Proteolytic activity and immunogenicity of oral bromelain within the gastrointestinal tract of mice

Bromelain is a mixture of proteinases derived from pineapple stem that is marketed by health food stores as a "digestive aid". A number of studies suggest that bromelain may also have anti-inflammatory activity in vivo, including an anecdotal report describing potential efficacy in inflammatory bowel disease. We and others have previously shown that proteolytically active bromelain removes certain cell surface molecules and affects leukocyte migration, activation, and production of cytokines and inflammatory mediators in vitro. The purpose of this study was to determine whether ingested bromelain retains proteolytic activity within the murine gastrointestinal tract in vivo. The proteolytic activity of bromelain was determined in vitro using model substrates or immunofluorescence assays after administration of various doses and formulations orally to mice. Immune responses against bromelain were detected by enzyme immunoassays. When formulated in antacid, oral bromelain retained substantial proteolytic activity throughout the gastrointestinal tract. Bromelain concentrations within the colon were dependent on both dose and formulation and were sufficient to remove bromelain-sensitive molecules from both leukocytes and colon epithelial cells. Peak activity in the stool was observed 4 h after oral dosing. Although anti-bromelain IgG was detected in both serum and stool after long-term oral therapy, these antibodies did not prevent bromelain proteolytic activity within the gastrointestinal tract. These studies demonstrate that bromelain enzymes can remain intact and proteolytically active within the murine gastrointestinal tract. They provide further support for the hypothesis that oral bromelain may potentially modify inflammation within the gastrointestinal tract via local proteolytic activity within the colonic microenvironment.     

Immunological implications of the use of plant lectins for drug and vaccine targeting to the gastrointestinal tract

Plant lectins are under consideration as targeting agents to enhance the efficacy of orally administered drugs and vaccines. A significant issue that must be considered is the immunogenicity of these molecules since an immune response to the targeting agent may interfere with its ability to interact with the epithelium. In contrast, the ability of certain lectins to activate the immune system may be exploited in the delivery of vaccines. We previously demonstrated that plant lectins vary widely in their immunogenicity and in particular that mistletoe lectins (ML) I, II and II (MLI, MLII, MLIII) are potent immunogens when administered nasotracheally. Here, we measured immune responses following oral delivery of the MLs and assessed their ability to enhance responses to a co-administered antigen to determine if the molecules possess adjuvant activity. Oral administration of the lectins induced potent lectin-specific systemic and mucosal antibody responses. In addition, each of the three lectins possessed adjuvant activity when delivered orally together with ovalbumin (OVA). The lectins enhanced both serum and mucosal antibody responses to the co-delivered antigen. This shows for the first time that MLI, MLII and MLIII possess adjuvant activity when administered orally and may provide a platform for the generation of effective mucosal adjuvants.     

Differences in allergenic potential of food extracts following oral exposure in mice reflect differences in digestibility: potential approaches to safety assessment.

An animal model for food allergy is needed to assess genetically modified food crops for potential allergenicity. The ideal model must produce allergic antibody (IgE) to proteins differentially according to known allergenicity before being used to accurately identify potential allergens among novel proteins. The oral route is the most relevant for exposure to food antigens, and a protein's stability to digestion is a current risk assessment tool based on this natural route. However, normal laboratory animals do not mount allergic responses to proteins administered orally due to oral tolerance, an immunologic mechanism which specifically suppresses IgE. To circumvent oral tolerance and evoke differential IgE responses to a panel of allergenic and nonallergenic food extracts, female C3H/HeJ mice were exposed subcutaneously or orally with cholera toxin as an adjuvant. All foods elicited IgE by the subcutaneous route. Oral exposure, however, resulted in IgE to allergens (peanut, Brazil nut, and egg white) but not to nonallergens (spinach and turkey), provided that the dose and exposures were limited. Additionally, in vitro digestibility assays demonstrated the presence of digestion-stable proteins in the allergenic food extracts but not in the nonallergenic foods. Our results suggest that the subcutaneous route is inadequate to distinguish allergens from nonallergens, but oral exposure under the appropriate experimental conditions will result in differential allergic responses in accordance with known allergenicity. Moreover, those foods containing digestion-resistant proteins provoke allergic responses in this model, supporting the current use of pepsin resistance in the decision tree for potential allergenicity assessment.     

