1: Cancer Immunol Immunother. 2006 Feb 17; [Epub ahead of print]

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Antitumor action and immune activation through cooperation of bee venom secretory phospholipase A2 and phosphatidylinositol-(3,4)-bisphosphate.

Putz T, Ramoner R, Gander H, Rahm A, Bartsch G, Thurnher M.

Department of Urology, University of Innsbruck, Anichstrasse 35, 6020, Innsbruck, Austria, martin.thurnher@uibk.ac.at.

We evaluated tumor cell growth modulation by bee venom secretory phospholipase A2 (bv-sPLA2) and phosphatidylinositol-(3,4)-bisphosphate as well as potential cooperative effects. In addition, the immunomodulatory impact of tumor cell treatment was examined by monitoring changes in phenotype and function of monocyte-derived dendritic cells (moDCs) cocultured with pretreated tumor cells. Bv-sPLA2 or phosphatidylinositol-(3,4)-bisphosphate alone displayed moderate effects on the proliferation of A498 renal cell carcinoma cells, T-47D breast cancer cells, DU145 prostate cancer cells and BEAS-2B transformed lung cells. However, when bv-sPLA2 was coadministered with phosphatidylinositol-(3,4)-bisphosphate a potent inhibition of [(3)H] thymidine incorporation into all tested cell lines occurred. This inhibition was due to massive cell lysis that reduced the number of cells with proliferative capacity. Importantly, tumor cell lysates generated with bv-sPLA2 plus phosphatidylinositol-(3,4)-bisphosphate induced maturation of human moDCs demonstrated by enhanced expression of CD83 and improved stimulation in allogeneic mixed leukocyte reactions. Our data demonstrate that bv-sPLA2 and phosphatidylinositol-(3,4)-bisphosphate synergistically generate tumor lysates which enhance the maturation of immunostimulatory human monocyte-derived dendritic cells. Such tumor lysates which represent complex mixtures of tumor antigens and simultaneously display potent adjuvant properties meet all requirements of a tumor vaccine.

PMID: 16485125 [PubMed - as supplied by publisher]

2: Clin Exp Allergy. 2005 Dec;35(12):1591-8.

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Toll-like receptor ligands as adjuvants in allergen-specific immunotherapy.

Johansen P, Senti G, Martinez Gomez JM, Storni T, von Beust BR, Wuthrich B, Bot A, Kundig TM.

Unit for Experimental Immunotherapy, University Hospital of Zurich, Zurich, Switzerland. pal.johansen@usz.ch

BACKGROUND: Allergen-specific immunotherapy (SIT) leads to long-term amelioration of T-helper type 2 (Th2)-mediated allergic symptoms and is therefore recommended as a first line therapy for allergies. The major disadvantage of SIT is its low efficiency, requiring treatment over years. OBJECTIVE: In this study, we evaluated the potential of Toll-like receptor (TLR) ligands to facilitate Th1-type immune responses. METHODS: The immunogenicity and therapeutic potential of the major bee venom allergen phospholipase A2 (PLA2) combined with various TLR ligands were tested in mice and compared with immune responses induced by conventional aluminium-based preparations. RESULTS: Regarding total IgG against PLA2, TLR2/4-binding lipopolysaccharide and TLR3-binding polyriboinosinic polyribocytidylic (PolyI:C) were the superior adjuvants for prophylactic vaccination. However, TLR9-binding phosphorothioate-modified cytosine-guanosine-rich oligonucleotide (CpG), TLR-3-binding PolyI:C, and TLR2/6-binding peptidoglycan skewed the immune responses more towards IgG2a isotype and Th1 cytokines. Furthermore, in a therapeutic approach, CpG, PolyI:C and TLR7/8-binding 3M003 had immune modulating properties as they suppressed established IgE titres. CONCLUSION: The potential of TLR ligands to adjuvate the immunogenicity of bee venom PLA2 and to skew the Th1-Th2 balance proved very heterogeneous. With respect to SIT, CpG, PolyI:C, and 3M003 were very promising. Hence, TLR ligands should be considered as adjuvants or immune modulators in SIT in human as to improve its efficiency regarding the Th1-Th2 balance of the immune response with a likely effect on therapy duration.

PMID: 16393325 [PubMed - in process]

3: Ann N Y Acad Sci. 2005 Nov;1056:279-92.

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Novel Drugs and Vaccines Based on the Structure and Function of HIV Pathogenic Proteins Including Nef.

Azad AA.

Faculty of Health Sciences, Medical School, University of Cape Town, Anzio Road, Observatory, 7925, Cape Town, South Africa. a_azad05@yahoo.com.au.

Evidence is presented to suggest that HIV-1 accessory protein Nef could be involved in AIDS pathogenesis. When present in extracellular medium, Nef causes the death of a wide variety of cells in vitro and may therefore be responsible for the depletion of bystander cells in lymphoid tissues during HIV infection. When present inside the cell, Nef could prevent the death of infected cells and thereby contribute to increased viral load. Intracellular Nef does this by preventing apoptosis of infected cells by either inhibiting proteins involved in apoptosis or preventing the infected cells from being recognized by CTLs. Neutralization of extracellular Nef could prevent the death of uninfected immune cells and thereby the destruction of the immune system. Neutralization of intracellular Nef could hasten the death of infected cells and help reduce the viral load. Nef is therefore a very important molecular target for developing therapeutics that slow progression to AIDS. The N-terminal region of Nef and the naturally occurring bee venom mellitin have very similar primary and tertiary structures, and they both act by destroying membranes. Chemical analogs of a mellitin inhibitor prevent Nef-mediated cell death and inhibit the interaction of Nef with cellular proteins involved in apoptosis. Naturally occurring bee propolis also contains substances that prevent Nef-mediated cell lysis and increases proliferation of CD4 cells in HIV-infected cultures. These chemical compounds and natural products are water soluble and nontoxic and are therefore potentially very useful candidate drugs.

PMID: 16387695 [PubMed - in process]

4: Eur J Immunol. 2005 Dec;35(12):3591-8.

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Heat denaturation, a simple method to improve the immunotherapeutic potential of allergens.

Johansen P, Senti G, Martinez Gomez JM, Wuthrich B, Bot A, Kundig TM.

Unit for Experimental Immunotherapy, University Hospital of Zurich, Zurich, Switzerland.

