtered ovalbumin solutions, at concentrations similar to those of endogenous xylem sap proteins, through the xylem of tobacco stem or olive twig segments resulted in the retention of c. 40% of the ovalbumin and reductions in the axial hydraulic conductance of the xylem. Smaller molecules such as Texas Red 3000 (MW 3 kDa) and Alexafluor 488–cadaverin conjugates (MW 0.64 kDa) did not show similar characteristics. The partial reduction in xylem hydraulic conductance appeared to be related to the accumulation of ovalbumin at xylem pit membranes and the consequent fouling of trans-membrane water-conducting pores with smaller diameters than those of the ovalbumin molecules. Potential implications of these novel findings for whole-plant water relations are considered.

Determining fouling‐independent component of critical flux in protein ultrafiltration using the free‐solvent‐based (FSB) model

In protein ultrafiltration (UF), critical flux is generally considered the point on the flux-pressure curve where the permeate flux begins to significantly deviate from the pure solvent flux line. This concept has generally been associated with membrane fouling. Recently, however, we showed that a free-solvent-based model (FSB model) demonstrated that osmotic pressure can be the primary and dominant factor for the nonlinear flux behavior observed in protein UF. Consequently, the critical flux may be the results of two phenomena, the osmotic pressure effect and potential irreversible fouling. Here we show that the fouling-independent component of the critical flux can be redefined as the critical point of the second derivative of the flux-pressure profile determined from the FSB model. This definition of the fouling-independent component of critical flux was compared to values traditionally determined qualitatively on the flux-pressure curve and was found to be in excellent agreement with these experimental observations. During protein ultrafiltration (UF), critical flux has been generally considered the mark of the deviation of the permeate flux from the linear pressure dependence of the pure solvent flux. It has been defined as the flux below which no fouling occurs. Critical flux has seen growing interest from the researchers over the past 10 years due to the importance in understanding the nonlinear flux-pressure dependence during UF processes. Bacchin et al. recently published a comprehensive review that summarizes the research on the critical flux. Despite the variation in theories, experimental methods, and mathematical models developed by different researchers, the general agreement is that the nonlinear flux behavior is the result of the hydraulic resistance associated with various membrane fouling phenomena. Hence, a family of critical fluxes (strong form, weak form, and critical flux for irreversibility) is defined based on different types of fouling behaviors. Recently, we have developed a new flux model, the freesolvent-based (FSB) model (based on the free-solvent model for osmotic pressure).The free-solvent model for concentrated solutions has been around of some time but has, surprisingly been overlooked for its application to concentrated protein solutions. As early as 1916, Frazer and Myrick analyzed the nonidealities in aqueous sucrose solutions using a free-solvent model. More recently, other researchers, in a similar approach, based their models on the van’t Hoff equation, but only had limited success for protein solutions up to moderate concentrations. However, we have subsequently demonstrated that a free-solvent model corrected for ion binding demonstrates excellent predictability for osmotic pressure of concentrated solutions of lysozyme, ovalbumin, albumin and immunogammaglobulins in a range of moderate salt solutions with varying pH. In fact, the free-solvent model demonstrated excellent prediction for protein concentrations up to saturation concentrations. We have recently shown that this model provides excellent prediction and characterization of the flux-pressure behavior in protein UF. We further showed that when membrane fouling contributions are experimentally minimized, the permeate flux continues to demonstrate nonlinear, reversible pressure dependence, and reaches limiting flux at sufficiently high pressure. These observations are in excellent agreement with the FSB model prediction. The unique feature of the free-solvent model for this work is that one Correspondence concerning this article should be addressed to V. G. J. Rodgers at victor.rodgers@ucr.edu.

An investigation into the adjuvanticity and immunogenicity of zein microspheres being researched as drug and vaccine carriers

We have determined whether zein microspheres could act as vaccine adjuvants i.e. increase the immune responses to co-administered immunogens. Ovalbumin (model antigen)-loaded zein microspheres, blank zein microspheres and ovalbumin solution were intramuscularly administered to mice and the sera antibody levels were determined by ELISA. Another group of mice was orally dosed with blank zein microspheres, and serum and faecal antibody levels were determined. As expected, negligible antibody titres were obtained with the ovalbumin solution. Surprisingly, intramuscular administrations of blank zein microspheres elicited high levels of serum IgG which bound to the ovalbumin antigen coated on ELISA microtitre plates. This indicated that anti-zein antibodies had been elicited by blank zein microspheres and that these antibodies were cross-reacting with ovalbumin antigen coated onto ELISA plates. Such cross-reactivity inhibited the determination of the adjuvant activity of zein microspheres, if any. Additional ELISA assays, where zein was used as the coating antigen, confirmed the generation of anti-zein antibodies by blank zein microspheres i.e. zein microspheres were immunogenic following intramuscular administration. Upon oral administration of blank zein microspheres, serum IgG levels remained low but intestinal IgA levels increased following booster doses i.e. systemic tolerance, but not mucosal tolerance, to oral zein particles was achieved. Zein microspheres were immunogenic when administered intramuscularly and orally.

