Extracellular matrix mimicking scaffold promotes osteogenic stem cell differentiation: A new approach in osteoporosis research.

BACKGROUND: Osteoporosis is a common metabolic disease, with mesenchymal stem cells discussed to play an important role in its pathomechanism. For in vitro osteoporosis studies, selection of adequate culture conditions is mandatory so as to preserve cell properties as far as possible. A suitable cell culture surface would ideally provide reproducible experimental conditions by resembling those in-vivo. OBJECTIVE: Generating an improved growth surface for osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs). METHODS: We modified electrospun gelatine meshes with hydroxyapatite nanopowder. The potential beneficial impact of the ensuing culture conditions were evaluated by cultivating and comparing the growth of cells from osteoporotic and non-osteoporotic donors on either hydroxyapatite-gelatine (HA) meshes, pure gelatine meshes, or 2D standard tissue culture surfaces. RESULTS: After 21 days of differentiation, cells grown on pure or HA-gelatine meshes showed significantly higher mineralization levels compared to cells cultured in standard conditions. The amount of mineralization varied considerably in hBMSC cultures of individual patients but showed no significant difference between stem cells obtained from osteoporotic or non-osteoporotic donors. CONCLUSIONS: Overall, these results indicate that the use of HA-gelatine meshes as growth surfaces may serve as a valuable tool for cultivation and differentiation of mesenchymal stem cells along the osteogenic lineage, facilitating future research on osteoporosis and related issues.

Osteoanabolic effect of alendronate and zoledronate on bone marrow stromal cells (BMSCs) isolated from aged female osteoporotic patients and its implications for their mode of action in the treatment of age-related bone loss.

SUMMARY: In the present study, we evaluated the potential for aminobisphosphonates to enhance the development of bone-forming osteoblasts from progenitor cells isolated from aged female osteoporotic patients. The aminobisphosphonates tested significantly enhanced osteoblast formation and thus lend further insights into their possible mode of action in the treatment of osteoporosis. INTRODUCTION: The primary aim of this study was to evaluate the influence of aminobisphosphonates on the osteogenesis of human bone marrow stromal cells (hBMSCs) and mineralization of differentiating bone-forming cells isolated from osteoporotic patients. METHODS: The influence of aminobisphosphonate treatment on hBMSC osteogenesis was assessed by the quantitative measurement of alkaline phosphatase (ALP) activity, in addition to quantitative reverse transcription polymerase chain reaction and Western blot analysis of known osteogenic markers. Mineralized matrix formation by hBMSC-derived osteoblasts was visualized and quantified using Alizarin red staining. RESULTS: hBMSC cultures treated with osteogenic medium supplemented with zoledronate demonstrated a significant increase in Alizarin red staining after 3 weeks as compared to cells cultured in osteogenic medium alone. Similarly, cultures of differentiating hBMSCs isolated from patients receiving alendronate treatment also demonstrated an increased propensity for mineralization, even in the absence of further in vitro stimulation by zoledronate. The stimulatory effects of aminobisphosphonate treatment on hBMSC-derived osteoblast-mediated mineralization were independent of any alterations in ALP activity, although significant decreases in the expression levels of osteopontin (SPP1) were evident in hBMSCs following exposure to aminobisphosphonates. Further analysis including Western blotting and loss-of-function studies revealed osteopontin as having a negative influence on the mineralization of differentiating osteoporotic bone-forming cells. CONCLUSIONS: The results presented here demonstrate for the first time that aminobisphosphonate treatment of osteoporotic hBMSCs enhances their capacity for osteoblast formation and subsequent mineral deposition, thus supporting the concept of aminobisphosphonates as having an osteoanabolic effect in osteoporosis.

Factors affecting long-term outcomes after thromboembolectomy for acute lower limb ischemia.

