Misleading Allergens in the Diagnosis of Latex Allergy: Profilin and Cross-Reactive Carbohydrate Determinants.

BACKGROUND: It has been suggested that latex-specific IgE analysis may lead to false-positive results, especially in patients with pollen allergy. In the present study, the reasons underlying clinically irrelevant latex-specific IgE positivity were investigated. METHODS: Thirty patients with latex allergy (group 1), 89 patients sensitised to aeroallergens (group 2a), and 98 healthy individuals without allergy (group 2b) were enrolled. Participants from all 3 groups were subjected to skin prick tests with aeroallergens including latex, latex-specific IgE analysis (ImmunoCAP), and nasal provocation test with latex. All cases demonstrating positive latex-specific IgE also underwent specific IgE tests (ImmunoCAP) with latex profilin, birch pollen profilin, peach lipid transfer protein, and pineapple bromelain as cross-reactive carbohydrate determinants. RESULTS: Comparison of the atopic and healthy control groups showed that the rate of positive latex-specific IgE was significantly higher in group 2a. Latex profilin-, birch pollen profilin-, and bromelain-specific IgE were remarkably higher in group 2a. CONCLUSION: False positivity to latex-specific IgE in ImmunoCAP analysis may be observed in approximately 19% of patients with pollen allergy. Profilins and bromelain are the main contributors to clinically irrelevant positive latex-specific IgE.

Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa.

Although type 2 immune responses to environmental Ags are thought to play pivotal roles in asthma and allergic airway diseases, the immunological mechanisms that initiate the responses are largely unknown. Many allergens have biologic activities, including enzymatic activities and abilities to engage innate pattern-recognition receptors such as TLR4. In this article, we report that IL-33 and thymic stromal lymphopoietin were produced quickly in the lungs of naive mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and thymic stromal lymphopoietin sensitized naive animals to an innocuous airway Ag OVA, which resulted in production of type 2 cytokines and IgE Ab, and eosinophilic airway inflammation when mice were challenged with the same Ag. Importantly, upon exposure to proteases, uric acid (UA) was rapidly released into the airway lumen, and removal of this endogenous UA by uricase prevented type 2 immune responses. UA promoted secretion of IL-33 by airway epithelial cells in vitro, and administration of UA into the airways of naive animals induced extracellular release of IL-33, followed by both innate and adaptive type 2 immune responses in vivo. Finally, a potent UA synthesis inhibitor, febuxostat, mitigated asthma phenotypes that were caused by repeated exposure to natural airborne allergens. These findings provide mechanistic insights into the development of type 2 immunity to airborne allergens and recognize airway UA as a key player that regulates the process in respiratory mucosa.

Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain.

Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)-bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle.

Rice-induced anaphylaxis: IgE-mediated allergy against a 56-kDa glycoprotein.

BACKGROUND: Although rice (Oryza sativa) is one of the most common cereals produced and consumed around the world, there have been only a few reports on immediate hypersensitivity reactions after ingestion of rice. Few clinical studies on rice allergy in Asia have been reported concerning rhinitis, asthma and atopic dermatitis. In this case study, we identify allergens presumably responsible for anaphylaxis after ingestion of rice in a German patient. METHODS: Prick-to-prick tests, determination of specific IgE and the basophil activation test (BAT) were performed to confirm IgE-mediated allergy. IgE reactivity was further analyzed by immunoblotting of protein extracts from cooked commercial rice products. Rice allergens were purified, subjected to N-terminal sequencing and characterized by IgE binding and IgE inhibition assays using additional sera from 8 subjects with sensitization to rice and/or a history of hypersensitivity symptoms after rice ingestion. RESULTS: Prick-to-prick tests were positive to raw and cooked rice (basmati rice and long-grain rice) and preparations of different rice extracts. Specific IgE against rice (f9) was 1.87 kU(A)/l. The BAT showed specific IgE-mediated activation of basophils after stimulation with rice extracts. Four IgE-reactive rice proteins with an apparent molecular weight of 49, 52, 56 and 98 kDa were identified. Interestingly, only binding to the 56-kDa glycoprotein was at least partially independent from cross-reactive carbohydrate determinants (CCD), whereas IgE binding to the other rice proteins was completely inhibited by pre-incubation with the CCD MUXF derived from bromelain. CONCLUSIONS: Yet unidentified high-molecular-weight allergens from rice seeds, predominantly a 56-kDa glycoprotein, seem to be responsible for anaphylaxis after consumption of rice in a German patient.