Allergen-specific immunotherapy (SIT) leads to a long-term amelioration of IgE- and Th2-mediated allergic diseases. However, SIT efficiency is low, with years of treatment along with frequent allergic side effects. The goal of this study was to reduce the side effects by destroying IgE-binding epitopes, i.e. by heat-denaturation, while preserving the therapeutic effect. Mice were immunised with bee venom, birch pollen, grass pollen or cat hair allergens, or with ovalbumin. Heat-denatured allergens bound less IgE but enhanced Th1-dependent IgG2a production as measured by ELISA. The strong IgG2a antibody responses also prevented allergic anaphylaxis in mice, as measured by body temperature drop after a challenge with a high allergen dose. We found that optimal heat-denaturation of allergens left a small proportion in the native conformation to sufficiently stimulate B cells, while non-B cell-mediated effects were probably amplified. The enhanced immunogenicity of heat-denatured allergens is likely explained by enhanced antigen presentation to T cells due to the particulate nature of heat-denatured proteins. This enables Th1 skewing of the immune response with strong production of IgG2a in mice. Therefore, heat-denaturation represents probably the simplest way to enhance the efficiency of SIT while reducing its side effects.

PMID: 16285011 [PubMed - in process]

5: J Allergy Clin Immunol. 2004 Oct;114(4):943-50.

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Modulation of allergic responses in mice by using biodegradable poly(lactide-co-glycolide) microspheres.

Jilek S, Walter E, Merkle HP, Corthesy B.

Department of Chemistry and Applied BioSciences, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland.

BACKGROUND: Biodegradable poly(lactide- co -glycolide) (PLGA) microspheres are a promising carrier for vaccine delivery capable of maturing antigen-presenting cells to stimulate T-cell-mediated immune responses. However, the potential of microspheres to downregulate an allergic response in vivo is unknown. OBJECTIVE: The aim of this study was to determine whether microspheres could potentiate DNA vaccination against allergy and to evaluate the immunomodulatory properties of microspheres alone. METHODS: Mice were treated prophylactically with DNA-loaded plain PLGA microspheres before sensitization with phospholipase A2 (PLA2), the major allergen of bee venom. PLA2-specific IgG1, IgG2a, IgE in serum were measured for 8.5 months, and splenocyte proliferative responses and cytokine profiles were determined. Protection against anaphylaxis was evaluated after injection of an otherwise lethal dose of PLA2. RESULTS: Phospholipase A2-specific IgG1 and IgG2a production turned out to be 2 times higher using cationic microspheres compared with anionic microspheres, but was not influenced by the presence of DNA. In contrast, reduction in IgE production and T-cell hyporesponsiveness were observed with all microsphere formulations. Recall challenge with PLA2 triggered combined expression of both IL-4 and IFN-gamma, together with sustained expression of IL-10 that can explain the protective effect against anaphylaxis. CONCLUSION: Our data suggest a dual mechanism that does initially rely on a TH2 to TH1 immune deviation and then on IL-10-mediated suppression. This is the first physiological demonstration that plain PLGA microspheres can induce tolerance in mice for as long as 6 months postsensitization.

PMID: 15480340 [PubMed - indexed for MEDLINE]

6: Am J Chin Med. 2004;32(3):361-7.

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Anti-inflammatory effect of bee venom on type II collagen-induced arthritis.

Lee JD, Kim SY, Kim TW, Lee SH, Yang HI, Lee DI, Lee YH.

Research Group of Pain and Neuroscience in Vision 2000 Project East-West Medical Research Institute, Kyung Hee University, Seoul, Korea. ljdacu@khmc.or.kr

Bee venom (BV) has been used to relieve pain and reduce inflammation in traditional Oriental medicine, especially in chronic inflammatory diseases such as rheumatoid arthritis (RA). We previously reported that the BV injection into a traditional acupuncture point (Zusanli) reduced arthritis-associated edema and nociceptive responses in Freund's adjuvant-induced arthritis in rats (Kwon et al., 2001). This study was designed to evaluate the anti-inflammatory and anti-cytokine effect of BV on a murine type-II collagen-induced arthritis (CIA) model. Male mice were immunized by spontaneous injection of 100 microg of an emulsion of bovine type-II collagen and complete Freund's adjuvant (CFA), with a booster injection after 2 weeks. In the experimental group, 0.1 ml BV was injected at acupuncture point (Zusanli) near both knees twice a week for a total of 5 times. In the control group, normal saline was injected at the same frequencies. These injections began 5 weeks after the first collagen injection. Starting the 3rd week after the first collagen injection, we examined limb swelling and severity of arthritis twice a week. At 8 weeks, mice were sacrificed and synovial tissue was examined with the light microscope and serum cytokines (IL-1beta and TNF-alpha) were measured by ELISA. The incidence of arthritis, the mean arthritis index and the number of arthritic limbs were significantly lower in the treatment compared to the control group (63% versus 75%, 3.4% versus 8.5%, 23% versus 75%, respectively). Among the serum proinflammatory cytokines, the production of TNF-alpha in the BV group was suppressed compared to the control group (59 +/- 4.5 versus 99.5 +/- 6.5, p < 0.05), but IL-1beta was not suppressed. The examination of the histopathology of the joints of murine CIA showed decreased inflammation signs and less lymphocyte infiltration after BV acupuncture therapy. Acupuncture therapy with BV suppressed the development of arthritis and caused inhibition of the immune responses in type-II collagen-induced arthritis.

PMID: 15344419 [PubMed - indexed for MEDLINE]

7: J Allergy Clin Immunol. 2003 Jun;111(6):1255-61.

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Induction of IL-10+CD4+CD25+ T cells by grass pollen immunotherapy.

Francis JN, Till SJ, Durham SR.

Upper Respiratory Medicine, National Heart and Lung Institute, Imperial College, Dovehouse Street, London SW3 6LY, UK.

BACKGROUND: Immunotherapy involves the modulation of allergen-specific T-cell responses, either T(H)2-to-T(H)1 immune deviation or, in bee venom-treated patients, induction of IL-10 production by CD4+CD25+ T cells. IL-10-producing CD4+CD25+ regulatory T cells have emerged as potential mediators of immune tolerance in numerous murine models of immunopathology. OBJECTIVE: The aim of this study was to evaluate the role of IL-10 production and CD4+CD25+ T cells in the response to grass pollen immunotherapy. METHODS: PBMCs were isolated from patients after 1 year of grass pollen immunotherapy and from matched untreated atopic and healthy control subjects. After 6 days of in vitro stimulation with Phleum pratense, production of IL-10, IL-5, IL-4, and IFN-gamma and proliferation and numbers of CD4+CD25+ T cells were measured. T cells were then stimulated for a further 5 hours with phorbol 12-myristate 13-acetate and ionomycin and assessed for intracellular IL-10 by means of flow cytometry. RESULTS: Patients undergoing immunotherapy produced significantly more IL-10 than atopic control subjects (patients undergoing immunotherapy, 116 +/- 21 pg/mL [n = 11]; atopic patients, 30 +/- 5 pg/mL [n = 11]; P <.001), and the number of CD4+CD25+ cells identified after allergen stimulation was also greater in the immunotherapy group. The numbers of CD4+CD25+ T cells correlated positively with activation as measured by proliferation in both of the control groups but not in the immunotherapy group. Moreover, only T cells from patients undergoing immunotherapy were positive for intracellular IL-10, and these were almost exclusively CD4+CD25+ cells. CONCLUSION: Grass pollen immunotherapy results in a population of circulating T cells that express the IL-10(+) CD4+CD25+ phenotype in response to allergen restimulation.