Pinellic acid from the tuber of Pinellia ternata Breitenbach as an effective oral adjuvant for nasal influenza vaccine

This study describes the isolation, purification, characterization, and adjuvant activity of an orally active adjuvant substance from the tuber of Pinellia ternata, as an active herbal component of the traditional Japanese herbal (Kampo) medicine, Sho-seiryu-to (SST, Chinese name: Xiao-Qing-Long-Tang), which has been reported to show oral adjuvant activity for nasally administered influenza HA vaccine [Int. J. Immunopharmacol. 16 (1994) 605]. The active compound was identified as 9S, 12S, 13S-trihydroxy-10E-octadecenoic acid using infrared spectra, proton magnetic resonance, mass spectrometry, and circular dichroism, and named pinellic acid. Oral administration of pinellic acid (1 microg) to BALB/c mice given primary and secondary intranasal inoculations of influenza HA vaccine (1 microg) enhanced antiviral IgA antibody (Ab) titers 5.2- and 2.5-fold in nasal and bronchoalveolar washes, respectively, and antiviral IgG Ab titers 3-fold in bronchoalveolar wash and serum. Intranasal administration of pinellic acid (1 microg) with influenza HA vaccine (1 microg) slightly enhanced antiviral IgG Ab titers in bronchoalveolar wash and serum but not antiviral IgA Ab titers in nasal and bronchoalveolar washes. Pinellic acid showed no hemolytic activity. The results of this study suggest that pinellic acid may provide a safe and potent oral adjuvant for nasal influenza HA vaccine.     

Comparison of the adjuvanticity of two adjuvant formulations containing de-<Emphasis Type="Italic">O</Emphasis>-acylated lipooligosaccharide on Japanese encephalitis vaccine in mice

Adjuvants are essential vaccine components used to enhance, accelerate, and/or prolong adaptive immunity against specific vaccine antigens. In this study, we compared the adjuvanticity of two adjuvant formulations containing de-O-acylated lipooligosaccharide (dLOS), a toll-like receptor 4 agonist, on the Japanese encephalitis (JE) vaccine in mice. Mice were immunized once or twice at a two-week interval with inactivated JE vaccine in the absence or presence of adjuvant. We found that both the alum- and the liposome-based formulation induced significantly faster and higher serum IgG antibody responses as compared with the non-adjuvanted vaccine after either one or two immunizations. The antibody titers of the mouse immune sera correlated with 50% plaque reduction neutralization test (PRNT₅₀) antibody titers. In addition, the dLOS/liposome formulation was more effective in inducing a Th1-type immune response than the dLOS/alum formulation, as suggested by a strong antigen-specific interferon (IFN)-γ response. Based on these results, we suggest that both alum- and liposome-based adjuvant formulations containing dLOS may be used for the development of JE vaccines with improved immunogenicity.     

Oral Tolerance Induction by Bothrops jararaca Venom in a Murine Model and Cross-Reactivity with Toxins of Other Snake Venoms

Oral tolerance is defined as a specific suppression of cellular and humoral immune responses to a particular antigen through prior oral administration of an antigen. It has unique immunological importance since it is a natural and continuous event driven by external antigens. It is characterized by low levels of IgG in the serum of animals after immunization with the antigen. There is no report of induction of oral tolerance to Bothrops jararaca venom. Here, we induced oral tolerance to B. jararaca venom in BALB/c mice and evaluated the specific tolerance and cross-reactivity with the toxins of other Bothrops species after immunization with the snake venoms adsorbed to/encapsulated in nanostructured SBA-15 silica. Animals that received a high dose of B. jararaca venom (1.8 mg) orally responded by showing antibody titers similar to those of immunized animals. On the other hand, mice tolerized orally with three doses of 1 µg of B. jararaca venom showed low antibody titers. In animals that received a low dose of B. jararaca venom and were immunized with B. atrox or B. jararacussu venom, tolerance was null or only partial. Immunoblot analysis against the venom of different Bothrops species provided details about the main tolerogenic epitopes and clearly showed a difference compared to antiserum of immunized animals.     

