The adhesion molecule ICAM-1 is overexpressed in patients with Hymenoptera venom allergy and decreases after ultrarush venom immunotherapy

Adhesion molecules, including ICAM-1, are an important factor in allergic inflammation caused by inhalant allergens, but there are no studies investigating their possible role in Hymenoptera venom allergy (HVA). We measured the level of ICAM-1 in 13 venom-allergic patients before and after ultra-rush venom immunotherapy (VIT). Eight patients were treated by yellow jacket venom and 5 were treated by honeybee venom. Serum ICAM-1 levels were assayed by an immunoenzymatic method, with a detection limit of 0.35 ng/ml. The mean level of ICAM-1 changed from 316.4±78.2 ng/ml before VIT to 294.7±77.9 after VIT. This difference was statistically significant (p = 0.019). These findings show that in patients with HVA there is an over-expression of ICAM-1, and that ultra-rush VIT significantly decreases ICAM-1 levels. It is likely that the known ability of VIT to correct the imbalance in T lymphocytes subpopulations and in the associated production of cytokines may account for this observation. In fact, such cytokines include IL-4 and TNF-alpha, that up-regulate adhesion molecules.     

Allergy to insect stings. IV. Diagnosis by radioallergosorbent test (R.A.S.T.)

Radioallergosorbent tests (RAST(s)) have been developed and assessed for the diagnosis of insect hypersensitivity by using a purified allergen from honeybee venom, phospholipase A, and crude yellow jacket venom. Sera from 193 patients positive both by history and skin test to one of these insects were compared with various groups of control sera. Eighty percent of sera from skin test-positive patients were RAST positive; positive RAST were found in 16% of sera tested from skin test-negative patients. A highly positive RAST correlates well with a positive skin test and clinical sensitivity, but serum IgE is not measurable in many patients with mast cell or basophil bound antibody. Since biologically important reactions of antigen with IgE require that the antibody be cell bound, skin testing would be preferred to RAST if one were limited to a single test for the diagnosis of insect allergy.     

Evaluation of different glycoforms of honeybee venom major allergen phospholipase A2 (Api m 1) produced in insect cells

Allergic reactions to hymenoptera stings are one of the major reasons for IgE-mediated anaphylaxis. However, proper diagnosis using venom extracts is severely affected by molecular cross-reactivity. In this study recombinant honeybee venom major allergen phospholipase A2 (Api m 1) was produced for the first time in insect cells. Using baculovirus infection of different insect cell lines allergen versions providing a varying degree of cross-reactive carbohydrate determinants as well as a non glycosylated variant could be obtained as secreted soluble proteins in high yields. The resulting molecules were analyzed for their glycosylation and proved to show advantageous properties regarding cross-reactivity in sIgE-based assays. Additionally, in contrast to the enzymatically active native protein the inactivated allergen did not induce IgE-independent effector cell activation. Thus, insect cell-derived recombinant Api m 1 with defined CCD phenotypes might provide further insights into hymenoptera venom IgE reactivities and contribute to an improved diagnosis of hymenoptera venom allergy.     

Inconsistent results of diagnostic tools hamper the differentiation between bee and vespid venom allergy

Background

Double sensitization (DS) to bee and vespid venom is frequently observed in the diagnosis of hymenoptera venom allergy, but clinically relevant DS is rare. Therefore it is sophisticated to choose the relevant venom for specific immunotherapy and overtreatment with both venoms may occur. We aimed to compare currently available routine diagnostic tests as well as experimental tests to identify the most accurate diagnostic tool.

Methods

117 patients with a history of a bee or vespid allergy were included in the study. Initially, IgE determination by the ImmunoCAP, by the Immulite, and by the ADVIA Centaur, as well as the intradermal test (IDT) and the basophil activation test (BAT) were performed. In 72 CAP double positive patients, individual IgE patterns were determined by western blot inhibition and component resolved diagnosis (CRD) with rApi m 1, nVes v 1, and nVes v 5.

Results

Among 117 patients, DS was observed in 63.7% by the Immulite, in 61.5% by the CAP, in 47.9% by the IDT, in 20.5% by the ADVIA, and in 17.1% by the BAT. In CAP double positive patients, western blot inhibition revealed CCD-based DS in 50.8%, and the CRD showed 41.7% of patients with true DS. Generally, agreement between the tests was only fair and inconsistent results were common.

Conclusion

BAT, CRD, and ADVIA showed a low rate of DS. However, the rate of DS is higher than expected by personal history, indicating that the matter of clinical relevance is still not solved even by novel tests. Furthermore, the lack of agreement between these tests makes it difficult to distinguish between bee and vespid venom allergy. At present, no routinely employed test can be regarded as gold standard to find the clinically relevant sensitization.     