Publication Types:

·       Clinical Trial

PMID: 12789226 [PubMed - indexed for MEDLINE]

8: Toxicon. 2003 Jun;41(7):861-70.

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Inhibition of mammary carcinoma cell proliferation in vitro and tumor growth in vivo by bee venom.

Orsolic N, Sver L, Verstovsek S, Terzic S, Basic I.

Department of Animal Physiology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10 000 Zagreb, Croatia. norsolic@yahoo.com

The possible tumor growth- and metastasis-inhibiting effects of bee venom in mice and in tumor cell cultures were studied. The tumor was a transplantable mammary carcinoma (MCa) of CBA mouse. Intravenous administration of bee venom to mice significantly reduced the number of metastases in the lung. However, subcutaneous administration of bee venom did not reduce the number of lung metastases, indicating that the antitumor effect of the venom could be highly dependent on the route of injection as well as close contact between the components of the venom and the tumor cells, as was shown by in vitro studies on MCa cells. We also observed variations in immunological parameter induced by bee venom. We proposed that bee venom has an indirect mechanism of tumor growth inhibition and promotion of tumor rejection that is based on stimulation of the local cellular immune responses in lymph nodes. Apoptosis, necrosis, and lysis of tumor cells are other possible mechanisms by which bee venom inhibits tumor growth.

PMID: 12782086 [PubMed - indexed for MEDLINE]

9: FASEB J. 2003 Jun;17(9):1026-35.

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T helper (Th) 2 predominance in atopic diseases is due to preferential apoptosis of circulating memory/effector Th1 cells.

Akdis M, Trautmann A, Klunker S, Daigle I, Kucuksezer UC, Deglmann W, Disch R, Blaser K, Akdis CA.

Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, CH-7270 Davos, Switzerland.

T cells constitute a large population of cellular infiltrate in atopic/allergic inflammation and a dysregulated, Th2-biased peripheral immune response appears to be an important pathogenetic factor. In atopic dermatitis, circulating cutaneous lymphocyte-associated antigen-bearing (CLA+) CD45RO+ T cells with skin-specific homing property represent an activated memory/effector T cell subset. They express high levels of Fas and Fas ligand and undergo activation-induced apoptosis. The freshly purified CLA+ CD45RO+ T cells of atopic individuals display distinct features of in vivo-triggered apoptosis such as pro-caspase degradation and active caspase-8 formation. In particular, the Th1 compartment of activated memory/effector T cells selectively undergoes activation-induced cell death, skewing the immune response toward surviving Th2 cells in atopic dermatitis patients. The apoptosis of circulating memory/effector T cells was confined to atopic individuals whereas non-atopic patients such as psoriasis, intrinsic-type asthma, contact dermatitis, intrinsic type of atopic dermatitis, bee venom allergic patients, and healthy controls showed no evidence for enhanced T cell apoptosis in vivo. These results define a novel mechanism for peripheral Th2 response in atopic diseases.

PMID: 12773485 [PubMed - indexed for MEDLINE]

10: J Allergy Clin Immunol. 2003 Apr;111(4):854-61.

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Allergen-specific T-cell tolerance induction with allergen-derived long synthetic peptides: results of a phase I trial.

Fellrath JM, Kettner A, Dufour N, Frigerio C, Schneeberger D, Leimgruber A, Corradin G, Spertini F.

Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.

BACKGROUND: There is a need to improve the safety and efficacy of allergen-specific immunotherapy. Long synthetic peptide-based immunotherapy was proven safe, immunogenic, and protective in preclinical trials. OBJECTIVE: To evaluate the safety and immunogenicity of an allergen-derived long synthetic overlapping peptide (LSP) immunotherapy, we designed a double-blind, placebo-controlled phase I clinical trial in patients hypersensitive to bee venom. METHODS: Patients from the active group were injected at day 0 with a mixture of 3 LSPs mapping the entire PLA2 molecule, a major bee venom allergen, in a dose-escalating protocol to a maintenance dose of 100 microg per peptide repeated at days 4, 7, 14, 42, and 70. The control group was injected with human albumin. RESULTS: Whereas specific T-cell proliferation in the peptide group increased up to day 14, a sharp decline was observed thereafter, ending in specific T-cell hyporesponsiveness at day 80. Serum-specific IgG4 response was enhanced, in contrast to anti-PLA2 IgE. Specific T-cell cytokine modulation was marked by increased IL-10 and IFN-gamma secretion. LSP injections were well tolerated in all patients except for mild, late allergic reactions in 2 patients at day 70. CONCLUSIONS: The results of this short-term study demonstrate that LSP-based allergen immunotherapy was safe and able to induce T(H)1-type immune deviation, allergen-specific IL-10 production, and T-cell hyporesponsiveness. LSPs, which offer the advantage of covering all possible T-cell epitopes for any HLA genotype, can be considered candidates for a novel and safe approach of specific immunotherapy.

Publication Types:

·       Clinical Trial

·       Clinical Trial, Phase I

·       Randomized Controlled Trial

PMID: 12704369 [PubMed - indexed for MEDLINE]

11: Eur J Immunol. 2002 Nov;32(11):3133-41.

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Reversal of the adult IgE high responder phenotype in mice by maternally transferred allergen-specific monoclonal IgG antibodies during a sensitive period in early ontogeny.

Lange H, Kiesch B, Linden I, Otto M, Thierse HJ, Shaw L, Maehnss K, Hansen H, Lemke H.

Biochemical Institute, Medical Faculty, Christian-Albrechts-Universitat at Kiel, Germany.

IgE is an important trigger in allergy and asthma, diseases whose development is suggested to depend on an initial sensitization in early life. While induction of murine IgE responses requires both a genetically based IgE high responder phenotype and defined experimental conditions, maternally transferred IgG can override these prerequisites and suppress IgE formation in an allergen-specific manner. Here, we show that maternally transferred monoclonal IgG, irrespective of their subclass and recognized epitopes, induce IgE unresponsiveness, which is effective for parenteral immunization with bee venom phospholipase A2 as well as for airway-immunization with nebulized ovomucoid-containing ovalbumin. This IgE suppression is detectable in the offspring during the first 4 months of life, but not thereafter and not in the dams. However, when the initial immunization at an age of 3 or 4 months was followed by further application of both allergens via their respective routes, IgE suppression persisted up to an age of more than one year. If applicable to man, these findings may allow the development of a new strategy for the prevention of allergy and asthma by maternally transferred or neonatally injected allergen-specific mAb in combination with natural or prophylactic exposure to the respective allergens during early childhood.

PMID: 12555658 [PubMed - indexed for MEDLINE]

12: Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 1999;(93):243-51; discussion 252.