Bromelain treatment leads to maturation of monocyte-derived dendritic cells but cannot replace PGE2 in a cocktail of IL-1ß, IL-6, TNF-α and PGE2.

Immunotherapy using dendritic cells (DC) has shown promising results. However, the use of an appropriate DC population is critical for the outcome of this treatment, and the search for an optimal DC subset is still ongoing. The DC used in immunotherapy today are usually matured with a cytokine cocktail consisting of TNF-α, IL-1ß, IL-6 and PGE(2). These cells have deficits in their cytokine production, particularly IL-12p70, mainly because of the presence of PGE(2). Bromelain is a pineapple stem extract containing a mixture of proteases that has been used clinically in adjuvant cancer treatment. In this study, we analysed the effect of bromelain on human monocyte-derived DC. We added bromelain to the cytokine cocktail and modified cytokine cocktails with either no PGE(2) or reduced amounts of PGE(2), respectively. Combining bromelain with the cytokine cocktails containing PGE(2) resulted in an increased surface expression of CD83, CD80 and CD86. The chemokine receptor CCR7 was also considerably upregulated in these DC populations compared with DC treated with the cytokine cocktail alone. Removal or reduction of PGE(2) from the cytokine cocktail did not increase the IL-12p70 secretion from stimulated DC, and addition of bromelain to the different cytokine cocktails resulted in only a minor increase in IL-12p70 production. Moreover, combining bromelain with the cytokine cocktails did not improve the T cell stimulatory capacity of the generated DC populations. In conclusion, bromelain treatment of monocyte-derived DC does not improve the functional quality compared with the standard cytokine cocktail.

Suitability of different glycoproteins and test systems for detecting cross-reactive carbohydrate determinant-specific IgE in hymenoptera venom-allergic patients.

BACKGROUND: In hymenoptera venom allergy, about 75% of detected in vitro double positivity to yellow jacket and honeybee venom is ascribed to specific IgE (sIgE) directed against cross-reactive carbohydrate determinants (CCDs). To date, for the detection of CCD-sIgE, different carbohydrate antigens and methods are used. The most suitable one still has to be identified. METHODS: Eighty-seven patients with confirmed hymenoptera venom allergy and venom sIgE values of ≥0.7 kU/l were investigated. Sixty-five patients showed sIgE reactivity to both yellow jacket and honeybee venom, 22 were venom mono positive and served as controls. Occurrence of CCD-sIgE was determined using bromelain, horseradish peroxidase (HRP) and MUXF(3) on system A, and ascorbic acid oxidase (AAO), bromelain and HRP on system B. Further, a reference standard for CCD-sIgE evaluation was created: CCD positivity was assumed when at least 4 of the 6 test results were positive. RESULTS: According to the defined reference standard, 45/65 venom double positive patients exhibited CCD-sIgE. Using system A, comparison with the reference standard revealed sensitivity and specificity values of 96 and 97%, respectively, for MUXF(3), 100 and 100%, respectively, for bromelain, and 96 and 97%, respectively, for HRP. Using system B, sensitivity and specificity was 98 and 97%, respectively, for AAO, 62 and 95%, respectively, for bromelain, and 96 and 69%, respectively, for HRP. Results of the 3 test allergens obtained with system A showed strong correlations (r = 0.932-0.976), whereas results with system B showed lower correlations (r = 0.714-0.898). CONCLUSIONS: All 3 test allergens used with system A are suitable for CCD-sIgE detection in hymenoptera venom allergy. With system B, only AAO seems to be a reliable tool.

Immunoglobulin E sensitization to cross-reactive carbohydrate determinants: epidemiological study of clinical relevance and role of alcohol consumption.

BACKGROUND: The determinants and biologic significance of IgE-mediated sensitization to cross-reactive carbohydrate determinants (CCDs) are not entirely known. An association between alcohol consumption and CCD sensitization has been reported in studies from Spain and Portugal. OBJECTIVE: To investigate the relationship of alcohol consumption with CCD sensitization, the role of alcohol dehydrogenase gene polymorphisms, and the clinical consequences of CCD sensitization. METHODS: Serum-specific IgE sensitization (> or =0.1 kU/l) to a CCD (the N-glycan from bromelain) was assessed in 1,197 adults participating in a population-based study in Copenhagen, Denmark. Alcohol consumption and atopic symptoms (rhinitis, asthma and oral allergy syndrome) were assessed by questionnaire. Examinations included skin prick tests (SPTs), qualitative multiallergen IgE test (Phadiatop), methacholine bronchial hyperreactivity, eosinophil cationic protein and alcohol dehydrogenase (ADH) gene polymorphisms. RESULTS: Alcohol consumption was significantly associated with CCD sensitization and this was particularly evident in SPT-negative individuals. The fast-metabolizing allele of the ADH1b polymorphism was significantly associated with CCD sensitization in alcohol drinkers. CCD sensitization was associated with atopic symptoms, but associations attenuated markedly when adjusting for SPT reactivity. CONCLUSIONS: Our results suggest that the positive association between alcohol consumption and CCD sensitization is universal and not specific to certain populations. The observed association between the ADH1b polymorphism and CCD sensitization may support that alcohol is causally related to the risk of CCD sensitization. The observed association between CCD sensitization and atopic phenotypes did not appear to be independent of SPT reactivity indicating limited significance of CCD sensitization per se.