AIM: The aim of this study was to analyze factors influencing outcomes of surgical management for lower limb acute ischemia. METHODS: A retrospective analysis of 490 thromboembolectomies performed in 468 patients was conducted. Perioperative and follow-up results were analyzed. Univariate and multivariate analysis of clinical variables and patients' characteristics for the risk of reocclusion, amputation and mortality at 2 years were performed. Statistical significance was defined as a P value <0.05. RESULTS: Cumulative reocclusion, amputation and mortality rates at 24 months were 22.6%, 14.3% and 42.8%, respectively. At univariate analysis, the factors associated with increased 2-year reocclusion rate were severity of clinical presentation, current smoking habit, arterial thrombosis rather than embolism, atrial fibrillation and the avoidance of completion angiography. All these factors except clinical presentation maintained significance at multivariate analysis. Factors associated with increased 2-year amputation rate at univariate analysis included severity of clinical presentation, smoke, arterial thrombosis, atrial fibrillation and valvulopathy. All these factors except clinical presentation and valvular defects maintained significance at multivariate analysis. Factors associated with increased 2-year mortality rate at univariate analysis included age >80 years, arterial thrombosis, history of peripheral arterial disease and antiplatelet drugs. The same factors, except antiplatelet treatment, were found to be significant at multivariate analysis. CONCLUSION: Surgical intervention for lower limb ischemia is associated with high 2-year mortality but offers good 2-year limb salvage. The pattern of risk factors for reocclusion and amputation rates is quite different from those affecting mortality. Only thrombotic aetiology is a significant risk factor for all the three outcomes.

Lower extremity traumatic vascular injury at a level II trauma center: an analysis of limb loss risk factors and outcomes.

AIM: The objectives were to review in our series the risk factors, management and outcomes of patients who sustained vascular injuries in the lower limbs and to determine the effect of risk factors and treatment on the outcome of the injured extremity. METHODS: Fifty-six patients submitted to surgical treatment were retrospectively reviewed. Results were analysed in terms of type of operation and reconstruction, intraoperative and 30 day complications, reconstruction occlusion, major amputation and mortality. RESULTS: The mechanism of trauma was blunt in 30.4% and penetrating in 69.6%. The overall primary amputation rate was 5.4%, the overall secondary amputation rate was 1.8%. The overall intraoperative and postoperative mortality were 1.8% and 5.4% respectively. At univariate analysis, the presence of compartment syndrome and ischemia time >6 hours were associated with a significantly higher risk of early reconstruction thrombosis (both P=0.03). It showed also that the number of patent vessels (P=0.0000) and the presence of a MESS score >7 (P=0.0000) significantly affected primary amputation, and that the occurrence of postoperative deep wound infection or sepsis (P=0.0000), of tibio-peroneal trunk injury (P=0.003) and of a MESS score >7 (P=0.004) significantly affected secondary amputation. CONCLUSION: The number of patent arteries (0-1), the presence of a MESS score >7, the incidence of tibio-peroneal trunk injury and the occurrence of postoperative deep wound infection are significant independent factors for limb loss. The presence of compartment syndrome and of ischemia time >6 hours are associated with a significantly higher risk of early reconstruction thrombosis.

Contrast media-induced nephropathy in patients undergoing angiography prior to or during vascular surgery: a systematic review.

Nephropathy induced after administration of contrast medium is an acute and severe complication that is of particular concern in vascular surgery. While patients undergoing coronary procedures have been extensively studied, there is a paucity of data on pre- and intraoperative prevention of contrast-induced nephropathy in vascular surgery patients. There is lack of a robust analysis exploring the additive effect of pre- or intraoperative administration of drugs such as N-acetylcysteine, dopamine and sodium bicarbonate in addition to adequate hydration in patients undergoing angiography prior to or after completion of vascular surgery. A systematic review of contrast-induced nephropathy after angiography in patients undergoing vascular surgery was carried out. Eligible trials were sought by multiple methods, and the pooled odds ratios for contrast-induced nephropathy were computed under a random effects model. Twenty-one publications were identified for screening and 6 studies were included for systematic review. All 6 studies investigated preoperative angiography-related contrast-induced nephropathy; one study also investigated completion angiography. The overall frequency of contrast-induced nephropathy in patients undergoing vascular surgery was 9.2% (79/862). Risk factors for contrast-induced nephropathy identified were age >70 years, high contrast volume, pre-existing renal disease and antihypertensive medication. Two studies found that administration of N-acetylcysteine prior to angiography does not provide added benefit in preventing contrast-induced nephropathy. Advanced age and pre-existing renal and vascular risk factors such as arterial hypertension expose vascular surgery patients to increased risk of contrast-induced nephropathy. Those undergoing completion angiography appear to be at even higher risk, particularly if severely azotemic. Further randomized clinical trials analyzing strategies for preventing contrast-induced nephropathy are needed.

Carotid endarterectomy: comparing anesthesia in awakened and intubated patients with general anesthesia.