Labrafil--a new adjuvant for peptide-specific oral tolerance in rat experimental autoimmune uveitis.

Application of soluble antigen via the oral route results in systemic antigen-specific tolerance, a therapeutic approach that has already been used for uveitis patients. In the Lewis rat experimental autoimmune uveitis (EAU) can be induced by active immunisation with retinal antigens such as retinal soluble antigen (S-Ag) or interphotoreceptor retinoid-binding protein (IRBP) and peptides thereof. These normally pathogenic antigens can also be used to induce oral tolerance. In order to optimize oral tolerance induction we analysed the effect of Labrafil M 2125 CS, an orally administrable composition for pharmaceutical use, consisting of fatty acid esters and glycerides and capable of forming micro emulsions. Feeding peptide emulsified in Labrafil M 2125 CS/PBS prior to immunisation significantly improved oral tolerance compared to feeding peptide in PBS only. We observed a delayed onset of disease, reduced intraocular inflammation and less retinal destruction. Application of Labrafil M 2125 CS without tolerogen had no effect. Combined feeding of peptide with Labrafil M 2125 CS even allowed 10-fold reduction of the tolerogenic peptide dose. Furthermore, the effect of Labrafil M 2125 CS upon oral tolerance was dose-dependent, a peptide emulsion containing 0.5-2% Labrafil M 2125 CS achieved a maximal enhancement of oral tolerance induction, suggesting that Labrafil M 2125 CS might be a useful adjuvant to enhance therapeutic use of oral tolerance.     

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.     

Design of Fully Synthetic, Self-Adjuvanting Vaccine Incorporating the Tumor-Associated Carbohydrate Tn Antigen and Lipoamino Acid-Based Toll-like Receptor 2 Ligand

Overexpression of certain tumor-associated carbohydrate antigens (TACA) caused by malignant transformation offers promising targets to develop novel antitumor vaccines, provided the ability to break their inherent low immunogenicity and overcome the tolerance of the immune system. We designed, synthesized, and immunologically evaluated a number of fully synthetic new chimeric constructs incorporating a cluster of the most common TACA (known as Tn antigen) covalently attached to T-cell peptide epitopes derived from polio virus and ovalbumin and included a synthetic built-in adjuvant consisting of two 16-carbon lipoamino acids. Vaccine candidates were able to induce significantly strong antibody responses in mice without the need for any additional adjuvant, carrier protein, or special pharmaceutical preparation (e.g., liposomes). Vaccine constructs were assembled either in a linear or in a branched architecture, which demonstrated the intervening effects of the incorporation and arrangement of T-cell epitopes on antibody recognition.     