Cross-Reactive Carbohydrate Determinant in Apis mellifera, Solenopsis invicta and Polybia paulista Venoms: Identification of Allergic Sensitization and Cross-Reactivity.

Allergic reactions to Hymenoptera venom, which could lead to systemic and even fatal symptoms, is characterized by hypersensitivity reactions mediated by specific IgE (sIgE) driven to venom allergens. Patients multisensitized to sIgE usually recognize more than one allergen in different Hymenoptera species. However, the presence of sIgE directed against Cross-Reactive Carbohydrate Determinant (CCD), which occurs in some allergens from Hymenoptera venom, hampers the identification of the culprit insects. CCD is also present in plants, pollen, fruits, but not in mammals. Bromelain (Brl) extracted from pineapples is a glycoprotein commonly used for reference to sIgE-CCD detection and analysis. In sera of fifty-one Hymenoptera allergic patients with specific IgE ≥ 1.0 KU/L, we assessed by immunoblotting the reactivity of sIgE to the major allergens of Apis mellifera, Polybia paulista and Solenopsis invicta venoms. We also distinguished, using sera adsorption procedures, the cases of CCD cross-reaction using Brl as a marker and inhibitor of CCD epitopes. The presence of reactivity for bromelain (24-28 kDa) was obtained in 43% of the patients, in which 64% presented reactivity for more than one Hymenoptera venom in radioallergosorbent (RAST) tests, and 90% showed reactivity in immunoblot analysis to the major allergens of Apis mellifera, Polybia paulista and Solenopsis invicta venoms. Sera adsorption procedures with Brl lead to a significant reduction in patients' sera reactivity to the Hymenoptera allergens. Immunoblotting assay using pre- and post-Brl adsorption sera from wasp-allergic patients blotted with non-glycosylated recombinant antigens (rPoly p1, rPoly p5) from Polybia paulista wasp venom showed no change in reactivity pattern of sIgE that recognize allergen peptide epitopes. Our results, using Brl as a marker and CCD inhibitor to test sIgE reactivity, suggest that it could complement diagnostic methods and help to differentiate specific reactivity to allergens' peptide epitopes from cross-reactivity caused by CCD, which is extremely useful in clinical practice.

Oral immunogenicity of the plant proteinase bromelain.

Bromelain is a natural mixture of proteolytic enzymes derived from pineapple stem that has been shown to have anti-inflammatory activity when administered orally. Although most proteins given orally without adjuvant (e.g., food) result in tolerance, we previously reported that long-term oral exposure to bromelain stimulated the development of high serum anti-bromelain antibody titers. The purpose of these studies was to further investigate the mechanisms responsible for the immunogenicity of oral bromelain. Results showed that repeated exposure was required for development of anti-bromelain antibodies, with strong antibody responses in all mice that received at least 12 doses of bromelain either orally or intragastrically over 3-6 weeks. Proteolytic activity was required for strong oral immunogenicity in the absence of conventional adjuvant, with strong serum antibody responses generated against proteolytically active bromelain and trypsin, but not against ovalbumin, lysozyme, or inactivated bromelain. Significantly higher anti-bromelain antibody titers were seen in IL-10-deficient versus wild-type mice, suggesting that simultaneous treatments that decrease IL-10 activity may further enhance systemic antibody responses following oral exposure. The antibodies generated did not affect the proteolytic activity of bromelain. The data demonstrate that proteolytically active antigens such as bromelain can stimulate both systemic and mucosal immune responses following repeated oral exposure. Further studies of the mechanisms involved in generation of immune responses following oral exposure to proteolytically active antigens can lead to a better understanding of mechanisms of oral tolerance and to the development of novel adjuvants for oral vaccines.