AIM: The purpose of this study was to determine utility and efficacy of anesthesia with remifentanil in awakened and intubated patients in comparison with general anesthesia and routine shunting. METHODS: Hundred patients were randomized for general anesthesia (group A) or anesthesia with remifentanil (group B). The two groups were compared in terms of postoperative outcome, neurological complications, arterial pressure during intervention and in during the following 36 hours. Group B patients answered a proposed questionnaire about intraoperative and postoperative degree of comfort, fear, pain and discomfort for the orotracheal tube. RESULTS: The two groups differed significantly only for age (group B>A); 10% of patients was not considered fit for the anesthesia with remifentanil (initial experience only). The two groups did not differ in terms of central neurologic complications (0%), general complications or technical problems in terms of intra-operative arterial pressure problems (the anesthesia with remifentanil caused less hypertensive problems (P>0,19) and (not technique-dependent) peripheral neurologic deficits. All patients accepted the method and did not report fear or discomfort. Only 3 patients (6.6%) described light pain during the intervention. In 2 cases (4.4%) the surgeon complained about the restlessness of the patient; this did not cause a change in procedure. CONCLUSIONS: General anesthesia with remifentanil produces an association between the advantage of locoregional anesthesia and those of general anesthesia, probably lowering the risk for the patients.

Late endosomal traffic of the epidermal growth factor receptor ensures spatial and temporal fidelity of mitogen-activated protein kinase signaling.

Mitogen-activated protein kinase (MAPK) signaling is regulated by assembling distinct scaffold complexes at the plasma membrane and on endosomes. Thus, spatial resolution might be critical to determine signaling specificity. Therefore, we investigated whether epidermal growth factor receptor (EGFR) traffic through the endosomal system provides spatial information for MAPK signaling. To mislocalize late endosomes to the cell periphery we used the dynein subunit p50 dynamitin. The peripheral translocation of late endosomes resulted in a prolonged EGFR activation on late endosomes and a slow down in EGFR degradation. Continuous EGFR signaling from late endosomes caused sustained extracellular signal-regulated kinase and p38 signaling and resulted in hyperactivation of nuclear targets, such as Elk-1. In contrast, clustering late endosomes in the perinuclear region by expression of dominant active Rab7 delayed the entry of the EGFR into late endosomes, which caused a delay in EGFR degradation and a sustained MAPK signaling. Surprisingly, the activation of nuclear targets was reduced. Thus, we conclude that appropriate trafficking of the activated EGFR through endosomes controls the spatial and temporal regulation of MAPK signaling.

Acute limb ischemia in elderly patients: can iloprost be useful as an adjuvant to surgery? Results from the ILAILL study.

OBJECTIVES: To evaluate the effects of iloprost, in addition to surgery, on the outcome of acute lower limb ischemia (ALLI). DESIGN: Post-hoc analysis of a randomized, double-blind, placebo-controlled study. METHODS: In the context of the ILAILL (ILoprost in Acute Ischemia of Lower Limbs) study, 192 elderly patients (>70 years old) undergoing surgery for ALLI were assigned to receive perioperative iloprost (intra-arterial, intra-operative bolus of 3000 ng, plus intravenous infusion of 0.5-2.0 ng/kg/min for six hours/day for 4-7 days following surgery), or placebo (iloprost: n=100; placebo: n=92). Patients were followed-up for three-months following surgical revascularization. RESULTS: The combined incidence of death and amputation (primary study end-point) was significantly reduced in patients treated with iloprost (16.0% vs 27.2% in the placebo group; hazard ratio 1.99, 95% confidence interval 1.05-3.75, p=0.03). A statistically significant lower mortality (6.0%) was reported in patients receiving iloprost, compared to controls (15.2%) (hazard ratio 2.93, 1.11-7.71, p=0.03). The overall incidence of death and major cardiovascular events was lower in patients receiving iloprost compared to those assigned placebo (24.0% and 35.9%, respectively), at the limits of statistical significance (relative risk 1.64, 0.97-2.79, p=0.06). CONCLUSIONS: These results confirm the poor outcome in elderly patients with ALLI. Based on a subgroup analysis iloprost, as an adjuvant to surgery, appears to reduce the combined end-point of death and amputation.

Allergy to millet: another risk for atopic bird keepers.

