Inhibition of IgE binding to cross-reactive carbohydrate determinants enhances diagnostic selectivity.

BACKGROUND: Allergy diagnosis by determination of allergen-specific IgE is complicated by clinically irrelevant IgE, of which the most prominent example is IgE against cross-reactive carbohydrate determinants (CCDs) that occur on allergens from plants and insects. Therefore, CCDs cause numerous false-positive results. Inhibition of CCDs has been proposed as a remedy, but has not yet found its way into the routine diagnostic laboratory. We sought to provide a simple and affordable procedure to overcome the CCD problem. METHODS: Serum samples from allergic patients were analysed for allergen-specific IgEs by different commercial tests (from Mediwiss, Phadia and Siemens) with and without a semisynthetic CCD blocker with minimized potential for nonspecific interactions that was prepared from purified bromelain glycopeptides and human serum albumin. RESULTS: Twenty two per cent of about 6000 serum samples reacted with CCD reporter proteins. The incidence of anti-CCD IgE reached 35% in the teenage group. In patients with anti-CCD IgE, application of the CCD blocker led to a clear reduction in read-out values, often below the threshold level. A much better correlation between laboratory results and anamnesis and skin tests was achieved in many cases. The CCD blocker did not affect test results where CCDs were not involved. CONCLUSION: Eliminating the effect of IgEs directed against CCDs by inhibition leads to a significant reduction in false-positive in vitro test results without lowering sensitivity towards relevant sensitizations. Application of the CCD blocker may be worthwhile wherever natural allergen extracts or components are used.

Determination of sIgE to rPhl p 1 is sufficient to diagnose grass pollen allergy.

BACKGROUND: New diagnostic tools such as the basophil activation test (BAT) and component-resolved diagnosis (CRD) are promising for Hymenoptera venom or food allergy. A clear benefit for inhalant allergens has not yet been shown. Our aim was to compare new and established tests for grass pollen allergy. METHODS: Forty-nine patients with grass pollen allergy and 47 controls were prospectively enrolled in the study. A symptom score was calculated for each patient. Conjunctival provocation tests (CPT), skin prick tests (SPT), BAT, and sIgE determination including CRD were performed. Sensitivity and specificity were compared and results were correlated with the symptom score. RESULTS: Single determination of sIgE to rPhl p 1 showed the best balance between sensitivity (98%) and specificity (92%). Use of additional components, such as rPhl p 2 and 5, did not increase sensitivity. Generally, sensitivity of tests was high: SPT 100%, ISAC-112 100%, sIgE to timothy grass 98%, BAT 98%, ISAC-103 84%, and CPT 83%. Specificity ranged from 79% (SPT) to 96% (CPT). All test results and calculated values (e.g. ratio sIgE/tIgE) did not correlate with symptom severity. Asymptomatic sensitization to timothy grass in controls was rare in the CAP (11%) and predominantly due to Phl p 1 sensitization. CONCLUSION: rPhl p 1 was sufficient to diagnose grass pollen allergy, and sIgE patterns were the same in symptomatically and asymptomatically sensitized subjects. The testing of multiple components was of minor importance, and no test correlated with symptom severity.

[GSTCVS certificate for cardiac pacemaker, ICD and CRT therapy]. / DGTHG-Zertifizierung zur Herzschrittmacher-, ICD- und CRT-Therapie.

Medical qualifications to perform operations with cardiac electronic implantable devices as well as for preoperative and postoperative therapy, including follow-up in this patient population are not well defined. Based on recommendations which have been worked out and published by an interdisciplinary consensus of cardiac surgeons, cardiologists and electrophysiologists, a certificate with three modules has been developed by the Working Group for Electrophysiologic Surgery of the German Society for Thoracic and Cardiovascular Surgery (GSTCVS, Deutsche Gesellschaft für Thorax-, Herz- und Gefäßchirurgie, DGTHG). First examinations for this certificate will be held in 2013 and transitional regulations apply until 1st April 2014. Further details are available on the homepage of the GSTCVS.