Immunostimulating complexes (ISCOMs) for nasal vaccination

The immunostimulating complex (ISCOM) is documented as a strong adjuvant and delivery system for parenteral immunization. Its effectiveness for mucosal immunization has also been proven with various incorporated antigens. L?vgren et al. were the first to demonstrate the capacity of influenza virus ISCOMs to induce mucosal immune response and protection after one comparatively low nasal dose. Further studies show that similar to Cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), ISCOMs break immunological tolerance and exert strong mucosal adjuvant activity, resulting in secretory IgA and systemic immune responses. Striking is the capacity of ISCOMs to induce CTL response also after nasal administration. In contrast to CT, ISCOMs initiate mucosal as well as systemic immune responses in an IL-12 dependent manner but independently of IL-4. The recombinant B subunit of cholera toxin (rCTB) was incorporated in the same ISCOM particle to explore symbiotic effects. The IgA response to rCTB in lungs was increased 100-fold when rCTB was administered nasally in ISCOMs and more than 10-fold in the remote mucosa of the genital tract. An enhanced IgA response to a passenger antigen OVA was recorded in the remote genital tract. After i.n. administration of the envelope proteins of respiratory syncytial virus in ISCOMs, high serum antibodies were induced, almost at the same levels as those following parenteral immunization and potent IgA responses were also evoked both at the local respiratory mucosa, and in the cases tested at the distant mucosae of the genital and intestinal tracts. Similar results have also been recorded with ISCOMs containing envelope proteins from Herpes simplex virus, Influenza virus and Mycoplasma mycoides. The mucosal targeting property of envelope proteins of RSV was utilized in an HIV-gp120 RSV ISCOM formulation. After nasal administration an enhanced mucosal IgA response to gp120 was observed in the female reproductive tract. In general, antigens derived from envelope viruses or cell membranes incorporated into ISCOMs retain their biological activity and conformation, encompassing the mucosal targeting and virus neutralizing properties.     

Association between immunogenicity and adsorption of a recombinant Streptococcus pneumoniae vaccine antigen by an aluminum adjuvant

The impact on immunogenicity of the degree of adsorption of three Streptococcus pneumoniae (Sp) vaccine antigens to aluminum adjuvants was studied. The three antigens evaluated (Sp1, Sp2 and Sp3) were highly adsorbed by aluminum hydroxide adjuvant, but not adsorbed by aluminum phosphate adjuvant. All of the Sp antigens adjuvanted with aluminum hydroxide elicited higher antibody responses in mice than formulations prepared with aluminum phosphate or non-adjuvanted antigen. Varying the percent aluminum-bound Sp antigen in the formulated vaccine affected the observed antibody responses. These observations suggest that the antibody response observed for Sp antigens in this study is stimulated by a depot effect of the antigen bound to an aluminum adjuvant.     

Therapeutic manipulation of the immune system: enhancement of innate and adaptive mucosal immunity

The mucosal immune system has evolved alongside, but separate, from the general systemic immune system. As a major consequence of this dichotomy, only immune responses initiated in mucosal inductive sites can result in effective immunity in mucosal tissues themselves. Oral tolerance, as usually assessed as orally-induced systemic unresponsiveness, contributes to mucosal homoeostasis by preventing unwanted immune reactions to food or environmental antigens. It is now established that tolerance can also be induced by the nasal route and mucosally-induced tolerance is being actively investigated for immune therapy against a number of diseases. Nontoxic derivatives of cholera toxin and the heat labile toxin of Escherichia coli as well as chimeric enterotoxins have been developed. These molecules retain the mucosal adjuvant activity of native enterotoxins and are effective at inducing targeted Th1 or Th2- type immune responses. Mucosal delivery of cytokines as adjuvants represents a safer alternative to parenteral cytokine injection. Nasally administered cytokines such as IL-1 and IL-12 or chemokines including RANTES, lymphotactin, MIP-1 beta, all act as mucosal adjuvants for co-administered antigens. Each of these cytokines promote specific pattern of CD4(+) T helper cell cytokine responses that could be exploited for targeted immune therapy. Although GALT and NALT are both parts of the Common Mucosal Immune System, there are major differences between orally and nasally induced immune responses. Nasal vaccines more effectively promote protective immunity in the genitourinary tract than do oral vaccines. In addition, aging affects mucosal tolerance or immunity in GALT more than is seen in NALT. Therapeutic manipulation of mucosal immunity involves regulation of CD4(+) T cell cytokine responses and thus, should require a careful examination of the host status, including the occurrence of inflammatory bowel diseases.     