Dose-dependence of protection from systemic reactions to venom immunotherapy by omalizumab

Background

Systemic reactions (SR) to venom immunotherapy (VIT) are rare but may occur, with a rate significantly higher for honeybee than for vespid VIT. In patients with repeated SRs to VIT it is difficult to reach the maintenance dose of venom and pre-treatment with omalizumab is indicated, as shown by some studies reporting its preventative capacity, when antihistamines and corticosteroids are ineffective.

Case presentation

We present the case of a 47 years old woman allergic to bee venom who experienced two severe SRs after bee stings and several SRs to VIT with bee venom. Pre-treatment with antihistamines and corticosteroids as well as omalizumab at doses up to 300 mg was unsuccessful, while an omalizumab dose of 450 mg finally achieved in our patient the protection from SRs to VIT with 200 mcg of bee venom.

Conclusions

The search of the dose of omalizumab able to protect a patient with repeated SRs to VIT may be demanding, but this search is warranted by the need to provide to this kind of patient, by an adequate VIT, the protection from potentially life-threatening reactions.

     

Preventive actions of allergen immunotherapy: the facts and the effects in search of evidence

Allergen immunotherapy (AIT) is the only treatment that works on the causes of allergy. Available AIT nowadays are subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) for allergic rhinitis and asthma, while for allergy to Hymenoptera venom only subcutaneous route is recommended. A bulk of trials and meta-analyses demonstrated that efficacy and safety of AIT in decreasing allergic clinical symptoms and use of rescue medications, while its preventive capacity is yet under investigation. The most important of these effects is the prevention of potentially fatal anaphylactic reactions to Hymenoptera stings by venom immunotherapy (VIT). A certain number of studies thus far available showed that AIT, in both forms, is able to prevent the progress of allergic rhinitis into asthma and the development of new sensitizations. These effects should be related to the mechanisms of action of AIT. In fact, it has been demonstrated that both SCIT and SLIT are able to modify the allergen presentation by dendritic cells, with result in modification of the phenotype of allergen-specific T cells, switching from the typical of allergic inflammation Th2-type response to a Th1-type one. Also allergen-specific T regulatory (Treg) cells play a pivotal role by producing suppressive cytokines, such as IL-10 and TGF-beta. However, the only plain evidence of a preventive effect concerns VIT, while the other outcomes need to be furtherly investigated.     

Radioallergosorbent testing for penicillin allergy in family practice.

OBJECTIVES: To determine (a) the prevalence of patients supposedly allergic to penicillin who have a positive radioallergosorbent test (RAST) result for penicillin G or V and (b) the predictive power of family physicians'' clinical judgement that a patient who is supposedly allergic to penicillin will have a positive RAST result. DESIGN: Prospective multicentre cross-sectional observational study. SETTING: Eleven primary care practices in Newfoundland; 10 were in a rural setting. PATIENTS: Of 110 consecutive adult patients with a supposed allergy to penicillin 97 agreed to participate in the study; 92 underwent RAST. INTERVENTIONS: Patients helped physicians complete a questionnaire and had a venous blood sample taken for the RAST. Physicians examined the clinical history and judged whether the patient was likely to have a positive RAST result. MEAN OUTCOME MEASURES: Rates of positive and negative RAST results for penicillin V and G. RESULTS: Of the 92 patients 8 had a positive RAST result and 84 a negative one. The positive predictive power of a "good" clinical history (e.g., urticaria, swollen eyes, tongue or lips, or an anaphylactic reaction witnessed by a physician) was low (10%); the negative predictive power of a "poor" clinical history (e.g., nausea, vomiting, diarrhea, fever, nonspecific rash or fainting) was 92%. CONCLUSIONS: Less than 10% of primary care patients with a supposed allergy to penicillin will have a positive RAST result. In addition, physicians'' predictions of allergy in such patients are imprecise.     

Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris

Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification.Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST). Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils.The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy.     