Where do the immunostimulatory effects of oral proteolytic enzymes ('systemic enzyme therapy') come from? Microbial proteolysis as a possible starting point.

Enteric-coated proteolytic enzyme preparations like Wobenzym and Phlogenzym are widely used for the so-called 'systemic enzyme therapy' both in humans and animals. Numerous publications reveal that oral proteolytic enzymes are able to stimulate directly the activity of immune competent cells as well as to increase efficiency of some of their products. But origins of the immunostimulatory effects of oral proteolytic enzymes are still unclear. The hypothesis described here suggests that it may be proteolysis of intestinal microorganisms that makes the immune competent cells to work in the immunostimulatory manner. The hypothesis was largely formed by several scientific observations: First, microbial lysis products (lipopolysaccharides, muropeptides and other peptidoglycan fragments, beta-glucans, etc.) are well known for their immunostimulatory action. Second, a normal human being hosts a mass of intestinal microorganisms equivalent to about 1 kg. The biomass (mainly due to naturally occurring autolysis) continuously supplies the host's organism with immunostimulatory microbial cell components. Third, the immunostimulatory effects resulting from the oral application of exogenously acting antimicrobial (lytic) enzyme preparations, such as lysozyme and lysosubtilin, are likely to be a result of the action of microbial lysis products. Fourth, cell walls of most microorganisms contain a considerable amount of proteins/peptides, a possible target for exogenous proteolytic enzymes. In fact, several authors have already shown that a number of proteases possess an ability to lyse the microbial cells in vitro. Fifth, the pretreatment of microbial cells (at least of some species) in vitro with proteolytic enzymes makes them more sensitive to the lytic action of lysozyme and, otherwise, pretreatment with lysozyme makes them more susceptible to proteolytic degradation. Sixth, exogenous proteases, when in the intestines, may participate in final steps of food-protein digestion. The resulting food-borne peptides have recently been shown to be potential activators of microbial autolysis. The main question that needs to be answered in order to verify the hypothesis is whether oral proteases are able (and to what extent) to lyse/mediate lysis of intestinal microorganisms in situ. Methods based on up-to-date molecular biology techniques to allow investigation of the influence of exogenous proteases on microbial lysis processes in vivo (in the intestines) need to be developed. Research testing of this hypothesis may have an important impact in development of novel preparations for the systemic enzyme therapy.

Binding of antibromelain monomeric Fab' improves the stability of stem bromelain against inactivation.

Antienzyme polyclonal antibodies against stem bromelain were raised in male albino rabbits and the Fab' monomers isolated from the IgG of the immune sera. Incubation of bromelain with the Fab' resulted in binding and gel filtration of the resulting complex suggested a 1:1 stoichiometry. Complexing with the Fab' resulted in significant stabilization of bromelain against thermal inactivation and alkaline pH.

Interaction of human IgG chimeric antibodies with the human FcRI and FcRII receptors: requirements for antibody-mediated host cell-target cell interaction.

Chimeric monoclonal antibodies (McAb), specific for the hapten 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP), expressing human IgG1, IgG2, IgG3 and IgG4 subclass constant domains, have been examined for their ability to interact with the human FcRII receptor. Human red blood cells (RBC) sensitized by each of these McAbs have been assayed for their ability to form rosettes with the human histiocytic lymphoma U937 cell line, human B cell line Daudi and erythroblastoid K562 cell line. IgG1 and IgG3 sensitized RBC formed significant rosettes with the FcR- and FcRII+ Daudi and K562 cell lines, the percentage of cells forming rosettes being directly proportional to the degree of sensitization of the RBC. Bromelin treating Daudi cells did not alter this pattern of reactivity, whereas bromelin treated FcRI+ and FcRII+ U937 cells formed significant resettes with IgG1, IgG3 and IgG4 sensitized RBC, demonstrating a difference in the IgG subclass specificity between human FcRI and FcRII. Murine IgG2b anti-NIP sensitized RBC did not form rosettes with any cell line tested; however, RBC sensitized by some members of a panel of murine IgG1 McAb, specific for the glycophorin A molecule, were able to form rosettes with Daudi, U937 and K562 cells. This interaction was enhanced by bromelin treating the Daudi or U937 cells and can be correlated to the disposition of the epitopes recognized, relative to the target cell membrane, those McAbs recognizing epitopes furthest from the RBC surface being most effective in interacting with FcRII. The data are interpreted in terms of a simple model for antibody-mediated cell--cell interaction.

