Improved recombinant Api m 1- and Ves v 5-based IgE testing to dissect bee and yellow jacket allergy and their correlation with the severity of the sting reaction.

BACKGROUND: No study has assessed the diagnostic sensitivity of rApi m 1 and rVes v 5 on Immulite testing system. OBJECTIVE: To compare the diagnostic sensitivity of commercially available venom recombinant allergens between the currently available immunoassays [ImmunoCAP (CAP) and Immulite (LITE)] and establish their correlation with the severity of the sting reaction. METHODS: This study evaluated 95 bee venom and 110 yellow jacket venom-allergic subjects. We measured the levels of sIgE to rApi m 1, rVes v 5 (LITE and CAP), rApi m 2 (LITE), rVes v 1 (CAP) and total IgE (CAP). Forty-nine healthy subjects served as controls. RESULTS: The diagnostic sensitivity of rApi m 1 and rVes v 5 was significantly higher with the LITE than with the CAP system (71% vs. 88% and 82% vs. 93%). The specificity of both assays for both allergens was between 94% and 98%. Twenty-nine patients that tested negative for rApi m 1 or rVes v 5 with CAP were positive with LITE, but none of the patients that tested negative with LITE were positive with CAP. The positive values of rApi m 1 and rVes v 5 were on average 2.7 and 2.3 times higher, with the LITE than with the CAP system. The combination of rApi m 1 and rApi m 2 (LITE) and the combination of rVes v 5 (LITE) and rVes v 1 (CAP) almost matched the sensitivity of native venoms (95% and 97%, respectively), whereas the diagnostic sensitivity of the combination of rVes v 5 and rVes v 1 (CAP) did not reach the sensitivity of rVes v 5 (LITE) alone (90% vs. 93%). IgE levels to venom recombinants and total IgE did not correlate with the severity of sting reaction. CONCLUSIONS & CLINICAL RELEVANCE: The use of rApi m 1 and rVes v 5 with the LITE system significantly enhanced diagnostic utility of venom recombinants and should improve the dissection of bee and yellow jacket venom allergy.

Up-regulated PLA2G10 in cancer impairs T cell infiltration to dampen immunity.

T cells are often absent from human cancer tissues during both spontaneously induced immunity and therapeutic immunotherapy, even in the presence of a functional T cell-recruiting chemokine system, suggesting the existence of T cell exclusion mechanisms that impair infiltration. Using a genome-wide in vitro screening platform, we identified a role for phospholipase A2 group 10 (PLA2G10) protein in T cell exclusion. PLA2G10 up-regulation is widespread in human cancers and is associated with poor T cell infiltration in tumor tissues. PLA2G10 overexpression in immunogenic mouse tumors excluded T cells from infiltration, resulting in resistance to anti-PD-1 immunotherapy. PLA2G10 can hydrolyze phospholipids into small lipid metabolites, thus inhibiting chemokine-mediated T cell mobility. Ablation of PLA2G10's enzymatic activity enhanced T cell infiltration and sensitized PLA2G10-overexpressing tumors to immunotherapies. Our study implicates a role for PLA2G10 in T cell exclusion from tumors and suggests a potential target for cancer immunotherapy.

Double positivity to bee and wasp venom: improved diagnostic procedure by recombinant allergen-based IgE testing and basophil activation test including data about cross-reactive carbohydrate determinants.

BACKGROUND: Specific IgE (sIgE) antibodies to both bee and wasp venom can be due to a sensitivity to both insect venoms or due to cross-reactive carbohydrate determinants (CCDs). OBJECTIVE: Investigating whether a basophil activation test (BAT) with both venoms as well as with bromelain and horseradish peroxidase (HRP) or recombinant allergen-based IgE testing can improve the diagnostic procedure. METHODS: Twenty-two Hymenoptera-venom allergic patients with sIgE antibodies to both bee and wasp venom were studied. sIgE antibodies to MUXF3 CCD, bromelain, HRP, rApi m 1, and rVes v 5 were determined, and a BAT (Flow2 CAST) with venom extracts, bromelain, and HRP was performed. Further recombinant allergen-based IgE testing was done by using an ELISA, if required. The reactivity of basophils was calculated from the insect venom concentration at half-maximum stimulation. RESULTS: Double positivity/double negativity/single positivity to rApi m 1 and rVes v 5 was seen in 12/1/9 patients. Further recombinant allergen-based IgE testing in the last ones revealed positive results to the other venom in all cases except one. BAT was double positive/double negative/single positive in 6/2/14 patients. Four patients with negative results in sIgE antibodies to CCDs had positive results in BAT. BAT with bromelain/HRP showed a sensitivity of 50%/81% and a specificity of 91%/90%. CONCLUSION: Component-resolved IgE testing elucidates the pattern of double positivity, showing a majority of true double sensitizations independent of CCD sensitization. BAT seems to add more information about the culprit insect even if the true clinical relevance of BAT is not completely determined because of ethical limitations on diagnostic sting challenges. BAT with HRP is a good method to determine sensitivity to CCDs.

