Specific Immunotherapy in Hymenoptera Venom Allergy and Concomitant Malignancy: A Retrospective Follow-up Focusing on Effectiveness and Safety.

BACKGROUND: Malignancies are often considered a contraindication for allergen-specific immunotherapy. Consequently, patients with severe Hymenoptera venom allergy and cancer require specific care. The aim of this retrospective study was to assess patients with Hymenoptera venom allergy and cancer undergoing venom immunotherapy (VIT). METHODS: The study population comprised all patients referred for evaluation of Hymenoptera venom allergy or for a routine check-up during VIT from January 1, 2004 to December 31, 2008. RESULTS: Of the patients assessed, 2% (51 of 2594) had a documented Hymenoptera venom allergy and cancer (25 female, 26 male; mean age 58 years). Of these, 42 patients received VIT (82%): 25 patients had a previously diagnosed malignancy, 16 were diagnosed with malignancy during VIT, and 1 patient was diagnosed with cancer after completion of VIT. The most frequent type of tumor was breast cancer in female patients (60%) and prostate cancer in male patients (39%). Systemic allergic reactions during VIT were recorded in 7% of patients. A total of 19 patients experienced a field sting or underwent a sting challenge test during VIT: 95% tolerated the sting well. VIT was halted definitively in 9 patients (new diagnosis of cancer in 7 patients, reactivation of cancer in 1, and progressive polyneuropathy in 1). CONCLUSIONS: The effectiveness and adverse effects of VIT in patients with Hymenoptera venom allergy and cancer in remission are comparable to those of patients without malignancy. Our findings show that patients with Hymenoptera venom allergy and cancer are eligible for VIT.

Influence of total and specific IgE, serum tryptase, and age on severity of allergic reactions to Hymenoptera stings.

BACKGROUND: The aim of this study was to analyze the influence of total serum IgE and other potential risk factors on severity of systemic allergic Hymenoptera sting reactions. METHODS: In a retrospective analysis of one thousand and two patients referred for insect allergy over 5 years, 865 reported systemic allergic sting reactions, most often by honey bees and wasps. In 758, total IgE, venom-specific IgE, and baseline tryptase levels were available and analyzed together with atopy state, age, and sex in relation to severity of sting reactions according to H. L. Mueller. RESULTS: In a binary logistic regression model considering, besides IgE, also other risk factors for severity, an influence of total and specific IgE on severity of systemic allergic sting reactions could not be shown, while high severity of systemic allergic sting reactions was significantly more often reported in patients with a baseline tryptase of ≥11.4 µg/l (P < 0.0001) and higher age (P = 0.026). In a bivariate analysis, however, in patients with grade IV reactions total IgE (P = 0.003) and honey bee venom-specific IgE (P = 0.001) were significantly lower than in lower severity grades. Bee venom-specific mean IgE rank was significantly higher in bee than in Vespula venom allergic patients (P = 0.0001). CONCLUSIONS: Connection of high severity sting reactions with lower IgE is mainly because of older age, which is associated with lower total IgE, and moreover with cardiovascular disease and elevated baseline serum tryptase, which are both risk factors for severe reactions.

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.

[Hymenoptera venom anaphylaxis and cardiovascular disease]. / Hymenopterengiftanaphylaxie und Herz-Kreislauf-Erkrankungen.

Preexisting cardiovascular disease may worsen the course of anaphylaxis. This is illustrated based on the example of Hymenoptera venom allergy. Fatal sting anaphylaxis is most often observed in elderly patients. During autopsy preexisting cardiovascular disease is frequently found. Preexisting cardiovascular disease in patients with anaphylaxis may also cause lasting morbidity, e.g. cerebral or myocardial infarction. Heart medications, notably beta-blockers und ACE-inhibitors may worsen the course of anaphylactic reactions due to their pharmacologic effects. Since cardiovascular diseases are much more frequent than anaphylaxis and these medications are very effective, these drugs cannot be substituted in patients with both diseases without a careful risk analysis. Epinephrine is the drug of first choice for treatment of anaphylaxis. It may however, especially following rapid intravenous administration, cause severe arrhythmias or myocardial infarction. Adrenaline should therefore preferably be given intramuscularly, or by slow intravenous infusion.

Eclipse kinetics as a probe of quaternary structure in bacteriophage phi X174.