[Cross-reactivity to honeybee and wasp venom]. / Kreuzreaktivität auf Bienen- und Wespengift.

About 30-40% of patients with insect venom allergy have IgE antibodies reacting with both honeybee and Vespula venom. Apart from true double sensitization, IgE against cross-reactive carbohydrate determinants (CCDs, alpha1,3-fucosylated N-glycans) with low clinical relevance is the most frequent and often only cause for the multiple reactivity. Venom hyaluronidases have been identified as the most important allergens displaying CCDs, whereas cross-reactions through the hyaluronidases' peptide backbones are less common. If IgE binding to CCDs is disregarded, Vespula venom hyaluronidase is only a minor allergen. In-vitro tests using fucosylated plant glycoproteins (e.g. assessment of specific IgE antibodies by CAP-FEiA to bromelain) are helpful in identifying sera containing CCD-specific IgE, although a positive result (occurring in 70-80% of all double-positive sera) does not reliably exclude true double-sensitization. Reciprocal in-vitro inhibition including non-venom inhibitor proteins rich in CCDs is the method of choice to discriminate between double-sensitization and cross-reactivity. Future in-vitro diagnosis will be markedly improved when recombinant allergens lacking CCDs become commercially available.

Identification by immunoblot of venom glycoproteins displaying immunoglobulin E-binding N-glycans as cross-reactive allergens in honeybee and yellow jacket venom.

BACKGROUND: IgE antibodies against carbohydrate epitopes have been identified recently as a major cause of in vitro double positivity to honeybee (HB) and vespid venom in patients with stinging-insect allergy. As these antibodies possibly have low clinical relevance they may be misleading in the diagnosis of venom allergy. OBJECTIVE: To confirm the role of carbohydrate epitopes in double positivity and to locate the responsible glycoallergens in HB and yellow jacket (YJ) venom by western blot. METHODS: Immunoblot inhibition using HB venom, YJ venom and two glycoprotein sources displaying 1-3-fucosylated N-glycans (i.e. oilseed rape (OSR) pollen, and the synthetic neo-glycoprotein fucosylated/xylosylated N-glycans from bromelain coupled to bovine serum albumin (MUXF-BSA)) as inhibitors were performed with sera from 15 double-positive patients with stinging-insect allergy. Additionally, reactivity with blotted hymenoptera venoms of a carbohydrate-specific rabbit antiserum against OSR pollen was investigated. RESULTS: Major venom glycoallergens binding with carbohydrate-specific human IgE and rabbit IgG were detected in HB venom at 42 (hyaluronidase (HYA)), 46, 65 and 95 kDa, and in YJ venom at 38 and 43 kDa (HYA). Antibody binding to these allergens was completely lost after periodate treatment. Glycans of HB phospholipase were bound by patients' IgE only after protein denaturation. In 10 of the 15 patients the reactivity was with the second venom because of carbohydrates alone. The high-molecular-weight glycoallergens identified in HB venom probably correspond to similar proteins described earlier, including allergens B and C. The 38-kDa YJ allergen might represent a homologue of V mac 3. CONCLUSIONS: The data confirm the proposed role of carbohydrate-specific IgE in double positivity to HB and YJ venom and shed new light on some previously described minor hymenoptera allergens of uncertain clinical significance. The consideration of carbohydrate-specific IgE may allow to discriminate between patients with potentially relevant and patients with non-relevant double sensitization.

Identification of oilseed rape (Brassica napus) pollen profilin as a cross-reactive allergen.