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Differential regulation of allergen-specific antibodies in allergy and specific immunotherapy.

Blaser K, Akdis CA, Faith A.

Allergen-specific immunotherapy (SIT) aims to specifically skew an allergic response into a normal immune reaction against an allergen. The response to bee venom (BV) provides an especially suited model to study the immunological mechanisms of SIT in human. The BV-phospholipase A2 (PLA) represents the major antigen/allergen of BV. In SIT of BV allergy both whole BV and T cell epitope peptides of PLA were successfully applied. It appeared that the induction of specific anergy in peripheral T cells and reactivation of the T cells by microenvironmental cytokines represent the basic key steps in the immunological mechanism of SIT. The proliferative and cytokine responses by specific T cells were significantly suppressed simultaneously with an increase in IL-10 after 7 days. The anergic state was fully established after 4 weeks. Neutralization of IL-10 in PBMC by a specific antibody reconstituted the original proliferative and cytokine responses. Intracytoplasmatic cytokine staining revealed that IL-10 was initially produced by activated allergen-specific T cells. IL-10-producing B cells and monocytes were involved at a later stage of SIT and in maintenance of the anergy. The addition of IL-10 to stimulated PBMC or purified B cells inhibited IgE synthesis and enhanced the IgG4 antibody formation. Thus, SIT generates IL-10, which in turn induces specific anergy by autokrine interaction in T cells and counter-regulates IgE and IgG4 production. Particular cytokines from the tissue microenvironment reactivate the T cells to produce distinct Th1 or Th2 cytokine patterns respectively and by this way direct SIT towards successful or unsuccessful treatment. High amounts of allergen administered in SIT preferentially generate Th1 cytokines in T cells and IgG4 antibodies in memory B cells. Further investigations demonstrated that suppression of T cells by IL-10 is an active process, which depends on the expression and participation of CD28.

PMID: 11487881 [PubMed - indexed for MEDLINE]

13: Ther Umsch. 2001 May;58(5):274-7.

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[Principles of specific immunotherapy of IgE-induced allergic reactions]

[Article in German]

Akdis CA, Blaser K.

Schweizerisches Institut fur Allergie- und Asthmaforschung (SIAF), Davos.

Allergen-specific immunotherapy (SIT) aims to selectively skew an allergic immune response into a normal immunity. It appeared that the induction of specific anergy in peripheral T cells and reactivation of anergized T cells by microenvironmental cytokines represent two key steps in the mechanism of SIT. In SIT of bee venom allergy the proliferative and cytokine responses were significantly suppressed within seven days, simultaneously with an increase in IL-10 production. IL-10 induces total anergy in T cells by autokrine interaction. In addition, it can counter-regulate IgE and IgG4 synthesis. The addition of blocking anti-IL-10 to stimulated PBMC fully reconstituted the proliferative and cytokine responses in anergized T-cells. Again, particular cytokines are able to reactivate anergic T cells to produce distinct IFN-gamma/IL-2 or IL-4/IL-13 dominated T cell cytokine patterns and direct by this way SIT towards successful or unsuccessful treatment. The suppression of T cells by IL-10 is an active biochemical process, which depends on the interaction of the ligated IL-10 receptor with the CD28 costimulatory signaling pathway in T cells.

PMID: 11407227 [PubMed - indexed for MEDLINE]

14: J Immunol. 2001 Mar 1;166(5):3612-21.

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Antigen-independent suppression of the allergic immune response to bee venom phospholipase A(2) by DNA vaccination in CBA/J mice.

Jilek S, Barbey C, Spertini F, Corthesy B.

Division of Immunology and Allergy, R & D Laboratory, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.

Phospholipase A(2) (PLA(2)) is one of the major honey bee venom allergens for humans. To assess the long-term prevention of allergic reactions by DNA vaccination, a PLA(2)-CBA/J mouse model was employed using empty or PLA(2) sequence-carrying DNA plasmids. Early skin application of either DNA construct before (prophylactic approach) or after (therapeutic approach) sensitization with PLA(2)/alum led to reduced PLA(2)-specific IgE and IgG1 titers at 7 mo, with concomitant rise in IgG2a and IgG3. Splenocytes recovered at 5-6 mo after the last DNA administration exhibited a sustained IFN-gamma and IL-10 secretion and reduced IL-4 production. Recall challenge with PLA(2) boosted IFN-gamma and IL-10 secretion, suggesting the reactivation of quiescent memory Th1 lymphocytes. Mice from the prophylactic groups were fully protected against anaphylaxis, whereas 65% of the animals recovered in the therapeutic groups. Th1-polarized immune responses were also active in mice vaccinated with an empty plasmid 32 wk before sensitization with another Ag (OVA). This is the first demonstration that the Ag-coding sequence in DNA vaccine is not necessary to promote immune modulation in naive and sensitized animals for a prolonged period, and has relevance for the understanding of the innate and induced mechanisms underlying gene immunotherapy in long-term treatment of allergy.

PMID: 11207323 [PubMed - indexed for MEDLINE]

15: J Immunol. 2000 Sep 15;165(6):3497-505.

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Inducing tolerance by intranasal administration of long peptides in naive and primed CBA/J mice.

Astori M, von Garnier C, Kettner A, Dufour N, Corradin G, Spertini F.

Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.

To assess the capacity of a peptide-based immunotherapy to induce systemic tolerance via the nasal route, we designed three long overlapping peptides of 44-60 aa covering the entire sequence of phospholipase A2 (PLA2), a major bee venom allergen. Both prophylactic and therapeutic intranasal administrations of long peptides to PLA2-hypersensitive CBA/J mice induced specific T cell tolerance to the native allergen. In prophylactic conditions, this tolerance was marked by a suppression of subsequent specific IgE response, whereas the therapeutic approach in presensitized mice induced a more than 60% decrease in PLA2-specific IgE. This decline was associated with a shift in the cytokine response toward a Th1 profile, as demonstrated by decreased PLA2-specific IgG1 and enhanced IgG2a levels, and by a decline in the specific IL-4/IFN-gamma ratios. T cell transfer from long peptide-tolerized mice to naive animals abrogated the expected anti-PLA2 IgE and IgG1 Ab response, as well as specific T cell proliferation, but enhanced specific IgG2a response upon sensitization with PLA2. These events were strongly suggestive of a clonal anergy affecting more profoundly Th2 than the Th1 subsets. In conclusion, these results demonstrate that allergen-derived long peptides delivered via the nasal mucosa may offer an alternative to immunotherapy with native allergens without the inherent risk of systemic anaphylactic reactions. Moreover, long peptides, in contrast to immunotherapy strategies based on short peptides, have the advantage of covering all potential T cell epitopes, and may represent novel and safe tools for the therapy of allergic diseases.