Development and application of PROVAX adjuvant formulation for subunit cancer vaccines

A major challenge facing the development of subunit vaccines comprised of well-defined recombinant antigens is their weak immunogenicity and inability to induce effective cytotoxic T cell (CTL) responses. Adjuvants aimed at increasing the immunogenicity of recombinant antigens remain a focus in vaccine development. The potency of an adjuvant is linked to specific stimulation of T cell responses, involving TH1 and TH2 subsets of CD4(+) T helper cells and CD8(+) CTL and B cell-mediated antibody responses. As a result of the existence of two distinct intra-cellular pathways for antigen processing, immunization with exogenous antigens often shows a greater propensity for T helper and antibody responses, but not CD8(+) CTL responses. However, existing experimental evidence suggests that CD8(+) CTLs, which are critical in the elimination of viral-infected and neoplastic cells, can be elicited with soluble antigens when delivered in appropriate formulations or adjuvants. This review focuses on the properties of PROVAX adjuvant in inducing antigen-specific CTL responses, antibody responses and tumor regression in experimental models and its potential application for the development of recombinant cancer vaccines.     

The role of the gut lymphoid tissue in induction of oral tolerance

The gut-associated lymphoid tissue (GALT) maintains a balance between immunological tolerance to dietary proteins and induction of active immune responses to pathogenic microorganisms. The oral administration of soluble protein antigens induces a state of systemic immunological unresponsiveness specific to the fed protein, termed oral tolerance. The two major mechanisms to explain oral tolerance are anergy/deletion of autoreactive lymphocytes and active suppression. This review will discuss the mechanisms of therapeutic oral tolerance in relation to events occurring at the site of antigen entry.     

Raccoon poxvirus as a mucosal vaccine vector for domestic cats

In the present study, we evaluated both the immunogenicity and safety of recombinant raccoon poxvirus (RCN) as a mucosal vaccine vector for domestic cats. RCN is an orthopoxvirus that was isolated from healthy raccoons and has been used experimentally as a vaccine vector for rabies and other antigens in a variety of species, including raccoons, skunks, foxes, bobcats, rabbits, domestic cats, piglets, sheep and non-human primates. We evaluated the antibody response induced by a recombinant RCN vaccine expressing the rabies-G glycoprotein (RCN/rabies-G) administered to cats by the oral (PO), intranasal (IN), conjunctival (CO) or intranasal/conjunctival (IN/CO) route (dose: 10 plaque forming units or PFU). The IN route, either alone or combined with the CO route, induced the highest rabies virus neutralizing antibody (RVNA) titers. The RVNA titers remained high when measured at six months post-vaccination, demonstrating that the recombinant vaccine administered via these routes is very efficient at inducing long-lasting immunity. A dose-response was observed following IN vaccination in cats. Doses of 10 PFU induced strong antibody responses in 4 of 5 animals [geometric mean titer: 3.2 (log)]. None of the animals vaccinated with 10 PFU developed detectable RVNA titers. In this study, RCN/rabies-G viral shedding was below detectable levels. Nasal, oral and fecal swabs collected from these cats were negative for RCN by both virus isolation and by nested-PCR. In addition, no horizontal transmission of the virus could be detected. Gang-housed sentinel animals for each group did not develop detectable anti-RVNA or -RCN antibodies. To study tissue tropism of recombinant raccoon poxvirus vaccines, a RCN that can express the lacZ gene (RCN/lacZ) was constructed. Expression of beta-galactosidase (beta-gal) was validated in vitro and in mice in vivo. Cats were vaccinated IN with 10 PFU of RCN/lacZ. No histopathological lesions were detected in any of the tissues collected from these cats at 1, 4, 7 and 15 days post-vaccination. In addition, no virus or beta-gal expression was detected in any of these tissues. Controls demonstrated that virus could be reisolated from nasal swabs immediately after administration of 10 PFU to cats. These results suggest that recombinant RCN vaccines undergo limited replication after intranasal administration in cats that is sufficient to elicit strong, long-lasting systemic antibody responses.     