BACKGROUND: Major allergens of oilseed rape (OSR) pollen with molecular weights of 6/8, 14 and between 27 and 69 kD have been described. The aim of the present study was to further characterize the 14-kD allergen. METHODS: The 14-kD protein was purified from OSR pollen extracts by poly-(L-proline) (PLP)-Sepharose affinity chromatography and characterized immunologically by means of allergic patients' IgE antibodies, profilin-specific rabbit antisera, Western blot and ELISA inhibition using recombinant birch profilin (rBet v 2), and skin prick testing. RESULTS: By PLP affinity chromatography, OSR pollen profilin was purified as a single protein of 14.5 kD and further identified as a profilin by three polyclonal rabbit antisera raised against ragweed and tobacco pollen profilin and the C-terminus of birch profilin. IgE binding of a human serum pool (n = 15) and four profilin-reactive sera to nitrocellulose-blotted OSR profilin was completely inhibited by 1 microg/ml rBet v 2 (birch profilin). Reciprocal ELISA inhibition using increasing concentrations of rBet v 2 and purified OSR profilin, respectively, showed that rBet v 2 strongly inhibits antibody binding to OSR profilin, whereas almost 100 times the amount of OSR profilin was needed to inhibit IgE binding to rBet v 2. Skin prick tests were positive (wheal >/=3 mm) with 5 microg/ml rBet v 2 in all three patients tested, and with OSR profilin in two patients at a concentration of 50 microg/ml. CONCLUSIONS: OSR pollen profilin shares IgE and IgG epitopes with Bet v 2 and other plant profilins and may represent a potentially relevant allergen for profilin-sensitized patients.

Cross-reactivity between Ficus benjamina latex and fig fruit in patients with clinical fig allergy.

BACKGROUND: Anaphylactic reactions to fig fruits (Ficus carica) have been reported from subjects sensitized to Ficus benjamina (FB) latex allergens. Figs may also be involved in the latex-fruit syndrome. OBJECTIVE: To study the immunologic relationship between fig fruit, Ficus benjamina, natural rubber latex (Hevea brasiliensis), and other tropical fruits. METHODS: RAST inhibition and Western blotting with FB and fruit extracts was performed in five patients with oral allergy syndrome (OAS) or anaphylaxis after the ingestion of figs and one patient with symptoms from exposure to FB trees. Co-sensitization to rubber latex and tropical fruits (kiwi, banana, avocado, papaya, pineapple, mulberry) was studied by skin testing. RESULTS: RAST to FB was inhibited >95% by FB extracts and 16-65% (mean 49%) by extracts from fresh fig. RAST to fig fruit was inhibited >95% by FB and fresh fig, 63-97% (mean 86%) by dried fig, and 0-84% (mean 35.5%) by kiwi fruit. FB and fig extracts lost most of their allergenicity when denatured by heat (95 degrees C) or reduced by dithiothreitol. Western blotting after non-reducing gel electrophoresis revealed IgE binding to proteins of 22 and 28-34 kDa in FB latex; however, no corresponding allergens could be detected in fig extracts. Positive skin tests were obtained most often with kiwi fruit, papaya, and avocado. Sensitization to rubber latex could not be demonstrated in any of the patients. RAST to papain was positive in three of five patients. CONCLUSIONS: Allergic reactions to fresh or dried figs can present as a consequence of primary sensitization to airborne FB allergens independent of sensitization to rubber latex allergens. Kiwi fruit, papaya, and avocado as well as pineapple and banana may be other fruits associated with sensitization to Ficus allergens.

Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy.

BACKGROUND: Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity. OBJECTIVE: We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity. METHODS: CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera. RESULTS: CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P < .00001, chi(2) test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSR > CAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSR < CAP score to second venom). CONCLUSIONS: The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy.

Ash (Fraxinus excelsior)-pollen allergy in central Europe: specific role of pollen panallergens and the major allergen of ash pollen, Fra e 1.