BACKGROUND: Millet has been reported to induce not very frequent but severe anaphylactic reactions following ingestion. Seven individuals who all kept cage birds experienced allergic reactions after ingestion of millet-containing food. METHODS: We investigated the immunoglobulin E (IgE)-reactivity of these individuals to millet employing immunoblotting, RAST and skin prick tests. As the sensitization possibly occurred via the inhalant route we investigated millet-specific IgE levels of 16 additional sera from bird keepers with proven atopy, in retrospect. RESULTS: All patients who had experienced reactions after ingestion of millet displayed millet-specific IgE. Sixty-three percent of the atopic bird keepers possessed millet-specific IgE. By means of immunoblotting three major allergens in millet extract were detected. CONCLUSIONS: Our results indicate that millet plays an important role as inhalant allergen for atopic bird keepers. A sensitization to millet may subsequently also elicit food allergy.

Incidence and costs of osteoporosis-associated hip fractures in Austria.

This study is an investigation into the epidemiologic and socioeconomic impact of osteoporosis-associated hip fractures in Austria. We determined age- and gender-specific incidence rates of osteoporosis-associated hip fractures for all patients treated in hospitals in 1995 and calculated mortality rates, hospitalization days and direct costs of hospitalization. The data were obtained from the hospital discharge statistics for all general hospitals and for all hospitals of the General Austrian Accident Insurance. To calculate the portion of hip fractures attributable to osteoporosis in a given age-group, a basic, non-osteoporotic incidence of hip fractures was determined for ages 20-39, using gender-specific regression models. 11,379 patients with osteoporotic hip fractures underwent treatment in Austrian hospitals in 1995, accounting for 79 percent of all hip fracture patients treated. 82 percent of those were female, with the highest incidence among women aged 95 years and older with a rate of 3,000/100,000. For male patients the highest incidence was observed for the age-group of 90-94 years with 1,743/100,000. International comparisons indicate these incidence rates to be similar to those reported for the Swiss population. In 1995, 778 patients or 6.8 percent of all patients with osteoporotic hip fractures died during hospitalization. Hospital care of patients with osteoporotic hip fractures required an overall 250,268 bed-days with an age-group-specific length of stay between 8.5-27 days for female and 16-23 days for male patients. The total cost of hospital treatment of osteoporotic hip fractures in Austria was ATS 1,043,379,000 (US$ 103,509,800), with average costs per patient of ATS 91,700 (US$ 9,097). Due to the aging of the population in the years to come, an increase of osteoporotic hip fractures among individuals aged 50 years and older must be expected. The economic importance of this development and its impact on the health care system must be considered as significant.

Systemic immunological changes induced by administration of grass pollen allergens via the oral mucosa during sublingual immunotherapy.

Twenty-four patients suffering from grass pollen allergy underwent sublingual immunotherapy (SLIT) with standardized grass pollen extract for 1 year. In order to investigate immunological changes induced by the administration of allergens via the oral mucosa, the SLIT-spit method was applied. The cumulative dose of approximately 80 microg of major allergen (grass group 5 allergen), was relatively low. During the time of treatment, we could observe a significant increase in the levels of specific IgG and IgG4 antibodies. However, the titers of allergen-specific IgE antibodies showed a significant increase in the course of SLIT as well. Analyzing lymphoproliferative responses, a significant decrease in reactivity in response to stimulation with complete grass pollen extract (p = 0. 001) and to recombinant Phl p 1 (a major allergen of timothy grass, p<0.001) could be observed, indicating the induction of immunological tolerance. Proliferative responses to a control antigen (tetanus toxoid) were not influenced by the treatment. At different time points during SLIT, allergen (Phl p 1)-specific T cell clones (TCC) were established from the peripheral blood of the patients. Cytokine production by allergen-stimulated T cells did not reveal any changes consistent with immune deviation, i.e. the ratio of Th1/Th2 TCC did not change during SLIT. In conclusion, we provide evidence that sublingual treatment leads to systemic changes in immunoreactivity to the administered allergen.

Serological characterization of allergens in poppy seeds.