PMID: 10975871 [PubMed - indexed for MEDLINE]

16: J Allergy Clin Immunol. 2000 Aug;106(2):228-38.

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Structural biology of allergens.

Aalberse RC.

Department of Allergy, CLB and the Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

One of the major challenges of molecular allergy is to predict the allergenic potential of a protein, particularly in novel foods. Two aspects have to be distinguished: immunogenicity and cross-reactivity. Immunogenicity reflects the potential of a protein to induce IgE antibodies, whereas cross-reactivity is the reactivity of (usually preexisting) IgE antibodies with the target protein. In addition to these two issues, the relation between IgE-binding potential and clinical symptoms is of interest. This is influenced by physical properties (eg, stability and size) and immunologic properties (affinity and epitope valence). Discussions on immunogenicity and cross-reactivity of allergens rely on the establishment of structural similarities and differences among allergens and between allergens and nonallergens. For comparisons between the 3-dimensional protein folds, the representation as 2-dimensional proximity plots provides a convenient visual aid. Analysis of approximately 40 allergenic proteins (or parts of these proteins), of which the protein folds are either known or can be predicted on the basis of homology, indicates that most of these can be classified into 4 structural families: (1) antiparallel beta-strands: the immunoglobulin-fold family (grass group 2, mite group 2), serine proteases (mite group 3, 6, and 9), and soybean-type trypsin inhibitor (Ole e 1, grass group 11); (2) antiparallel beta-sheets intimately associated with one or more alpha-helices: tree group 1, lipocalin, profilin, aspartate protease (cockroach group 2); (3) (alpha+beta) structures, in which the alpha- and beta-structural elements are not intimately associated: mite group 1, lysozyme/lactalbumin, vespid group 5; and (4) alpha-helical: nonspecific lipid transfer protein, seed 2S protein, insect hemoglobin, fish parvalbumin, pollen calmodulin, mellitin from bee venom, Fel d 1 chain 1, serum albumin. Allergens with parallel beta-strands (in combination with an alpha-helix linking the two strands, a motif commonly found in, for example, nucleotide-binding proteins) seem to be underrepresented. The conclusion is that allergens have no characteristic structural features other than that they need to be able to reach (and stimulate) immune cells and mast cells. Within this constraint, any antigen may be allergenic, particularly if it avoids activation of T(H)2-suppressive mechanisms (CD8 cells and T(H)1 cells).

Publication Types:

·       Review

PMID: 10932064 [PubMed - indexed for MEDLINE]

17: J Am Vet Med Assoc. 1999 Apr 1;214(7):1026-7, 1021.

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Bee sting envenomation resulting in secondary immune-mediated hemolytic anemia in two dogs.

Noble SJ, Armstrong PJ.

Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St Paul 55108, USA.

Immune-mediated hemolytic anemia secondary to bee envenomation developed in 2 dogs. Clinical signs included lethargy, hematuria, ataxia, and seizures; 1 dog died. Clinicopathologic data included nonregenerative anemia, spherocytosis, positive results for Coombs' test, and occult hematuria. Treatment included oral administration of corticosteroids at immunosuppressive dosages and supportive care. The surviving dog initially responded to corticosteroids, but hemolysis recurred as the dosage was tapered. Hemolysis resolved with prolonged administration of corticosteroids. Bee venom contains hyaluronidase, histamines, and hemolysins that cause toxic and hemolytic effects. Envenomation should be considered in any dog with hemolytic anemia in which other causes are ruled out and exposure to bees is known.

Publication Types:

·       Case Reports

PMID: 10200797 [PubMed - indexed for MEDLINE]

18: Int Arch Allergy Immunol. 1998 Sep;117(1):1-10.

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Determinants and mechanisms of human immune responses to bee venom phospholipase A2.

Blaser K, Carballido J, Faith A, Crameri R, Akdis C.

Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland. kblaser@siaf.unizh.ch

The elicitation of an immune response to protein antigens depends on the specific recognition of antigenic determinants (epitopes) by T and B lymphocytes. Bee venom phospholipase A2 (PLA) represents the major antigen/allergen of honey bee venom. It displays three dominant immunogenic peptide and one glycopeptide T cell recognition sites. These epitopes are equally recognized by both allergic and nonallergic individuals. A mixture of the three epitope containing peptides was successfully used in specific immunotherapy of bee venom-allergic patients. Both peptide and whole bee venom immunotherapy induced a state of specific anergy in T cells. The production of specific IgE and IgG4 antibodies directly correlated with the secreted interleukin-4:gamma-interferon (IL-4:IFNgamma) ratio, which itself depended on the concentration of available antigen and the strength of the T cell-activating signal. This signal comprises accumulated molecular interactions delivered by engagement of the antigenic peptide/MHC class II complex with the T cell receptor (TcR). Indeed the thermodynamic laws of chemical equilibrium reactions reveal that the antigen concentration, together with the equilibration constant Ki and the related Gibbs standard free energy DeltaG degrees of the MHC-II/Ag/TcR complex reaction, may govern the secreted IL-4:IFNgamma ratio, and in consequence, differential IgE and IgG4 antibody formation. Ki includes epitope and MHC-II haplotype variability and therefore represents a measure of immunological individuality. A major B cell epitope was determined by using point-mutated PLA. Specific antigen recognition by B cells can trigger distinct cytokine profiles in T cells and contribute to the differential regulation of specific IgE and IgG4 antibodies. Our results indicate that distinct cytokine profiles inducing allergic and nonallergic responses can be attributed to thresholds of T cell activation generated by the specific binding properties of individual MHC-II molecules to immunogenic T cell epitopes and their presentation to TcR.

Publication Types:

·       Review

PMID: 9751842 [PubMed - indexed for MEDLINE]

19: Eur J Immunol. 1998 Jul;28(7):2124-30.

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Antigen-independent suppression of the IgE immune response to bee venom phospholipase A2 by maternally derived monoclonal IgG antibodies.

Seeger M, Thierse HJ, Lange H, Shaw L, Hansen H, Lemke H.

Biochemisches Institut, Medizinische Fakultat, Christian-Albrechts-Universitat, Kiel, Germany.

The IgE immune response to ovalbumin in rats can be suppressed by prior immunization of the dams. The results reported in this paper extend this observation to include a different antigen and another species, namely the IgE immune response to bee venom phospholipase A2 (PLA2) in CBA/J mice. The degree of suppression seemed to depend on the amount of IgG antibodies transferred to the offspring. Moreover, we found that the maternally mediated suppression of the IgE response could be achieved in a completely antigen-free system in which exogenous monoclonal anti-PLA2 IgG antibodies were transferred from the dams to the offspring. The following results were obtained: (i) The IgE suppression by monoclonal IgG antibodies was induced as efficiently with one single anti-PLA2 IgG1 antibody as with a mixture of ten antibodies (nine IgG1, one IgG2b). (ii) Even after several immunizations up to an age of 6 months with a dose of PLA2 that normally induces IgE production, none of the F1 mice developed an IgE response. (iii) This long-lasting suppression was observed in mice which were first immunized at an age of 4 weeks (i.e. when low amounts of maternally derived monoclonal IgG were still present), as well as in mice which were first immunized at an age of 8 weeks, when no such maternal antibodies could be detected in their sera. The corresponding IgG responses showed, compared to normal mice, a transient enhancement in the maternally influenced mice. It is concluded that the immunological experience of the mother is of particular importance for the isotype regulation in the newborns, especially with respect to the ability to elicit an IgE response. The possible implications for the development of allergic diseases in humans are discussed.