Clinical Immunogenicity of rHuPH20, a Hyaluronidase Enabling Subcutaneous Drug Administration

Recombinant human PH20 hyaluronidase (rHuPH20) is used to facilitate dispersion of subcutaneously delivered fluids and drugs. This report summarizes rHuPH20 immunogenicity findings from clinical trials where rHuPH20 was co-administered with SC human immunoglobulin, trastuzumab, rituximab, or insulin. Plasma samples were obtained from evaluable subjects participating in ten different clinical trials as well as from healthy plasma donors. A bridging immunoassay and a modified hyaluronidase activity assay were used to determine rHuPH20-reactive antibody titers and neutralizing antibodies, respectively. rHuPH20-binding antibody populations from selected subjects with positive titers were affinity-purified and subjected to further characterization such as cross-reactivity with endogenous PH20. Among individual trials, the prevalence of pre-existing rHuPH20-reactive antibodies varied between 3 and 12%, excepting the primary immunodeficiency (PID) studies. Incidence of treatment-induced rHuPH20 antibodies was 2 to 18%, with the highest titers (81,920) observed in PID. No neutralizing antibodies were observed. Within most trials, the kinetics of antibody responses were comparable between pre-existing and treatment-induced antibody responses, although responses classified as persistent were more common in subjects with pre-existing titers. There was no association between antibody positivity and either local or systemic adverse events. Pre-existing and treatment-induced antibody populations were of similar immunoglobulin isotypes and cross-reacted to endogenous PH20 to similar extents. No cross-reactivity to PH20 paralogs was detected. rHuPH20 induces only modest immunogenicity which has no association with adverse events. In addition, antibodies purified from baseline-positive individuals are qualitatively similar to those purified from individuals developing rHuPH20-reactive antibodies following exposure to the enzyme.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-015-9782-0) contains supplementary material, which is available to authorized users.KEY WORDS: anti-drug antibodies, clinical trial, immunogenicity, rHuPH20, subcutaneous drug delivery     

Inhibition of interleukin-12 expression by alpha-thrombin in human peripheral blood mononuclear cells: a potential mechanism for modulating Th1/Th2 responses

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a powerful regulator of inflammatory responses and is known to affect cell-mediated immunity. Interleukin (IL)-12 is a strong promoter of the development of Th1-type lymphocytes and its downregulation implies a positive feedback mechanism for development of Th2 responses. We have previously shown that thrombin enhances the release of IL-6, a Th2-related cytokine, in human peripheral blood mononuclear cells (PBMC). Here we show that thrombin downregulates IL-12 production at both protein and mRNA levels in human PBMC. The inhibition of IL-12 production was accompanied by an enhanced release of IL-10, which inhibits Th1-related processes and promotes Th2-type responses. The use of proteolytically inactive thrombin and of the specific thrombin receptor agonist peptide, SFLLRN, reveals that this downregulation is thrombin-specific and requires thrombin proteolytic activity. In addition, activation of coagulation inhibits IL-12 production in whole blood cultures, confirming the tight relationship between the coagulation pathway, where thrombin is a key enzyme, and inflammation. Decreased IL-12 production appears to be related also to IL-10 production, since the addition of an anti-IL-10 monoclonal antibody to thrombin-treated PBMC resulted in a partial restoration of IL-12 production. In conclusion, the observation that thrombin significantly affects the production of IL-12, as well as of IL-10, implies a concerted role orchestrated by thrombin in PBMC that could be crucial to effective immunity and inflammation.     

Immunogenic properties of rapidly digested food proteins following gavage exposure of mice: a comparison of ovalbumin with a potato acid phosphatase preparation.