The extracellular form of bacteriophage phi X174 consists of single-stranded DNA within an icosahedral capsid, which has short spikes at each of its vertices. Each spike is composed of gene G and H proteins, while the capsid itself consists of gene F protein. Since several molecules of gene H protein are injected into the cell along with the DNA, specific protein--protein and DNA--protein interactions must be broken when the genome exits and leaves an intact capsid structure at the receptor site. To demonstrate this we examined the eclipse (DNA ejection) reaction with two types of phi X174 mutants. The first contains missense mutations in a capsid or spike protein gene, and the second involves insertions or deletions in non-coding regions of the DNA. Using an improved procedure, the eclipse rate in vivo of the eclipse mutants Fcs70 has been redetermined over a larger temperature range than in previous studies. The three- to fivefold decrease in rate between 37 degrees C and 25 degrees C is due to an increase in both the enthalpy and entropy of activation when compared to the wild-type values of these kinetic parameters. This missence mutation also confers an increase in virus stability in 2 to 3 M-urea. In contrast to this, inserting 163 bases into the length of DNA packaged within the phi X174 capsid does not lead to a detectable change in eclipse rate over the same temperature range. yet this insertion into the J--F intercistronic region imparts a significant decrease in virus stability in urea. These results suggest that a specific set of non-covalent interactions is involved in phi X174 DNA ejection. This is supported by the small (50%), but significant, increase in eclipse rate that occurs when 27 bases are deleted from the J--F intercistronic region. The latter effect must be base-sequence-specific since no change in rate is observed when only seven of the 27 bases are deleted. Thus, the kinetics of the phi X174 eclipse reaction can be used as a sensitive probe of quaternary structure by correlating the change in reaction rate with alterations in amino acid and base sequences in the structural components of the virus.

Effects of palindrome size and sequence on genetic stability in the bacteriophage phi X174 genome.

By inserting palindromes of varying length and sequence into a non-essential region of the bacteriophage phi X174 genome we have investigated the effect of palindrome size and sequence on their genetic stability. Multimers of increasing size of the EcoRI linker CCGAATTCGG (E), the BamHI linker CCGGATCCGG (B) or mixtures of both (E, B) were inserted into the PvuII site of a previously constructed bacteriophage strain phi X174 J-F ins6. The largest inserts that could be maintained in the genome without significant loss of genetic stability were 2B, 4E, and 4(E, B), respectively. Polymers exceeding this size could be inserted but resulted in rapid and precise deletion from the phage genome, whereby nB was more unstable than nE, and nE was more unstable than n(E, B). Analysis of the resulting deletion mutants provided evidence for two different types of deletions. The more frequent deletion arose from either type palindrome and removed nucleotides in blocks of ten base-pairs (one linker unit), but only from the palindromic sequence, and always left at least an 18 base-pair long palindrome (one linker plus 8 neighboring base-pairs) behind. The less frequently occurring deletions arose only from nB type palindromes, removing the complete palindromic sequence plus adjacent nucleotides. At least the first type of deletion occurred in the absence of recA activity. Our results show a correlation between the sequence, as well as size, and the genetic stability of palindromes, i.e. sequences that could decrease the stability of a cruciform increased their genetic stability. This supports the theory that palindrome deletion occurs via extrusion of the palindrome into a cruciform or cruciform-like structure.

Insertions of palindromic DNA sequences into the J-F intercistronic region of bacteriophage phi X174 interfere with normal phage growth.

The effect of hairpin (cruciform) size on the regulation of gene expression was investigated by cloning a series of palindromic sequences into the non-essential J-F intercistronic region of the bacteriophage phi X174 ins6 genome. Genetic stability of the insert sequence and its effect on the growth efficiency of the phage was used as an initial measure of the biological consequence of hairpin insertions. Multimers of increasing size of the BamHI linker sequence C-C-G-G-A-T-C-C-G-G were inserted into the PvuII site of the parental strain ins6. The largest hairpin that could be constructed and maintained in the phi X174 genome had a stem length of 22 base-pairs and a loop size of four nucleotides (linker tetramer). However, this structure proved to be disadvantageous to the phage and was rapidly deleted from its genome. Trimer inserts were more stable, but were eventually deleted also. Monomer and dimer inserts, though genetically stable, decreased the growth efficiency of the phage as judged by competitive growth experiments and measurements of burst size. The physical formation of these hairpins was shown by restriction digests of single-stranded DNA with BamHI and HpaII. We argue that these secondary structures form in vivo, at least in the single-stranded genome and the polycistronic mRNAs, and were responsible for the observed growth defects.