PMID: 9692881 [PubMed - indexed for MEDLINE]

20: J Clin Invest. 1998 Jul 1;102(1):98-106.

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Role of interleukin 10 in specific immunotherapy.

Akdis CA, Blesken T, Akdis M, Wuthrich B, Blaser K.

Swiss Institute of Allergy and Asthma Research, CH-7270 Davos, Switzerland. akdisac@isac@siaf.unizh.ch

The induction of allergen-specific anergy in peripheral T cells represents a key step in specific immunotherapy (SIT). Here we demonstrate that the anergic state results from increased IL-10 production. In bee venom (BV)-SIT the specific proliferative and cytokine responses against the main allergen, the phospholipase A2 (PLA), and T cell epitope-containing PLA peptides were significantly suppressed after 7 d of treatment. Simultaneously, the production of IL-10 increased during BV-SIT. After 28 d of BV-SIT the anergic state was established. Intracytoplasmic cytokine staining of PBMC combined with surface marker detection revealed that IL-10 was produced initially by activated CD4(+)CD25(+), allergen-specific T cells, and followed by B cells and monocytes. Neutralization of IL-10 in PBMC fully reconstituted the specific proliferative and cytokine responses. A similar state of IL-10-associated T cell anergy, as induced in BV-SIT, was found in hyperimmune individuals who recently had received multiple bee stings. The addition of IL-10 to soluble CD40 ligand IL-4-stimulated PBMC or purified B cells inhibited the PLA-specific and total IgE and enhanced the IgG4 formation. Accordingly, increased IL-10 production by SIT causes specific anergy in peripheral T cells, and regulates specific IgE and IgG4 production toward normal IgG4-related immunity.

PMID: 9649562 [PubMed - indexed for MEDLINE]

21: J Allergy Clin Immunol. 1998 Jun;101(6 Pt 1):747-54.

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Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom.

Muller U, Akdis CA, Fricker M, Akdis M, Blesken T, Bettens F, Blaser K.

Medical Division, Zieglerspital, Bern, Switzerland.

BACKGROUND: Specific immunotherapy with honeybee venom (BV) is highly effective, but allergic side effects can occur during treatment. Immunotherapy with peptides containing major T-cell epitopes of the relevant allergen or allergens provides an alternative strategy without these problems. OBJECTIVE: The study investigates the immunologic mechanisms and clinical effects of immunotherapy with T-cell epitope peptides of the major BV allergen, the phospholipase A2 (PLA). METHODS: Five patients with IgE-mediated systemic allergic reactions to bee stings were treated with a mixture of three T-cell epitope peptides of PLA. Ten patients allergic to BV receiving whole BV immunotherapy served as control subjects. Increasing doses of the peptide mixture, up to a maintenance dose of 100 microg, were administered subcutaneously within 2 months. The patients were then challenged with PLA and 1 week later with a bee sting. The cellular and humoral immune response was measured in vitro. RESULTS: No allergic side effects were caused by the peptide immunotherapy, and all patients tolerated the challenge with PLA without systemic allergic symptoms. Two patients developed mild systemic allergic reactions after the bee sting challenge. After peptide immunotherapy, specific proliferative responses to PLA and the peptides in peripheral blood mononuclear cells were decreased in successfully treated patients. The production of TH2 and TH1 cytokines was inhibited, and B cells were not affected in their capacity to produce specific IgE and IgG4 antibodies. Their levels increased after allergen challenge in favor of IgG4. CONCLUSIONS: Immunotherapy of BV allergy with short T-cell peptides of PLA induces epitope-specific anergy in peripheral T cells and changes the specific isotype ratio in a fashion similar to that of conventional immunotherapy in successfully treated patients.

PMID: 9648701 [PubMed - indexed for MEDLINE]

22: Immunotechnology. 1995 Aug;1(2):115-25.

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Isolation and characterization of allergen-binding cells from normal and allergic donors.

Irsch J, Hunzelmann N, Tesch H, Merk H, Maggi E, Ruffilli A, Radbruch A.

Institute for Genetics, University of Cologne, Germany.

BACKGROUND: Flow cytometry of the immune system so far has been limited to the analysis of subpopulations according to lineage markers. The cells involved in a particular immune response could not be assayed due to their low frequency. Here we show the potential of antigen-specific high gradient magnetic cell sorting to enrich cells for visualisation in multiparameter cytometry, functional studies and immortalization. OBJECTIVES: The aim of this study was the development of an efficient technology for staining and isolation of antigen-binding cells from human peripheral blood. In particular, allergen-specific cells from normal and allergic donors should be analysed and compared to develop a cellular diagnosis of allergy. STUDY DESIGN: The rare antigen-specific cells were sorted by high-gradient magnetic cell sorting with MACS. Haptenized phospholipase A2 (PLA2), the major allergen of bee venom, or haptenized ParoI, the major allergenic component of Parietaria officinalis, were used as antigens. The cells from normal and allergic donors, binding to the allergen were characterized phenotypically by immuno-fluorescence. Allergen-specific B-cells were immortalized by EBV transformation. RESULTS AND CONCLUSION: Allergen-specific cells can be enriched from blood of both allergic and normal donors to purities of up to 75%, by high gradient magnetic cell sorting. The specificity of labelling with allergen was confirmed by establishing allergen-specific EBV-transformed B-cell lines from the sorted cells. Clear differences exist in the cellular composition of allergen-binding cells from normal compared to allergic donors. In normal donors the allergen-binding cells are B-cells expressing CD19 and CD21. In allergic donors, in addition to allergen-binding B-cells, occurring in about equal absolute numbers as in normal donors, basophilic granulocytes are labeled by allergen. These cells express CD38, CD9 and CD25 on their surface, and stain for IgE.

PMID: 9373340 [PubMed - indexed for MEDLINE]

23: Clin Exp Allergy. 1997 May;27(5):578-83.

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Sensitivity to bee and wasp venoms: association with specific IgE responses to the bee and wasp venom and HLA DRB1 and DPB1.

Faux JA, Moffatt MF, Lalvani A, Dekker J, Warrell DA, Cookson WO.

Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, UK.