BACKGROUND AND OBJECTIVE: Poppy seeds in food can induce immediate-type allergic reactions ranging from mild local symptoms to severe anaphylactic reactions. Previous publications showed that poppy seeds cross-react with other plant-derived allergens. The IgE-binding components have not been defined so far. METHODS: We analysed sera from 11 patients with adverse reactions after ingestion of poppy seed-containing food by IgE-immunoblotting. Nine of 11 patients showed concomitant IgE binding to allergens of birch, mugwort or grass pollen in RAST-CAP, and suffered from characteristic seasonal symptoms. RESULTS: Ten of 11 patients showed IgE binding to a 45-kDa protein, 4/11 to a 34-kDa, 5/11 to a 17-kDa, 5/11 to a 14-kDa, and 3/11 to a 5-kDa component. Furthermore, individual IgE binding to proteins of 20, 25, 30 and 40 kDa proteins could be observed. Periodate treatment of blots markedly reduced the IgE binding capacity of the 40- and 45-kDa compounds, indicating the existence of IgE epitopes of the carbohydrate type. Inhibition studies indicated the presence of homologues of pollen allergens in extracts from poppy seeds, i.e. Bet v 1 and Bet v 2. CONCLUSION: The serological analysis showed IgE binding to protein and sugar components of poppy seeds. The 40- and 45-kDa allergens are glycoproteins and contain IgE binding carbohydrate moieties. Moreover, cross-reacting homologues of pollen allergens including Bet v 1 and profilin were detected in poppy seed extract.

Clinical value of cervical mediastinoscopy in the staging of bronchial carcinoma.

Whether mediastinoscopy is indicated in the preoperative staging of bronchogenic carcinoma is still a controversial issue. It may be performed routinely (to exclude locally inoperable patients from surgery), selectively, or it may be regarded as superfluous (in centers which prefer an extended lympho adenectomy at the time of thoracotomy). We regard mediastinoscopy as indicated for the following purposes: 1) staging of NSCLC and SCLC; 2) diagnostic (mediastinal masses or lung tumors without previous histology); 3) restaging after primary chemotherapy; 4) assessment of prognosis in patients with borderline operability. The indication for 224 mediastinoscopies performed at our institution in the period from September 1991 through March 1999 was mainly for staging (59.2%) or diagnostic (30.6%). Eight (5.4%) patients underwent mediastinoscopy for the assessment of operability, and 7 (4.8%) after primary chemotherapy for the restaging of loco-regionally advanced lung cancer. Sensitivity and specificity rates were 87% and 100%, respectively, with an accuracy of 93% for the mediastinoscopy performed for the staging of lung cancer at all stages. If we consider the N2 tumors (42 cases) alone, the sensitivity was 76.7% and the specificity 100%, with an accuracy of 83.3%. Overall positive and negative predictive value resulted 100% and 87%, respectively, according to the data reported in literature. Our data confirm the role of mediastinoscopy as the gold standard for regional staging of lung cancer.

Allergens in pepper and paprika. Immunologic investigation of the celery-birch-mugwort-spice syndrome.

Mugwort and birch pollen allergy are frequently associated with IgE-mediated hypersensitivity to celery and spices. We analyzed 22 sera from patients with the mugwort-birch-celery-spice syndrome for IgE binding to the spices pepper and paprika by immunoblotting. Immunoblot results revealed two major allergens of 28 and 60 kDa in pepper and a 23-kDa allergen together with allergens of higher molecular weight in paprika. In immunoblot-inhibition studies, crude mugwort, birch pollen, and celery extracts significantly reduced the IgE binding to pepper and paprika allergens. However, no inhibition was achieved with rBet v 1 and rBet v 2, suggesting that no homologs of these birch proteins act as allergens in pepper or paprika extracts. N-terminal sequence analysis of the 14- and 28-kDa pepper and 23-kDa paprika allergens revealed no homology to known allergens. The 28-kDa pepper allergen showed homology to a wheat germin protein, and the 23-kDa paprika allergen was identified as a homolog of a osmotin-like or pathogenesis-related protein in tomato. Therefore, we conclude that the IgE cross-reactivity in the mugwort-birch-celery-spice syndrome to the spices pepper and paprika is not caused by homologs of Bet v 1 and profilin. N-terminal amino acid sequence analysis of the main allergens in pepper and paprika indicate a relation to frequently occurring plant proteins.

Food allergy to pumpkinseed--characterization of allergens.

In recent years, pumpkinseed has become increasingly popular as a foodstuff. Here we report the occurrence of allergic reactions (itching and swelling of oral mucosa, and asthma) to this member of the Cucurbitaceae family. We investigated three patients suffering from symptoms after ingestion of roasted pumpkinseed. All the patients fished for sport and used pressed pumpkinseed flour as bait. Sera were tested by the immunoblot technique for IgE reactivity with proteins of pumpkinseed extract. The immunoblot revealed pumpkinseed allergens of 13, 14, 36, 48, 77, and 87 kDa. Inhibition experiments with recombinant birch profilin were performed: IgE binding to the 14-kDa allergen was completely blocked by preincubation of the sera with recombinant birch profilin. In conclusion, type I allergy to pumpkinseed is rare, and the patients' histories suggest inhalation of pumpkinseed flour during fishing to be the relevant route of sensitization, leading to food allergy to pumpkinseed.