BACKGROUND: The role of ash (Fraxinus excelsior) pollen as a cause of spring pollinosis in central Europe has received little attention. It is not clear whether ash pollen is a primary cause of sensitization or whether it is implicated through cross-sensitization to other pollens. METHODS: Over a 22-month period, ash pollen was included in a screening series for inhalant allergies. Pollen data were documented from 1976 through 1999. The frequency of IgE-binding to the ash-specific allergen Fra e 1 and pollen panallergens, respectively, was compared by Western blot between mono- (n = 6), oligo- (n = 16), and polysensitized (n=25) patients. RESULTS: Of 5,416 consecutive patients sensitized to any pollen, 920 (17.6%) had a positive skin prick test to ash. Total pollen counts varied extensively between years (229-5,351) as did peak concentrations (23-837 grains/m3/24 h). Western blotting revealed Fra e 1 sensitization in 100% of monosensitized, 93% of oligosensitized, but only 44% of polysensitized patients. IgE against profilins (Fra e 2), Ca-binding proteins (Fra e 3), and carbohydrate epitopes in the three groups was found in 0/0/17%, 0/19/31%, and 32/72/60%, respectively. At least 50% of sera from patients with Fra e 1 sensitization did not bind with the protein in Western blots under reducing conditions. CONCLUSIONS: Ash pollen should be considered a relevant factor and distinct entity in spring pollinosis. In all, only 20% of positive skin tests to ash appear to result from cross-sensitization to pollen panallergens.

Identification of allergens in oilseed rape (Brassica napus) pollen.

BACKGROUND: Pollen from oilseed rape (OSR), Brassica napus, an increasingly cultivated oilplant from the Brassicaceae, has been recognized as a potential cause of allergic sensitization. Allergens have been hardly investigated. METHODS: We characterized IgE binding proteins in OSR pollen by immunoblot, immunoblot inhibition and specific monoclonal antibodies using sera from 89 patients sensitized to OSR. RESULTS: Two low-molecular-weight allergens of 6/8 kD and 14 kD as well as a high molecular-weight cluster (27-69 kD) comprising six cross-reactive peptides could be identified. The three allergens were recognized by 50, 34 and 80% of patients, respectively. Immunoblot IgE binding to OSR could be totally inhibited by rye pollen and moderately by birch pollen (6/8 and 14 kD) while mugwort had little effect. An anti-profilin-specific monoclonal antibody bound specifically to a 14-kD protein in OSR. Binding to the 6/8-kD rape allergen could be effectively inhibited by rAln g 2, a calcium-binding protein from alder. Periodate treatment led to a significant reduction in IgE binding to the 27 to 69-kD OSR allergens indicating that carbohydrate determinants are involved in IgE binding. OSR proteins were capable to quench IgE binding to timothy grass pollen proteins of >/=60 kD suggesting that grass pollen group 4 allergens cross-react with the 27 to 69-kD cluster in OSR. CONCLUSIONS: The data demonstrate that OSR pollen is allergenic and indicate that the identified allergens represent cross-reacting homologues of well-known pollen allergens, i.e. calcium-binding proteins, profilins, and high-molecular-weight glycoproteins. Via cross-reactivity, exposure to OSR pollen may be a prolonging and aggravating factor in underlying birch and grass pollen allergy.

High-dose diltiazem prevents migration and proliferation of vascular smooth muscle cells in various in-vitro models of human coronary restenosis.