Focus: Allergic Diseases and Type II Immunity: Development of New White Fish Allergy after Bone Marrow Transplantation from a Non-atopic Donor

Background: Transplant-acquired food allergy has become increasingly recognized in solid organ and bone marrow transplantation. As food allergy has no cure and causes considerable impact on the lives of patients who require strict avoidance of foods to avoid potentially severe or fatal reactions, it is crucial for physicians to better understand the risk factors and mechanisms driving development of food allergy post-transplant. We report a case of new food allergy to whitefish in an elderly patient post-bone marrow transplant in which neither donor nor recipient had a history of atopy. Methods: A 70-year-old man experienced an anaphylactic reaction to Swai whitefish (Pangasius hypophthalmus) 6 months post-transplant that he had previously tolerated on multiple occasions both pre-transplant and in the preceding months post-transplant. This allergy was investigated by commercial serum specific IgE testing and fresh prick-to-prick skin test to Swai whitefish. Results: Fresh prick-to-prick demonstrated large positive reaction to the Swai whitefish with wheal of 10 mm and flare of 22 mm compared to positive histamine control with a wheal/flare of 5x8mm. Serum specific IgE testing to commercial whitefish was negative (specific IgE <0.10kU/L). The patient continues to strictly avoid Swai whitefish but tolerates all other fish and shellfish. Conclusions: The unique development of specific Swai whitefish allergy in an elderly man after bone marrow transplant where both donor and recipient had no prior history of atopy strongly supports transplant-related immunomodulation as a major mechanism for transplant-acquired allergy and suggests that that absence of atopy or advanced age may not necessarily be protective.     

Induction of Allergic Responses to Peanut Allergen in Sheep

Peanut allergy is the leading cause of deaths due to food-induced anaphylaxis but despite continued research, there are currently no specific treatments available. Challenge testing is limited in patients due to the high risk of adverse reactions, emphasising the need for an appropriate animal model. In the present study we examine the induction of allergic responses in a sheep model for peanut allergy. Sheep were sensitised with peanut (PN) extract and in separate injections with ovalbumin (OVA) or house dust mite (HDM) extract. Serum PN-specific IgE responses were detected in 40–50% of immunised sheep, while only 10% (1 of 10 sheep) showed detectable OVA-specific IgE. All PN-allergic sheep tested showed an Ara h 1-specific IgE response, while four out of five allergic sheep showed an Ara h 2-specific IgE response. Animals with high serum IgE levels to HDM were also PN IgE-positive. Of the PN-sensitised animals with high PN-specific IgE, 80% also showed an immediate hypersensitivity reaction following an intradermal PN injection. This new large animal model of peanut allergy may provide a useful tool for future investigations of allergen-associated immune mechanisms and specific immunotherapy.     

Skin testing versus radioallergosorbent testing for indoor allergens

BACKGROUND: Skin testing (ST) is the most common screening method for allergy evaluation. Measurement of serum specific IgE is also commonly used, but less so by allergists than by other practitioners. The sensitivity and specificity of these testing methods may vary by type of causative allergen and type of allergic manifestation. We compared ST reactivity with serum specific IgE antibodies to common indoor allergens in patients with respiratory allergies. METHODS: 118 patients (3 mo-58 yr, mean 12 yr) with allergic rhinitis and/or bronchial asthma had percutaneous skin testing (PST) supplemented by intradermal testing (ID) with those allergens suspected by history but showed negative PST. The sera were tested blindly for specific IgE antibodies by the radioallergosorbent test (Phadebas RAST). The allergens were D. farinae (118), cockroach (60), cat epithelium (90), and dog epidermal (90). Test results were scored 0-4; ST >/= 2 + and RAST >/= 1 + were considered positive. RESULTS: The two tests were in agreement (i.e., either both positive or both negative) in 52.2% (dog epidermal) to 62.2% (cat epithelium). When RAST was positive, ST was positive in 80% (dog epidermal) to 100% (cockroach mix). When ST was positive, RAST was positive in 16.3% (dog epidermal) to 50.0% (D. farinae). When RAST was negative, ST was positive in 48.5% (cat epithelium) to 69.6% (D. farinae). When ST was negative, RAST was positive in 0% (cockroach) to 5.6% (cat epithelium). The scores of ST and RAST showed weak to moderate correlation (r = 0.24 to 0.54). Regardless of history of symptoms on exposure, ST was superior to RAST in detecting sensitization to cat epithelium and dog epidermal. CONCLUSION: For all four indoor allergens tested, ST was more sensitive than RAST. When both tests were positive, their scores showed poor correlation. Sensitizations to cat epithelium and dog epidermal are common, even in subjects who claimed no direct exposure.     