BACKGROUND: Stings from bees and wasps can cause systemic reactions which can be fatal in some individuals. In these venom-sensitive patients, specific IgE to the venom is produced and is considered to participate in the adverse reactions. This immune response requires antigen presentation by human leucocyte antigens (HLA) class II molecules, which includes DR and DP, which are present on antigen presenting cells. OBJECTIVE: To test for associations between HLA class II DRB1 and DPB1 alleles and life-threatening sensitivity to both bee and wasp venoms. To establish further whether any associations are independent of the atopy phenotype. METHODS: A total of 33 bee- and 44 wasp-venom-sensitive patients was studied. DRB1 genotypes were determined by single stranded oligonucleotide (SSO) probing of PCR products, and DPB1 genotypes by amplified fragment length polymorphism (AFLP) analysis. Total and specific IgE were measured using the Pharmacia Immunocap, FEIA. Patients with specific IgE to the venom antigens only were termed monosensitive and those with additional specific IgE to HDM and/or GP were termed polysensitive. RESULTS: Allele frequencies were compared to an unrelated control population. The 33 bee-sensitive patients had a greater prevalence of DRB1*07 alleles than the control subjects, 26% vs 14%, with an odds ratio (OR) of 2.1 (95% CI, 1.2-3.7, P = 0.015, corrected for multiple comparisons, Pc = ns). This association was confined to the 15 monosensitive bee patients, who had a 43% DRB1*07 allele frequency when compared with 11% in the 18 polysensitive bee patients, OR 6.1 (95% CI, 1.73-22, P = 0.004, Pc = 0.05), and when compared with a control group of non-venom subjects, 43% vs 16%, OR 3.9 (95% CI, 1.72-9.0, P = 0.002, Pc = 0.02). The 44 wasp-sensitive patients had an increase in the DRB1*11 allele when compared with the control subjects, 13% vs 6%, with an OR 2.2 (95% CI, 1.0-4.6, P = 0.04, Pc = NS), and a decreased prevalence of DRB1*04 alleles, 10% vs 19%, with an OR 0.33 (95% CI, 0.24-0.99, P = 0.04, Pc = NS), but these were not significant when multiple comparisons were taken into account. The DPB1 alleles were not significantly different between the venom sensitive patients and the controls. CONCLUSION: Patients monosensitive to bee venom had a significantly greater prevalence of DRB1*07 alleles than the non-venom, control population suggesting that IgE responses in these patients may, in part be controlled by immune response HLA class II genes. These results are also suggestive of wasp-sensitive patients having a higher prevalence of DRB1*11 and a lower prevalence of DRB1*04 than the control population.

PMID: 9179434 [PubMed - indexed for MEDLINE]

24: Eur J Immunol. 1996 Dec;26(12):2972-80.

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Potential allergens stimulate the release of mediators of the allergic response from cells of mast cell lineage in the absence of sensitization with antigen-specific IgE.

Machado DC, Horton D, Harrop R, Peachell PT, Helm BA.

Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, GB.

A number of structurally diverse antigens preferentially stimulate the synthesis of IgE antibodies, but no unifying principle has been proposed that explains the nature of isotype selection. In the present study, we show that common allergens present in bee venom, house dust mite emanations and parasite proteins induce mast cell and basophil degranulation and stimulate interleukin-4 synthesis, and secretion in the absence of antigen-specific IgE. These data point to a linkage between the initial activation of cells of the innate immune system and subsequent adaptive immune responses. They suggest that IgE-independent mast cell and basophil degranulation is predictive of potential allergenicity and can be evaluated by means of a cellular assay. Our study indicates that non-immunological degranulation by prototypic allergens, such as bee venom phospholipase A2 or proteases associated with house dust mite emanations, is critically dependent on enzymatic activity. These findings have potentially important implications for vaccine design in allergic and parasitic disease.

PMID: 8977293 [PubMed - indexed for MEDLINE]

25: Pediatr Allergy Immunol. 1996 Aug;7(3):109-16.

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Fetal peripheral blood mononuclear cell proliferative responses to mitogenic and allergenic stimuli during gestation.

Jones AC, Miles EA, Warner JO, Colwell BM, Bryant TN, Warner JA.

Department of Child Health, University of Southampton, England.

Blood samples were obtained from fetuses and premature babies (n = 51) (15-34 weeks gestation) to determine at what stage the fetal immune system was able to produce a positive proliferative response to common allergens. Peripheral blood mononuclear cells (PBMC) were stimulated with the mitogen, phytohaemagglutinin (PHA), and the allergens, house dust mite, cat fur, birch tree pollen, beta-lactoglobulin, ovalbumin and bee venom (mellitin). Results were expressed as ratios of stimulated to unstimulated 3H thymidine incorporation, and as percent positive responders. There was an increase in proliferation ratio which correlated with increasing gestational age for PHA (p < 0.0001), cat fur (p = 0.042), birch pollen (p = 0.022) and beta-lactoglobulin (p = 0.006). The point in gestation when cells from some individuals began responding to the allergens with a ratio of 2.0 was at approximately 22 weeks. PBMC proliferative response ratios were higher from samples from babies > 22 weeks gestation compared to < 22 weeks for the mitogen and all allergens, except mellitin. There was also a greater proportion of positive responders from samples > 22 weeks compared to < 22 weeks for the mitogen and all allergens, except mellitin. Maternal exposure to birch pollen, which has a discrete season, was assessed to determine whether exposure had occurred at 22 weeks gestation or beyond. Results showed a higher proliferative response in infant cells stimulated with birch pollen (p = 0.005) and higher proportion of positive responders (p = 0.01) in the group of babies whose mothers had been exposed to birch pollen beyond 22 weeks, compared to those whose mothers had not been so exposed. These results suggest that in utero fetal exposure to an allergen from around 22 weeks gestation may result in primary sensitisation to that allergen, leading to positive proliferative responses, at birth.

PMID: 9116874 [PubMed - indexed for MEDLINE]

26: Am J Trop Med Hyg. 1996 Aug;55(2):197-201.

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Neutralization of bee venom lethality by immune serum antibodies.

Schumacher MJ, Egen NB, Tanner D.

Steele Memorial Children's Research Center, University of Arizona, Tucson, USA.

The lethal effects of Africanized honey bee venom depend on the absorption of venom delivered during simultaneous sting attacks by large numbers of bees. The hypothesis that antibodies to whole bee venom and bee venom components could neutralize the lethal effect of bee venom was tested. Antibodies from beekeepers and immunized rabbits were incubated with bee venom and neutralization was studied by survival of intravenously injected mice. Beekeeper serum antibodies were found effective in protecting mice challenged with whole venom, and serum from rabbits immunized with phospholipase A2 (PLA2) was effective in protection against lethal effects of PLA2. Serum antibodies from rabbits immunized with whole venom or melittin were ineffective in neutralizing whole venom in vivo and had low titers in a venom enzyme-linked immunosorbent assay. The results suggest the need for development of more effective methods for raising antitoxic antibodies to bee venom components in other animals as a means of developing an antiserum that would be effective for treatment of human victims of multiple bee stings.