Monoclonal antibodies against Dermatophagoides group I allergens as pseudo-cystatins blocking the catalytic site of cysteine proteinases.

The enzyme allergens Der p I and Der f I produced by the house dust mites Dermatophagoides pteronyssinus and D. farinae display partial sequence homology with other members of the cysteine proteinase superfamily. We report that certain widely used mouse mAbs against these Group I allergens indeed crossreact with the plant enzymes papain, bromelain and ficin. The recognition sites of these anti Group I mAbs comprise conformational and thermolabile epitopes involved in molding the catalytic center of the proteinases. Thus, the mAbs inhibit the enzymatic hydrolysis of specific chromogenic substrates by the Group I allergens, while specific cysteine proteinase inhibitors abolish the recognition of the enzymes by the mAbs. Similarly, activation of the thiol-proteases with L-cysteine abrogates their binding in the two-site mAb system, indicating that the mAbs recognize a proenzyme conformational peptide epitope. It follows that mAb-based assays for mite Group I components can neither detect the allergens after inactivation, nor in their fully activated forms.

B cells specific for bromelain-treated erythrocytes are not derived from adult rat bone marrow.

As part of an evolutionary layered hematopoietic system, the B lymphocyte compartment consists of different lineages of B lymphocytes, which evolve sequentially during ontogeny. In mice, there is ample evidence for the existence of at least two lineages, a layer of B-1 cells (Ly-1 B cells) and the evolutionary more advanced layer consisting of conventional B cells. In a previous study we were unable to detect B-1 cells in the rat as determined by phenotypic markers. Here we studied the possible existence of putative B-1 cells in the rat based on some functional and developmental characteristics as have been described for mouse B-1 cells. We show that B cells secreting antibodies that recognize bromelain-treated mouse red blood cells (BrMRBC) can be identified in rat spleen, whereas these cells (in contrast to DNP-specific B cells) are virtually absent in lethally X-irradiated and bone marrow (BM) reconstituted animals. The number of anti-rMRBC-secreting B cells could not be restored to control levels by reconstitution with fetal liver cells or by cotransfer of 10(7) cells from peritoneal cavity, lymph node or Peyer's patches or up to 2 x 10(8) splenocytes. Although our findings thus suggest that B-1 cells (or B-1 like cells) may be present in rats, formal proof for the existence of such a lineage in rats awaits definition of these cells at the progenitor level.

The influence of mouse housing density on autoimmune reactivity.

Splenic cells from C57BL/6 mice housed in groups (six per cage) or individually (one per cage) were analyzed for their ability to synthesize autoantibodies against autologous bromelain-treated erythrocytes. Group-housed male mice had a significantly lower number of autoimmune plaque forming cells (APFC) in their spleens than age matched female mice housed under similar conditions. However, when male mice were housed individually for 4 to 44 weeks, a marked increase in numbers of APFC were found in their spleens, approaching those of female control mice. Our results demonstrate that a simple environmental manipulation such as housing conditions can profoundly modulate autoimmune reactivity. Thus, alteration of the psychophysiological state determined by the quality of housing may significantly affect the development of autoimmune disease.

Antigenicities of stem bromelain. Contribution of three-dimensional structure and individual amino acid residues.

The contribution of three-dimensional structure and individual amino acid residues to the antigenicities of macromolecular protein was investigated for a thiol protease stem bromelain as antigen. The extent of the participation was demonstrated by a decrease in antigenicity when the enzyme was denatured in 8 M urea before and after reductive cleavage of intrapeptide disulfide bonds or modified in particular amino acid residues. The results showed that the enzyme treated with 8 M urea without reductive cleavage of disulfide bonds preserved about 90% of antigenicity to antibodies against native stem bromelain, while the enzyme denatured after the reductive cleavage of disulfide bonds brought about almost 80% disappearance of the antigenicity. Modification of individual amino acid side chains revealed that lysine was the most immunodominant amino acid, showing 2.5% contribution per residue, and tyrosine followed with 1.2%. However, acidic amino acids such as flutamic and aspartic acids were found to be as low as 0.3%, and tryptophan was 0.2%. These data suggest that most of the antigenic determinants of stem bromelain are of the steric conformation in which lysine and/or tyrosine are most frequently involved as immunodominant amino acids.

Autoantibodies to phosphatidylcholine. The murine antibromelain RBC response.