The ability of food proteins to resist digestion in simulated gastric fluid (SGF) correlates with allergenic potential. The purpose of the current investigations was to determine whether this association is due solely to the failure of unstable proteins to elicit an immune response when administered orally. We have examined immune responses induced in BALB/c mice by gavage administration of ovalbumin (OVA) and a crude potato protein extract (PPE) containing acid phosphatase activity. The stability of OVA and PPE in SGF was measured using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The ability of these proteins to stimulate specific IgG and IgE antibody production in mice following parenteral (intraperitoneal; ip) or oral (gavage) exposure was compared using enzyme-linked immunosorbent and homologous passive cutaneous anaphylaxis assays, respectively. Both OVA and PPE induced specific IgG antibody responses when administered either by gavage or by ip injection. Parenteral, but not gavage, exposure to OVA was associated with robust IgE antibody responses. Administration of PPE failed to stimulate strong IgE production via either route of exposure. Differential stability in SGF was observed, with PPE being digested extremely rapidly (within 1 min), whereas OVA was more resistant. The strong association reported by others between stability in SGF and allergenic potential is unlikely to be solely due to orally-ingested labile proteins failing to provoke immune responses due to degradation in the stomach.     

Proteinase activity and stability of natural bromelain preparations

Bromelain is a complex mixture of proteinases typically derived from pineapple stem. Similar proteinases are also present in pineapple fruit. Beneficial therapeutic effects of bromelain have been suggested or proven in several human inflammatory diseases and animal models of inflammation, including arthritis and inflammatory bowel disease. However, it is not clear how each of the proteinases within bromelain contributes to its anti-inflammatory effects in vivo. Previous in vivo studies using bromelain have been limited by the lack of assays to control for potential differences in the composition and proteolytic activity of this naturally derived proteinase mixture. In this study, we present model substrate assays and assays for cleavage of bromelain-sensitive cell surface molecules can be used to assess the activity of constituent proteinases within bromelain without the need for biochemical separation of individual components. Commercially available chemical and nutraceutical preparations of bromelain contain predominately stem bromelain. In contrast, the proteinase activity of pineapple fruit reflects its composition of fruit bromelain>ananain approximately stem bromelain. Concentrated bromelain solutions (>50 mg/ml) are more resistant to spontaneous inactivation of their proteolytic activity than are dilute solutions, with the proteinase stability in the order of stem bromelain>fruit bromelain approximately ananain. The proteolytic activity of concentrated bromelain solutions remains relatively stable for at least 1 week at room temperature, with minimal inactivation by multiple freeze-thaw cycles or exposure to the digestive enzyme trypsin. The relative stability of concentrated versus dilute bromelain solutions to inactivation under physiologically relevant conditions suggests that delivery of bromelain as a concentrated bolus would be the preferred method to maximize its proteolytic activity in vivo.     

人甲型H7N9禽流感全病毒灭活疫苗和裂解疫苗在小鼠中的免疫原性研究

 目的   比较人甲型H7N9禽流感全病毒灭活疫苗和裂解疫苗在小鼠中的免疫原性,为该疫苗的类型选择提供初步依据。 方法   采用相同血凝素含量（5 μg）的H7N9全病毒灭活和裂解疫苗（含或不含氢氧化铝佐剂共4种类型）,分别对BALB/c小鼠进行1针或2针免疫。免疫后,用血凝抑制（hemagglutination inhibition,HI）试验检测血清抗体滴度,比较不同类型疫苗的免疫效果。 结果   小鼠免疫1针全病毒灭活疫苗后,全部血清阳转,HI抗体几何平均滴度（geometric mean titer,GMT）为149;免疫2针后,抗体GMT为243。小鼠免疫1针裂解疫苗后无抗体阳转;免疫2针后全部抗体阳转,GMT为139。两种疫苗添加铝佐剂后,诱导的HI抗体GMT仅略有增加。 结论   H7N9全病毒灭活疫苗在小鼠中的免疫原性较强。在同样类型和剂量的情况下,裂解疫苗需要免疫两次才能达到与全病毒疫苗相同的效果。铝佐剂对免疫原性提升不明显。