Effects of CD80 and CD86 on cytokine production in patients with wasp-venom allergy who receive venom immunotherapy

Several studies have provided evidence that activation of antigen-specific T cells requires interactions between CD28 on T cells and its ligands, CD80 and CD86, on antigen-presenting cells (APCs). However, the effects of CD80 and CD86 on cytokine production in patients with Hymenoptera venom allergy who receive venom immunotherapy remain unclear. We examined the effects of CD80 and CD86 on Th1- and Th2-cytokine production before and after venom immunotherapy in patients with wasp-venom allergy. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with wasp-venom allergy before and after three months of venom immunotherapy. CD4+ T cells and monocytes were isolated as APCs from PBMCs and were cocultured with wasp venom in the presence of anti-CD80 or -CD86 blocking antibodies. Interleukin (IL)-4, IL-10, and interferon (IFN)-gamma were measured by enzyme-linked immunosorbent assay. The expression of CD80 and CD86 on CD14+ PBMCs was detected by fluorescence-activated cell-sorter analysis. The expression of CD86, but not that of CD80, on CD14+ PBMCs cocultured with venom increased after three months of venom immunotherapy, but not before venom immunotherapy. Blockade of CD86 reduced IL-10 production after three months of venom immunotherapy. IL-10 production promoted by CD86 costimulation may be involved in the mechanism of venom immunotherapy in patients with venom allergy.     

Dydrogesterone in threatened abortion: pregnancy outcome

OBJECTIVE: To determine whether therapy with dydrogesterone in threatened abortion during the first trimester of pregnancy will improve pregnancy outcome. DESIGN: Prospective open study. SUBJECTS: Pregnant women presenting to the obstetric and gynaecology clinic admitting center with vaginal bleeding before 13 weeks gestation were evaluated for entry into the study. Women were excluded if they had a history of recurrent miscarriage. METHOD: Eligible subjects were randomized to receive either dydrogesterone 40 mg stat dose followed by 10 mg twice a day for one week or conservative therapy. RESULTS: One hundred and 54 women were recruited. There was no statistically significant differences between the two groups with regard to pre-treatment status. The continuing pregnancy success rate was significantly (p=0.037) higher in women treated with dydrogesterone (95.9%) compared with women who received conservative treatment (86.3%). The odds ratio of the success rate between dydrogesterone treatment and non-treatment was 3.773 (95% confidence interval: 1.009-14.108). CONCLUSION: Corpus luteal support with dydrogesterone has been shown to reduce the incidence of pregnancy loss in threatened abortion during the first trimester in women without a history of recurrent abortion.     

Vitellogenins Are New High Molecular Weight Components and Allergens (Api m 12 and Ves v 6) of Apis mellifera and Vespula vulgaris Venom

Background/Objectives

Anaphylaxis due to hymenoptera stings is one of the most severe clinical outcomes of IgE-mediated hypersensitivity reactions. Although allergic reactions to hymenoptera stings are often considered as a general model for the underlying principles of allergic disease, venom immunotherapy is still hampered by severe systemic side effects and incomplete protection. The identification and detailed characterization of all allergens of hymenoptera venoms might result in an improvement in this field and promote the detailed understanding of the allergological mechanism. Our aim was the identification and detailed immunochemical and allergological characterization of the low abundant IgE-reactive 200 kDa proteins of Apis mellifera and Vespula vulgaris venom.

Methods/Principal Findings

Tandem mass spectrometry-based sequencing of a 200 kDa venom protein yielded peptides that could be assigned to honeybee vitellogenin. The coding regions of the honeybee protein as well as of the homologue from yellow jacket venom were cloned from venom gland cDNA. The newly identified 200 kDa proteins share a sequence identity on protein level of 40% and belong to the family of vitellogenins, present in all oviparous animals, and are the first vitellogenins identified as components of venom. Both vitellogenins could be recombinantly produced as soluble proteins in insect cells and assessed for their specific IgE reactivity. The particular vitellogenins were recognized by approximately 40% of sera of venom-allergic patients even in the absence of cross-reactive carbohydrate determinants.

Conclusion

With the vitellogenins of Apis mellifera and Vespula vulgaris venom a new homologous pair of venom allergens was identified and becomes available for future applications. Due to their allergenic properties the honeybee and the yellow jacket venom vitellogenin were designated as allergens Api m 12 and Ves v 6, respectively.     

Radioimmunologic tests in children with pruritus and with a combination of pruritus and allergic respiratory disease

Radioimmunological tests were carried out in 180 children with pruritus and 167 children with pruritus and allergic respiratory diseases. Difference between these groups was statistically insignificant in case of food allergy. Results of RAST were more often positive in case of respiratory allergy in the patients with pruritus and coexisting allergic respiratory diseases than in patients with skin involvement only. Reaction was more intense and allergy polyvalence measured with RAST was higher. All differences were statistically significant (p less than 0.001 or 0.01). Mean IgE levels was higher in children of all age groups in case of pruritus coexisting with allergic respiratory diseases.     