The observation that murine B-cell populations can contain relatively large numbers of cells that produce IgM with the ability to lyse bromelain-treated mouse erythrocytes (BrMRBC), but not normal untreated MRBC, was made nearly 20 years ago. The major observations regarding the antigen specificity, the cells that produce this IgM, and the immunoglobulin V genes that encode them are summarized in this report. The epitope on BrMRBC that is recognized has been identified as the head group of phosphatidylcholine (PtC); B cells whose IgM has this specificity can be easily identified by their ability to bind fluorescent synthetic liposomes whose membrane contains PtC. The cells producing IgM specific for PtC all derive from the Ly-1 B-cell subset, and they use primarily two VH/VL gene pairs to encode the anti-PtC antibodies. The VH genes used describe two new VH gene families, VH11 and VH12. The genes encoding anti-PtC are unmutated and have characteristics and restricted VDJ constructions. The cells with this specificity, within individual mice, are polyclonal. These criteria are consistent with a primary antigen-driven clonal selection mechanism as the basis for the development of this immune specificity.

Bromelain is an accelerator of phagocytosis, respiratory burst and Killing of Candida albicans by human granulocytes and monocytes.

OBJECTIVE: The aim of this study was to examine the influence of immuno modulating agents like bromelain and trypsin (e.g. Wobenzym on granulocyte and monocyte functions in healthy volunteers and patients with disorders of the humoral immuno system X-linked agammaglobulinaemia (XLA) and common variable immuno deficiency (CVID) and to find out whether the unspecific immunity could be improved by these enzymes. METHODS: In a whole-blood assay kinetics of phagocytosis, respiratory burst and killing (PBK) were measured in blood samples incubated with and without bromelain and trypsin (B/T) using Candida albicans as target organism. The time-reaction curves were analysed determining their gradient (T1) and their onset (T2) as well as the half effect time (HET). RESULTS: Phagocytes from patients with XLA showed a significantly accelerated basal phagocytosis (reduction of HET by 24% p < 0.001) compared to healthy controls. After incubation with B/T (10 microg/ml each) speed of phagocytosis was nearly doubled (phagocyte activity p < 0.0001, Candida uptake p < 0.003), T2 of respiratory burst was reduced by 65 % (p < 0.0001) and killing was accelerated by 27% (p < 0.046). However, the maximal activities of all kinetics were not altered. Incubation of phagocytes from healthy controls with B/T accelerated phagocytosis to a level comparable to that of untreated phagocytes from patients with XLA and also accelerated reactive oxygen species (ROS) production (reduction of HET by 28%, p < 0.012). In contrast to phagocytes from patients with XLA, phagocytes of patients with CVID showed a similar stimulation by B/T like healthy controls. Further experiments with the single substances showed that bromelain was the active compound. CONCLUSION: Our data suggest, that bromelain possesses immuno stimulatory properties. Phagocytes of XLA patients appear to be particularly susceptible to this stimulation.

Inhibition of influenza virus haemolytic and haemagglutination activities by monoclonal antibodies to haemagglutinin glycopolypeptides HA1 and HA2.

Acid treatment of influenza virus enhanced haemagglutination inhibiting (HI) activity of some anti-HA1 monoclonal antibodies (MoAbs). These changes in the HI-activity could be either due to alteration in the mutual orientation of MoAb (e.g. IC8, IB8) binding epitope to receptor site or to an increase in the number of epitopes accessible to the corresponding MoAbs (e.g. IVA1). HI test with pH 5-virus revealed similar (although not identical) antigenic differences among related virus strains as the HI test with pH 7-virus. Anti-HA2 MoAbs were negative in the HI test with both pH 5- and pH 7-virus. Anti-HA1 MoAbs showed a HI activity with pH 5-treated BHA similar to that with pH 5-treated virus. Surprisingly one out of eight anti-HA2 MoAbs (IIF4) exhibited a relatively high HI activity to pH 5-BHA-mediated haemagglutination. Virus-induced red blood cell haemolysis was efficiently inhibited with several anti-HA1 MoAbs (e.g. IC8, IB8, and IIB4) while other anti-HA1 antibodies, including IVA1 and IVG6 with preferential reactivity with pH 5-treated antigens in RIA, gave no inhibition. As a rule, anti-HA2 MoAbs were poor haemolysis inhibitors.

[Cross-reactivity between bromelain and soluble fraction from wheat flour].

It is revealed that patients allergic to a water-soluble fraction from wheat flour were sensitive to pineapple enzyme, bromelain. Since bromelain has a high similarity to other SH-proteases such as papain, it may imply that the patients may recognize varieties of other SH-proteases as the epitope.