Extraction and differentiation of the Su autoantigen from calf thymus nuclei.

Antibodies to Su antigen have been reported previously as a distinct antigen-antibody system associated with connective tissue diseases; most specifically systemic lupus erythematosus and undifferentiated connective tissue disease. The Su antigen was first identified by double immunodiffusion using calf thymus nuclear extract (CTNE) as a source for Su antigen. In this report, enhanced extraction of Su antigen was achieved using deoxyribonuclease I (DNase) for preparation of CTNE. Only the Sm antigen was found in comparable quantities in the DNase CTNE. Western immunoblotting and immunoprecipitation employing DNase CTNE and extracts of [35S]methionine-labeled HeLa cells respectively were used for further characterization and differentiation of the Su antigen. Sera from patients positive for Su antibodies by double immunodiffusion were found to react most specifically with antigen components in a molecular weight range of approximately 50-55 kDa. These methods should assist in further understanding the biochemical properties of the Su antigen.

Novel surface and multicolor charge coupled device-based fluorescent imaging system for DNA microarrays.

We report a novel support, concomitant attachment chemistry, and a fluorescent imaging system for DNA microarrays. The support consists of soda lime glass coated with a layer of chromium, which eliminates any autofluorescence from the underlying glass substrate and reduces nonspecific probe binding. Attachment of DNA fragments exceeding 300 nucleotides in length is achieved without chemical modifications of either the chrome surface or the DNA itself. The charge coupled device (CCD)-based imaging system employs a 175 W xenon arc lamp as the light source, allowing the use of many different fluorophors. A 14 mm x 9 mm sample area is imaged with a single exposure, which takes between 5 and 20 s for each color plane in typical genomic comparative genomic hybridization type assays. The spatial resolution is limited only by the pixel size of the CCD chip (9 microm). The oblique illumination geometry combined with effective background reduction afforded by the chromium surface enables the system to achieve a detection limit of <5 x 10(7) fluorophors/cm(2) with 10 s integration. In a model system with arrayed lambda DNA targets a dose response was observed over four orders of magnitude in response to hybridizations with increasing amounts of the fluorescent labeled lambda probe.

Simultaneous multiple analyte detection using fluorescent peptides and capillary isoelectric focusing.

Analyte-specific detection based on the isoelectric point of the detection moiety is a new concept that is under investigation at Vysis. We have developed methods for the synthesis of of fluorescent synthetic peptides that can be conjugated to bioanalytes such as nucleic acids and antibodies, processed in a hybridization or binding assay, and then chemically released prior to detection by capillary isoelectric focusing (cIEF)-laser-induced fluorescence (LIF) detection. A two-step cIEF method in coated capillaries using salt mobilization has been used that produces high peak efficiencies and good assay reproducibility. The concentration by focusing aspect of cIEF, which allows for the entire capillary to be filled with sample, enables detection limits in the pM as opposed to sub-nM level for conventional capillary electrophoresis (CE)-LIF. The simultaneous multiple detection of eleven different focusing entities has been achieved.

Discriminating two classes of toxicants through expression analysis of HepG2 cells with DNA arrays.

Microarray technology provides a rapid and cost-effective method to associate specific cellular responses with unique gene expression patterns. If characteristic expression patterns of a small number of genes could be associated with drug toxicity, this association may be used for toxicity prediction, and thereby to reduce the need for traditional toxicity testing. To test this hypothesis, we have designed an array composed of 92 known human genes of toxicological interest (including seven housekeeping genes) and eight bacterial controls. HepG2 cells were treated with either ethanol or one of two quinone containing anticancer drugs, mitomycin C or doxorubicin. RNA was isolated from treated and untreated cells, differentially labeled with fluorescent dyes, and then hybridized to the array. Our results show that the expression patterns induced by ethanol and the anticancer drugs are different. Both of the anticancer drugs, but not ethanol had a differential effect on the regulation of several genes, including CYP4F2/3, CYP3A3, TNFRSF6 and CHES1, demonstrating that the two drugs might function through a similar mechanism, which differs from that of ethanol. These results suggest that microarray-based expression analysis may offer a rapid and efficient means for assessing drug toxicity.