IgE to recombinant allergens Api m 1, Ves v 1, and Ves v 5 distinguish double sensitization from crossreaction in venom allergy.

BACKGROUND: Diagnostic tests in patients with Hymenoptera venom allergy are frequently positive to venoms of both honey bee and wasp (Vespula). Component-resolved analysis with recombinant species-specific major allergens (rSSMA) may help to distinguish true double sensitization from crossreactivity. METHODS: Included were 121 patients with systemic allergic reactions to Hymenoptera stings, 76 with double positivity of serum-specific IgE (sIgE) to both venoms, 45 with single positivity to bee or wasp venom, and 32 controls without history of systemic reactions to Hymenoptera stings and no sIgE to whole venoms. In venom-allergic patients and controls, sIgE to rSSMA Api m 1 of bee venom and to Ves v 1 and Ves v 5 of wasp venom were tested by ImmunoCAP. RESULTS: Only 47% of 76 patients with double positivity to whole venoms reacted also to rSSMA of both species. Specificity of sIgE to the 3 rSSMA was very high, with no sIgE to rSSMA of the other species in single-positive venom-allergic patients and only one control with low sIgE to Ves v 1. All wasp-allergic single-positive patients had sIgE to Ves v 5 and/or Ves v 1, and 78.3% of single-positive bee venom-allergic patients had sIgE to Api m 1. CONCLUSION: Specificity of sIgE to rSSMA of both species is excellent. Sensitivity of sIgE to rSSMA was optimal for wasp venom. Sensitivity of bee venom Api m 1 could be increased by adding rSSMA of other important bee venom allergens.

Value of component based diagnostics in IgE-mediated hymenoptera sting reactions.

BACKGROUND: Stings by insects can precipitate many signs and symptoms of dermatological and ocular diseases. Of particular importance is the anaphylaxis after Hymenoptera stings. Selection of the appropriate venom for immunotherapy requires a precise diagnosis, which is frequently difficult to confirm since the history presented by the patient is many times not conclusive and diagnostic tests are often positive for bee venom (BV) and vespula venom (VV). This double positivity is either caused by true double sensitization or by antibodies cross-reactive to homologous peptide sequences or to cross-reactive carbohydrate determinants (CCDs). In this study, we analyzed in 39 patients, tested positive for specific immunoglobulin E (sIgE) against BV and VV and CCDs whether the routine detection of sIgE against the recombinant species-specific major allergens (SSMAs) rApi m1 and rVes v5 enables the discrimination between genuine double sensitization and cross reactivity and therefore may be superior to other in vitro assays such as IgE-inhibition test or the basophil activation test. MATERIALS AND METHODS: Thirty-nine patients each with allergic reactions to vespula and/or honey bee stings and tested positive for sIgE antibodies against CCDs were analyzed for sIgE against BV, VV, CCDs (MUFX3) and SSMAs by UNICAP (CAP) and to BV, VV, bromelain, horseradish peroxidase and ascorbat oxidase by Immulite 2000 (IMMU). In 12 cases results from a basophil activation test, in nine cases results from IgE-inhibition assays and in 10 cases an unambiguous history of the patient were taken into consideration. RESULTS: A definite diagnosis could be assigned to each patient: sensitization to BV n = 7, sensitization to VV n = 29 and true double sensitization to both venoms n = 3. Detection of sIgE against BV and VV by CAP leads in three cases to the diagnosis BV allergy, in 35 cases to the diagnosis double sensitization and in one case to the diagnosis VV allergy. Detection of sIgE against BV and VV by IMMU leads in five cases to the diagnosis BV allergy, in 27 cases to the diagnosis double sensitization and in seven cases to the diagnosis VV allergy. Detection of sIgE against rApi m1 and rVes v5 by CAP leads in six cases to the diagnosis BV allergy, in eight cases to the diagnosis double sensitization, in 21 cases to the diagnosis VV allergy and in four cases to a false double-nagative result implicating no allergy. DISCUSSION AND CONCLUSION: Detection of sIgE to rApi m 1 and rVes v 5 by CAP is the most reliable diagnostic procedure to discriminate between true double sensitization and cross reactivity in patients with double-positive IgE results to venom extracts in the presence of sIgE against CCDs. In this study, however, we demonstrate that in nine of 39 patients tested positive for sIgE against CCDs, even the allergen component based diagnostic produces false double-positive and also false double-negative test results. Thus, we conclude that especially in hard to diagnose CCD positive patients beside the detection of sIgE, in vitro assays such as the IgE-inhibition test or the basophil activation test are still of importance. Detection of sIgE against only two SSMAs is not sufficient for a precise diagnosis. We propose inclusion of further SSMAs in diagnostic procedures.