BACKGROUND: Restenosis after coronary angioplasty is considered to be caused mainly by increased migration and proliferation of smooth muscle cells (SMC). The concept of local, site-specific delivery of pharmacologic therapies has opened the door for new, high-dose drug regimes. METHODS AND RESULTS: SMC were isolated by enzymatic disaggregation with collagenase/elastase from human coronary plaque tissue of 29 patients (pSMC) and post mortem from the coronary media of 33 corpses (mSMC). Endothelial cells were isolated from human umbilical veins by enzymatic disaggregation with collagenase/dispase. By positive reaction with antibodies against smooth muscle alpha-actin and von Willebrand factor cells were identified as SMC or endothelial cells. In proliferation studies 5-150 micrograms/ml diltiazem was added to the culture media of pSMC, mSMC and endothelial cells. After 5 days there was a significant dose-dependent inhibition of cell proliferation (for pSMC with > 50 micrograms/ml, for mSMC with > 25 micrograms/ml, and for endothelial cells with > 5 micrograms/ml). In migration studies the effect of 5-150 micrograms/ml diltiazem on the velocity of migration of pSMC was investigated over a period of 48 h. Administration of diltiazem at concentrations of 100 and 150 micrograms/ml caused a significant inhibition of the migration of pSMC. The cytoskeletal components smooth muscle alpha-actin, vimentin, and alpha-tubulin of pSMC and the expression of von Willebrand factor of endothelial cells were investigated after an incubation period of 5 days with 50 and 150 micrograms/ml diltiazem. In the transfilter coculture model the effect of 50 micrograms/ml diltiazem on mSMC was investigated after mechanical injury of cocultured endothelial cells. Administration of diltiazem at a concentration of 50 micrograms/ml inhibited the development of a neointimal proliferate in the transfilter coculture model significantly (P < 0.001). CONCLUSIONS: A high dose of diltiazem inhibited the migratory and proliferative activities of coronary SMC significantly. In further experimental studies the effect of locally applied high doses of diltiazem on postangioplasty restenosis should be elucidated.

Shed mediastinal blood in 500 elective cardiac surgery patients: is there enough for retransfusion routinely?

OBJECTIVE: Since an increased use of several blood salvaging measures has contributed to a reduction in perioperative blood loss and the requirement for banked blood in recent years, the aim of this study was to establish current postoperative drainage losses in order to evaluate whether homologous retransfusion may be a useful measure to reduce autologous transfusion in elective cardiac surgery. DESIGN/SETTING: This prospective clinical investigation was performed at a University Intensive Care Unit during the first six hours following cardiac surgery. PATIENTS: 373 men and 127 women undergoing elective cardiac surgery were investigated. MEASURES: The amount of shed blood was measured four and six hours postoperatively. RESULTS: The average blood loss was higher in men than in women both in all operations as a whole (men, four hours: 223+/-73 ml, six hours 270+/-95 ml; women, four hours: 156+/-25 ml, six hours 195+/-22 ml), in valve replacement (men, four hours: 299+/-87 ml, six hours 350+/-101 ml; women, four hours: 187+/-30 ml, six hours: 219+/-31 ml) and in coronary artery bypass grafting (men, four hours: 197+/-69 ml, six hours: 242+/-83 ml; women, four hours: 128+/-15 ml, six hours: 173+/-18 ml). A blood loss of 400 ml was exceeded in 13% of men after valve replacement four and six hours postoperatively. In all other groups, less than 8% of patients had a loss of more than 400 ml both after four and after six hours. CONCLUSIONS: Postoperative drainage losses in elective cardiac surgery patients are small and a measurable advantage from retransfusion seems to be unlikely. We therefore endorse the routine use of shed mediastinal blood retransfusion in these patients.

Oilseed rape pollen is a potentially relevant allergen.

Oilseed rape (Brassica napus) (OSR) is a partly wind-pollinated crop which has been increasingly cultivated both in Europe and overseas. Allergological data about OSR is scarce and controversial. We evaluated the frequency of sensitization to OSR pollen by skin prick test and RAST over a period of 1 yr. Airborne OSR pollen load and the agricultural role of this crop were analysed. Furthermore, six patients were investigated by immunoblot. In 4468 patients with suspect inhalant allergy investigated between June 1994 and May 1995, routine skin prick testing revealed OSR sensitivity in 7.1% of pollen-allergic patients. In all, monovalent sensitization was detected in nine patients. Routine pollen counts showed daily maxima not exceeding 50 grains/m3/24 h, but airborne OSR pollen has continuously increased during the last decade correlating with the increasing acreage. Characterization of OSR allergens by immunoblot revealed major allergens of 6/8 kD, 12/14 kD and in the high molecular weight range at 33, 42, 51, 58/61 and 70 kD. Some OSR proteins may cross-react with birch pollen allergens. In summary, the results suggest that OSR pollen is a moderate but true source of allergy and may sensitize despite low pollen exposure.