[Insect venom allergy]. / Insektengiftallergie.

Systemic allergic reactions to hymenoptera stings are most often mediated by venom-specific IgE antibodies. The most frequent symptoms are urticaria, angioedema, asthma and anaphylactic shock. Rarely, cerebral or myocardial ischemia with permanent damage may result from severe anaphylactic shock. Each year several individuals die in Switzerland from allergic reactions following hymenoptera stings. The most valuable diagnostic tests are skin tests and estimation of venom-specific serum-IgE antibodies. The most important drugs for emergency treatment are adrenaline and antihistamines. During anaphylactic shock, volume substitution may be essential. All patients with a history of severe systemic reactions should receive venom immunotherapy which induces complete protection in a high percentage of the patients.

[The value of immunotherapy in treatment of IgE-induced allergic diseases]. / Der Stellenwert der Immunotherapie in der Behandlung IgE-vermittelter allergischer Krankheiten.

Until today, immunotherapy is the only causal treatment of IgE-mediated allergic disease. Its efficacy has been demonstrated in controlled studies for rhinitis and bronchial asthma due to various pollens, house-dust mite and animal dander as well as for systemic allergic reactions following hymenoptera stings. This treatment is especially successful in younger patients with seasonal allergy of rather short duration and with sensitization only to few allergens. In order to avoid allergic side effects of this treatment, measures of precautions must be strictly observed. A close cooperation between patient, family practitioner and allergist is important for the success of this treatment.

Enzymatic construction and selection of bacteriophage G4 mutants with modifications of a DNA secondary structure in the J-F intercistronic region.

The J-F intercistronic region of bacteriophage G4 has the potential to form a perfectly base-paired hairpin structure, thought to act as a terminator of transcription. To investigate this proposed structure-function relationship, viable mutants were constructed by site-specific mutagenesis with small deletions of 2 to 4 base pairs in the center of the corresponding palindromic sequence. These sequence modifications had a small positive effect on the growth efficiency of the phage. The approach of biochemical rather than biological selection of these mutant phages is generally applicable to the construction of virus and plasmid vectors.

New developments in the diagnosis and treatment of hymenoptera venom allergy.

According to most textbooks, diagnostic tests with Hymenoptera venoms are reliable, and immunotherapy with these venoms in Hymenoptera-venom-allergic patients leads in near to 100% to full protection. Careful analysis of the literature shows however that the specificity of diagnostic tests is far from perfect and that both efficacy and tolerance, especially in patients receiving honeybee venom immunotherapy, are still suboptimal. The major allergens of honeybee and vespid venoms are now available in recombinant form. Preliminary trials analyzing diagnostic tests with recombinant allergens in honeybee venom allergy are promising: the specificity is clearly increased in both skin testing and in determining venom-specific IgE antibodies when compared to natural venom allergens. An important recent finding is the frequent association of severe Hymenoptera venom allergy and elevated basal serum levels of the mast-cell-specific enzyme tryptase. Elevated levels are found in up to 30% of the patients with a history of severe shock reactions following Hymenoptera stings. The current findings indicate that basal tryptase levels indicating an increased mast cell load are much more frequent than previously thought and are a risk factor for severe or even fatal sting reactions. Premedication with antihistamines in the initial phase of venom immunotherapy reduced both local and systemic allergic side effects in several controlled studies. In a retrospective analysis of one of these trials it was found that reexposure during immunotherapy resulted in significantly more systemic allergic reactions in patients on placebo than on antihistamine premedication, suggesting that initial antihistamine premedication might increase the efficacy of venom immunotherapy. Different ways of allergen modification for venom immunotherapy have been proposed. While the results with chemical modifications were not convincing, recent studies with T-cell epitope peptides from the major bee venom allergen phospholipase A(2) look promising. Patient-tailored cocktails of recombinant venom allergens or isoforms thereof may be another possibility in the future. A number of prospective studies analyzing the duration of venom immunotherapy required for long-term protection have been published in the last decade. While most patients are still fully protected 1 year after discontinuation of therapy, relapses may occur in up to 20% of patients reexposed many years after treatment. Various risk factors for such relapses have been identified.