Recombinant allergen-based IgE testing to distinguish bee and wasp allergy.

BACKGROUND: The identification of the disease-causing insect in venom allergy is often difficult. OBJECTIVE: To establish recombinant allergen-based IgE tests to diagnose bee and yellow jacket wasp allergy. METHODS: Sera from patients with bee and/or wasp allergy (n = 43) and patients with pollen allergy with false-positive IgE serology to venom extracts were tested for IgE reactivity in allergen extract-based tests or with purified allergens, including nonglycosylated Escherichia coli-expressed recombinant (r) Api m 1, rApi m 2, rVes v 5, and insect cell-expressed, glycosylated rApi m 2 as well as 2 natural plant glycoproteins (Phl p 4, bromelain). RESULTS: The patients with venom allergy could be diagnosed with a combination of E coli-expressed rApi m 1, rApi m 2, and rVes v 5 whereas patients with pollen allergy remained negative. For a group of 29 patients for whom the sensitizing venom could not be identified with natural allergen extracts, testing with nonglycosylated allergens allowed identification of the sensitizing venom. Recombinant nonglycosylated allergens also allowed definition of the sensitizing venom for those 14 patients who had reacted either with bee or wasp venom extracts. By IgE inhibition studies, it is shown that glycosylated Api m 2 contains carbohydrate epitopes that cross-react with natural Api m 1, Ves v 2, natural Phl p 4, and bromelain, thus identifying cross-reactive structures responsible for serologic false-positive test results or double-positivity to bee and wasp extracts. CONCLUSION: Nonglycosylated recombinant bee and wasp venom allergens allow the identification of patients with bee and wasp allergy and should facilitate accurate prescription of venom immunotherapy.

Hymenoptera venom allergy: analysis of double positivity to honey bee and Vespula venom by estimation of IgE antibodies to species-specific major allergens Api m1 and Ves v5.

BACKGROUND: In patients with hymenoptera venom allergy diagnostic tests are often positive with honey bee and Vespula venom causing problems in selection of venoms for immunotherapy. METHODS: 100 patients each with allergic reactions to Vespula or honey bee stings and positive i.e. skin tests to the respective venom, were analysed for serum IgE to bee venom, Vespula venom and crossreacting carbohydrate determinants (CCDs) by UNICAP (CAP) and ADVIA Centaur (ADVIA). IgE-antibodies to species specific recombinant major allergens (SSMA) Api m1 for bee venom and Ves v5 for Vespula venom, were determined by ADVIA. 30 history and skin test negative patients served as controls. RESULTS: By CAP sensitivity was 1.0 for bee and 0.91 for Vespula venom, by ADVIA 0.99 for bee and 0.91 for Vespula venom. None of the controls were positive with either test. Double positivity was observed in 59% of allergic patients by CAP, in 32% by ADVIA. slgE to Api m1 was detected in 97% of bee and 17% of Vespula venom allergic patients, slgE to Ves v5 in 87% of Vespula and 17% of bee venom allergic patients. slgE to CCDs were present in 37% of all allergic patients and in 56% of those with double positivity and were more frequent in bee than in Vespula venom allergic patients. CONCLUSIONS: Double positivity of IgE to bee and Vespula venom is often caused by crossreactions, especially to CCDs. IgE to both Api m1 and Ves v5 indicates true double sensitization and immunotherapy with both venoms.

Localization of phospholipase A2 in normal and ras-transformed cells.

The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed.

Intracellular phospholipase A2 expression and location in human macrophages: influence of synthetic material surface chemistry.

Phospholipase A(2) (PLA(2)) enzymes participate in a potent inflammatory pathway through the liberation of arachidonic acid upon hydrolysis of membrane glycerophospholipids. The presence of implanted polycarbonate-urethane (PCNU) materials, used in several medical applications, has the ability to influence inflammatory responses of human macrophages that are recruited to a tissue-material interface; however, the specific inflammatory pathways that are activated upon macrophage attachment to PCNU are largely unknown. Previous studies suggested the participation of PLA(2) pathways in material degradation with the use of chemical inhibitors, such as aristolochic acid (ARIST), however not accurately defining the specific PLA(2) enzymes involved. The current study aimed to establish specific groups of PLA(2) involved in the macrophage foreign body response to PCNU. ARIST was assessed for specific effects on secretory PLA(2) (sPLA(2)) protein expression and non-specific effects on key proteins, beta-actin and monocyte-specific esterase, implicated in the macrophage attack on PCNU materials. Macrophage attachment to PCNU materials induced increased intracellular expression of cytosolic PLA(2) (cPLA(2)), but not sPLA(2), relative to tissue culture polystyrene (TCPS) as detected by immunoblot analysis, demonstrating an early and delayed stimulation during the time course of increased cPLA(2) protein expression. Laser scanning confocal microscopy images indicated a change in location of cPLA(2) in macrophages adherent to PCNU surfaces compared to TCPS. This study has illustrated changes in macrophage cPLA(2) expression in response to cell-attachment to PCNU surfaces, demonstrating that the macrophage foreign body response to biomaterials induces a potent inflammatory pathway, which may lead to tissue damage near the site of material implantation.