The Cannabinoid Receptor Agonist WIN 55,212-2 Inhibits Antigen-Induced Plasma Extravasation in Guinea Pig Airways

Background: Although neurogenic inflammation of the airways via activation of C-fibers is thought to be important in the pathogenesis of asthma, the mechanisms regulating C-fiber activity remain uncertain. Objective: The influence of a cannabinoid receptor agonist, WIN 55,212-2, on C-fiber activation in guinea pig airways was investigated, as was the mechanism by which cannabinoids regulate antigen-induced airway inflammation. Methods: The inhibitory effect of WIN 55,212-2 on antigen-induced plasma extravasation was assessed in guinea pig tracheal tissues by photometric measurement of extravasated Evans blue dye after extraction with formamide. Results: Pretreatment with WIN 55,212-2 (0.001, 0.01 or 0.1 mg/kg) significantly and dose-dependently reduced tracheal plasma extravasation induced by inhaling a 5% ovalbumin solution for 2 min after pretreatment with a neutral endopeptidedase inhibitor (phosphoramidon at 2.5 mg/kg i.v.). A cannabinoid CB2 receptor antagonist (SR144528) blunted the inhibitory effect of WIN 55,212-2, while a cannabinoid CB1 antagonist (SR141716A) did not. Pretreatment with a neurokinin-1 receptor antagonist (FK888) significantly reduced ovalbumin-induced extravasation of Evans blue dye. Pretreatment with the combination of WIN 55,212-2 and FK888 reduced antigen-induced plasma extravasation more markedly than FK888 alone. Conclusions: These findings suggest that WIN 55,212-2 inhibits C-fiber activation via the cannabinoid CB2 receptor and thus suppresses antigen-induced inflammation in guinea pig airways.

Inhibitory Effects of Cyclosporine A Eye Drops on Symptoms in Late Phase and Delayed-Type Reactions in Allergic Conjunctivitis Models

We investigated the efficacy of cyclosporine A (CyA) eye drops on ocular symptoms in late phase and delayed-type reactions in guinea pig allergic conjunctivitis models. An emulsion of ovalbumin (OVA) and Freund's complete adjuvant (FCA) was intraperitoneally injected into guinea pigs, and 15% OVA solution was applied topically to the eyes to elicit late phase reactions. Following the early phase reaction, increased scores for hyperemia, swelling, edema, and discharge were detected 6 h after antigen challenge, and CyA eye drops significantly inhibited the increase in scores for edema and discharge, the increase in the number of infiltrating inflammatory cells, and the percentage of eosinophils among polymorphonuclear leukocytes in conjunctival tissue. To induce delayed-type reactions, guinea pigs were sensitized by injecting FCA into the footpad, followed by injections of purified protein derivative into palpebral conjunctivae 24 d later. Increased scores for hyperemia, swelling, and discharge were detected 6 h after the induction of delayed-type allergy, and CyA eye drops significantly inhibited the increase in scores for hyperemia and swelling. In contrast, betamethasone sodium phosphate eye drops showed a tendency to inhibit the symptoms in both late phase and delayed-type reactions, or inflammatory cell infiltration in the late phase reaction, but the inhibition was not significant. These results suggest that CyA eye drops are useful for suppressing ocular symptoms in both late phase and delayed-type reactions in allergic conjunctivitis models.

Experiment and numerical simulation of heat and mass transfer during a spray freeze-drying process of ovalbumin in a tray

Spray freeze-drying is a promising technology for producing high-quality porous particles primarily for pharmaceutical uses. The advantages of freeze-drying in the production of pharmaceuticals and biomedical products, such as minimization of thermal and chemical degradation, retention of volatile components, preservation of high porosity, and a very low content of residual water after drying, are mostly retained in spray freeze-drying. In this study, we performed spray freeze-drying of a 3% (w/w) chicken egg ovalbumin solution in a tray with a batch-type spray freeze-dryer that we developed. The physical characteristics of the spray freeze-dried particles were qualitatively evaluated by means of scanning electron microscopy. The freeze-drying behavior of spray-frozen particles was experimentally investigated by measuring the histories of product temperatures and numerically studied by developing an analysis model based on the finite volume method in a fixed grid system.