Influence of Specific Immunotherapy on the Activity of Human T Lymphocyte Kv1.3 Voltage-Gated Potassium Channels in Insect Venom Allergic Patients

Kv1.3 channels play an important role in T lymphocytes function. CD4⁺ and CD4⁺CD25⁺ T cells are two broad categories of T cells that are critically involved in the immunoresponse to allergens and that are also a major target for allergen immunotherapy. The aim of the study was to evaluate the effects of venom immunotherapy (VIT) on the activity of Kv1.3. channels on noncultured subsets: CD4⁺ and CD4⁺CD25⁺ T cells of insect venom allergic patients. Eleven patients with allergic reactions to bee or wasp venoms participated in the study. The patients were provided VIT according to the ultrarush protocol. CD4⁺ and CD4⁺CD25⁺ T cells were isolated from peripheral blood mononuclear cells of VIT-treated patients by an immunomagnetic method. We used the whole-cell patch clamp technique to investigate the whole potassium chord conductance (gK) of Kv1.3. channels in CD4⁺ and CD4⁺CD25⁺ T cells of venom-sensitive patients before and during the course of VIT. The conductance of Kv1.3. channels on CD4⁺CD25⁺ T cells decreased during the course of VIT. On day 0 it was 0.054 ± 0.07 [nS], and on day 70 it was 0.008 ± 0.09 [nS] (P = 0.03). The observed decrease of the gK of the Kv1.3 channels in the subpopulation of activated T cells may contribute to T cell tolerance and functional unresponsiveness of these cells to allergen in the early stages of VIT.     

Does suppressing luteinising hormone secretion reduce the miscarriage rate? Results of a randomised controlled trial.

OBJECTIVE--To determine whether prepregnancy pituitary suppression of luteinising hormone secretion with a luteinising hormone releasing hormone analogue improves the outcome of pregnancy in ovulatory women with a history of recurrent miscarriage, polycystic ovaries, and hypersecretion of luteinising hormone. DESIGN--Randomised controlled trial. SETTING--Specialist recurrent miscarriage clinic. SUBJECTS--106 women with a history of three or more consecutive first trimester miscarriages, polycystic ovaries, and hypersecretion of luteinising hormone. INTERVENTIONS--Women were randomised before conception to receive pituitary suppression with a luteinising hormone releasing hormone analogue followed by low dose ovulation induction and luteal phase progesterone (group 1) or were allowed to ovulate spontaneously and then given luteal phase progesterone alone or luteal phase placebo alone (group 2). No drugs were prescribed in pregnancy. MAIN OUTCOME MEASURES--Conception and live birth rates over six cycles. RESULTS--Conception rates in the pituitary suppression and luteal phase support groups were 80% (40/50 women) and 82% (46/56) respectively (NS). Live birth rates were 65% (26/40) and 76% (35/46) respectively (NS). In the luteal phase support group there was no difference in the outcome of pregnancy between women given progesterone and those given placebo pessaries. Live birth rates from an intention to treat analysis were 52% (26/50 pregnancies) in the group given pituitary suppression and 63% (35/56) in the controls (NS). CONCLUSIONS--Prepregnancy suppression of high luteinising hormone concentrations in ovulatory women with recurrent miscarriage and hypersecretion of luteinising hormone does not improve the outcome of pregnancy. The outcome of pregnancy without pituitary suppression is excellent.     

Kinetics of plasma cytokine levels in children hyposensitized with wasp venom

Specific immunotherapy shifts immune responses towards a Th0/Th1 response. Production of chemokines is also decreased early after the initiation of rush venom immunotherapy. We aimed to investigate in vivo whether cytokine plasma levels reflect the shift towards a Th0-Th1 pattern of immune response as seen in vitro in lymphocytes from patients undergoing venom immunotherapy. Therefore, we studied plasma levels of various cytokines before (day 1), during (day 2), and after (day 4) rush immunotherapy in nine wasp-allergic children. The levels of interleukin-5 and IFN-gamma were below the detection threshold. No variations were observed in levels of interleukin-4, interleukin-10, and TNF-alpha. In contrast, mean levels of RANTES transiently increased at day 2, and decreased below the pretreatment levels at day 4. Those alterations were significant in five children with high levels of RANTES at day 1, and reporting severe anaphylaxis. In the four other children, the levels of RANTES were not significantly increased at day 4. These results suggest that RANTES plasma levels are positively correlated with the severity of the reaction to hymenoptera venom, and that a significant decrease in the levels of RANTES occurs only in children with high pretreatment levels. However, the kinetics of RANTES plasma levels correlated neither with the results of allergological tests, nor with the outcome of venom immunotherapy.