The non-venom insect phospholipases A2.

Phospholipases A(2) (PLA(2)s) are responsible for releasing the fatty acid moiety from the sn-2 position of phospholipids. These enzymes are virtually ubiquitous proteins known from all major biological taxa. Various PLA(2)s act in a wide array of biological processes, including digestion of dietary lipids, cellular homeostasis, intra- and intercellular signaling, host defense and at least a few ecological interactions. PLA(2) activities have been recorded in a small number of insect species, which can be taken to represent the broad group, Insecta. Within insects, PLA(2)s act in functions expected from the background on these enzymes. So far, we know PLA(2)s act in lipid digestion, cellular host defense signaling, reproduction and in organismal-level metabolism. Additional PLA(2) actions are certain to emerge. This is the first article devoted to assembling the known information on insect PLA(2)s. I review the scant information available on the biological actions of PLA(2)s in insects, relate new findings on insect pathogens that disrupt insect immune functions by inhibiting PLA(2)s and mention the few reports of sequence information on insect PLA(2)s. Finally, I offer a brief prospectus on future research into insect PLA(2)s. There are two overarching points in this paper. One, there remains a great deal to learn about insect PLA(2)s and two, some of the findings on insect PLA(2)s will have meaningful practical significance.

Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom.

Phospholipases A(2) are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. This study used a large nonimmune human scFv library named Griffin.1 (MRC, Cambridge, UK) for selection of recombinant antibodies against antigens present in Bothrops jararacussu venom and identification of specific antibodies able to inhibit phospholipase activity. Four clones were identified as capable of inhibiting this activity in vitro. These clones were able to reduce in vivo the myotoxic activity of BthTX-I and BthTX-II PLA(2), but had no effect on the in vitro anticoagulant activity of BthTX-II. This work shows the potential of using recombinant scFv libraries in the search for antibodies that neutralize relevant venom components.

Phospholipase A2 and phospholipase B activities in fungi.

As saprophytes or disease causing microorganisms, fungi acquire nutrients from dead organic material or living host organisms. Lipids as structural components of cell membranes and storage compartments play an important role as energy-rich food source. In recent years, it also has become clear that lipids have a wide range of bioactive properties including signal transduction and cell to cell communication. Thus, it is not surprising that fungi possess a broad range of hydrolytic enzymes that attack neutral lipids and phospholipids. Especially during infection of a mammalian host, phospholipase A(2) (PLA(2)) enzymes released by fungi could play important roles not only for nutrient acquisition and tissue invasion, but for intricate modulation of the host's immune response. Sequencing of fungal genomes has revealed a wide range of genes encoding PLA(2) activities in fungi. We are just beginning to become aware of the significance these enzymes could have for the fungal cells and their interaction with the host.

Activation of human inflammatory cells by secreted phospholipases A2.

Secreted phospholipases A(2) (sPLA(2)s) are enzymes detected in serum and biological fluids of patients with various inflammatory, autoimmune and allergic disorders. Different isoforms of sPLA(2)s are expressed and released by human inflammatory cells, such as neutrophils, eosinophils, T cells, monocytes, macrophages and mast cells. sPLA(2)s generate arachidonic acid and lysophospholipids thus contributing to the production of bioactive lipid mediators in inflammatory cells. However, sPLA(2)s also activate human inflammatory cells by mechanisms unrelated to their enzymatic activity. Several human and non-human sPLA(2)s induce degranulation of mast cells, neutrophils and eosinophils and activate exocytosis in macrophages. In addition some, but not all, sPLA(2) isoforms promote cytokine and chemokine production from macrophages, neutrophils, eosinophils, monocytes and endothelial cells. These effects are primarily mediated by binding of sPLA(2)s to specific membrane targets (heparan sulfate proteoglycans, M-type, N-type or mannose receptors) expressed on effector cells. Thus, sPLA(2)s may play an important role in the initiation and amplification of inflammatory reactions by at least two mechanisms: production of lipid mediators and direct activation of inflammatory cells. Selective inhibitors of sPLA(2)-enzymatic activity and specific antagonists of sPLA(2) receptors are current being tested for pharmacological treatment of inflammatory and autoimmune diseases.