Characterization of allergens from deer: cross-reactivity with allergens from cow dander.

BACKGROUND: Animal hair/dander proteins frequently cause Type I hypersensitivities. Species-specific and broadly cross-reacting allergens have been characterized in the past. METHODS: Sera from eight individuals suffering from symptoms due to exposure to deer and deer-derived products were investigated by immunoblotting. Extracts from deer, dog, cat, horse, rabbit and cow, respectively, were tested for IgE-binding. To reveal cross-reactivities patients' sera were preadsorbed with these extracts prior to testing with deer extract. RESULTS: Deer allergens with the molecular mass of 22 and 25 kD (major allergens), as well as 60 kD were identified. The 22 and 25 kD allergens are cross-reactive with the corresponding cow allergens. CONCLUSION: Deer allergy is a rare sensitization mainly affecting persons exposed to deer, who displayed an atopic disposition. From our results it can be assumed that this hypersensitivity is partly associated with allergy to cow dander.

Immunological changes during specific immunotherapy of grass pollen allergy: reduced lymphoproliferative responses to allergen and shift from TH2 to TH1 in T-cell clones specific for Phl p 1, a major grass pollen allergen.

BACKGROUND AND OBJECTIVE: The mechanisms operative in specific immunotherapy (SIT) of Type I allergy are not completely understood. In the present study we evaluated immunological changes during SIT in pollinosis. METHOD: Eight patients suffering from pollinosis (monosensitized to grass pollen) were treated with conventional SIT. All subjects had IgE specific for Phl p 1, a major allergen of timothy grass. In vitro changes in the immunological reactivity to grass pollen extract and to recombinant Phl p 1 were evaluated. Subjects were examined at three occasions: before, after 3 months and after 1 year of SIT. RESULTS: Serological analysis revealed a marked increase of grass pollen- and Phl p 1-specific IgG, titres of specific IgE did not change significantly. Lymphoproliferative responses to grass pollen extract and rPhl p 1 were reduced already after 3 months of treatment. Accordingly, the cloning efficiency for Phl p 1-specific T-cell clones (TCC) dropped markedly in all patients. The majority of allergen-specific TCC raised before SIT revealed a TH2-like pattern of cytokine production, TCC established after SIT revealed TH1 characteristics. This shift was due to a decrease in IL-4 rather than an increase in IFN-production by T cells. Investigations of the epitopes recognized by T cells before and after SIT did not reveal the outgrowth of new ('protecting') specificities. We could not observe induction of allergen-specific CD8+ lymphocytes (supressor cells). CONCLUSION: Our data indicate that -- on the level of TH lymphocytes -- SIT induces tolerance to the allergen and a modulation of the cytokine pattern produced in response to allergen stimulation.

Food allergy to honey: pollen or bee products? Characterization of allergenic proteins in honey by means of immunoblotting.

OBJECTIVE: To characterize the allergenic components of honey, 23 patients allergic to honey were investigated. All displayed allergic symptoms after ingestion of honey or honey-containing products, ranging from itching in the oral mucosa to severe systemic symptoms to anaphylactic shock. METHODS AND RESULTS: Immunoblot analyses of the patients' sera revealed IgE binding to proteins at a molecular mass of 54 kd, 60 kd, 72 kd, or to a 30 kd/33 kd double band, or to both in sunflower honey extracts. The three bands corresponding to higher molecular mass proteins could also be detected in the three other kinds of honey (locust tree, European chestnut and forest honey) that were tested and represented bee products because IgE binding to these proteins was inhibited by extracts of honeybee heads and extracts of isolated bee venom sacs. The 30 kd/33 kd bands could be identified as sunflower honey-specific. When testing sera from patients allergic to bee venom with honey extracts, in seven of 10 cases IgE binding to bee-specific components could be observed. CONCLUSION: Both proteins derived from secretions of pharyngeal and salivary glands of honeybee heads and pollen proteins contained in the honey cause allergic reactions to honey.