PMID: 8780460 [PubMed - indexed for MEDLINE]

27: Toxicon. 1996 Feb;34(2):183-99.

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Synaptosomal binding of 125I-labelled daboiatoxin, a new PLA2 neurotoxin from the venom of Daboia russelli siamensis.

Maung-Maung-Thwin, Gopalakrishnakone P, Yuen R, Tan CH.

Venom & Toxin Research Group, Department of Anatomy, Faculty of Medicine, National University of Singapore, Republic of Singapore.

Daboiatoxin (DbTx), the PLA2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca2+. Scatchard analysis of biphasic plateaus revealed Kdl 5 nM and Bmax1, 6 pmoles/mg protein, and Kd2 80 nM and Bmax2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, Co2+ and Sr2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding (IC50 12.5 nM, KI 5.5 nM). Two other crotalid PLA2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and nontoxic C. Atrox PLA2 enzyme, were relatively weaker inhibitors, while two viperid PLA2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at microM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and alpha-bun-garotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at microM concentration range compared to the Kd (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca2+ and K+ channels modulators were examined, Ca2+ channel blockers (omega-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendiprene) did not affect the binding even at high concentrations, while charybdotoxin was the only K+ channel effector that could partially displace 125I-DbTx synaptosomal binding amongst the K+ channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethylammonium), suggesting that neither K+ nor Ca2+ channels are associated with DbTx binding sites.

PMID: 8711753 [PubMed - indexed for MEDLINE]

28: Adv Exp Med Biol. 1996;409:295-303.

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Allergen dose dependent cytokine production regulates specific IgE and IgG antibody production.

Blaser K.

Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland.

The elicitation of a specific immune response against allergens depends on the recognition of antigenic determinants (epitopes) by specific T and B lymphocytes. In order to determine the relevant epitopes for human T and B cells and their features in the regulation and production of specific IgE and/or IgG antibodies, we have investigated the immune response to bee venom phospholipase A2 (PLA) in allergic and non-allergic subjects. This enzyme represents the major allergen in bee sting allergy. It consists of 134 amino acid residues with a carbohydrate side chain at position 13 and is available as recombinant protein. We have developed PLA-specific T-cell clones from bee sting allergic and non-allergic human subjects. Using a panel of dodecapeptides overlapping in 10 residues and a large set of 18-25 mer overlapping peptides, we detected three epitopes that were recognized by peripheral blood T-cells and T-cell clones. A fourth determinant involved the carbohydrate moiety on Asn13 of PLA. Whereas the CHO-depending epitope seems to be mostly active in allergics, the other three epitopes are equally recognized by peripheral blood mononuclear cells (PBMC) of both allergic and non-allergic individuals. In T-cell clones, the ratio of IL-4/IFN gamma cytokines and the quality of the activating signal depend on the strength of the binding of the MHC-II/Ag/TcR complex between APC and T-cells. The number of antigen-specific APC-T-cell contact sites can be varied in vitro by changing the dose of antigen added to the cell culture. While isotype switch for both IgE and IgG4 requires IL-4, this cytokine suppresses antigen-specific IgG4 production by already switched B-cells. Therefore, IL-4 and IFN gamma display counter-regulatory effects on the production of IgE being responsible for atopic states and IgG4 antibodies which are signs of a normal immune response to allergen and act as protective antibodies. The combination of this counter-regulation of IgE and IgG4 antibodies with the fundamental law of mass action for chemical equilibrium reactions revealed that the antigen concentration governs to a great part the ratio of IL-4/IFN gamma secretion and therefore the formation of IgE and IgG and allergy or protection, together with the equilibrium constant K, which represents immunological individuality and a measure of Ag presentation.

Publication Types:

·       Review

PMID: 9095257 [PubMed - indexed for MEDLINE]

29: Clin Exp Allergy. 1995 Dec;25(12):1205-10.

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Ultra rush bee venom immunotherapy does not reduce cutaneous weal responses to bee venom and codeine phosphate.

Jutel M, Skrbic D, Pichler WJ, Muller UR.

Zieglerspital Bern, Switzerland.

BACKGROUND: The rapid administration of bee venom in cumulative doses exceeding the quantity contained in one bee sting is well tolerated by most of the patients during 3.5 h of ultra-rush bee venom immunotherapy (VIT). The mechanism of this tolerance is unknown. OBJECTIVE: The aim of the study was to verify the hypothesis that either slow mediator depletion of mast cells or blockade of their surface receptor mechanisms by increasing doses of allergen might be the major mechanisms of tolerance induced by ultra-rush VIT. METHODS: Nine bee venom allergic patients with a history of severe systemic reactions after a bee sting, positive skin tests and bee venom specific serum IgE antibodies were treated as follows: on the first day a cumulative dose of 111 micrograms was administered over 3.5 h under intensive care conditions. Further injections were given on day 7, day 21 and thereafter at 4 week intervals. Intradermal tests with codeine phosphate (non-specific mast cell degranulation) and bee venom were performed before the initiation of VIT and 30 min after the last injection on the same day as well as before the subsequent bee venom injections. RESULTS: No significant changes of skin reactivity to both codeine phosphate and bee venom were observed on day 1 (before initiation of VIT and after the last injection on the same day). CONCLUSIONS: Ultra-rush VIT does not induce mediator depletion or surface receptor blockade in skin mast cells.

PMID: 8821301 [PubMed - indexed for MEDLINE]

30: Int Immunol. 1995 Oct;7(10):1649-57.

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Administration of IL-12 during ongoing immune responses fails to permanently suppress and can even enhance the synthesis of antigen-specific IgE.

Germann T, Guckes S, Bongartz M, Dlugonska H, Schmitt E, Kolbe L, Kolsch E, Podlaski FJ, Gately MK, Rude E.

Institut fur Immunologie, Mainz, Germany.

The synthesis of antibodies of the IgE isotype in mice largely depends on IL-4, a cytokine that is released by T lymphocytes of the Th2 subtype. IL-12 is a cytokine considered to direct Th cell development into a Th1 direction and to suppress Th2 responses including the synthesis of IgE. Here we report about the influence of IL-12 on the IgE responses of mice immunized with protein antigens adsorbed to aluminum hydroxide. To avoid problems with the detection of IgE caused by an excess of competitive IgG antibodies produced in IL-12-treated mice, serum IgE was first extracted from the serum by plate-bound anti-IgE mAb and then determined either as total IgE or as antigen-specific IgE by using biotinylated anti-IgE or biotinylated antigen. Depending on the strain of mice and the dose of IL-12 injected together with the antigen, IL-12 can either temporarily suppress or augment the synthesis of (antigen-specific) IgE antibodies. This applies for CBA/J mice immunized six times in biweekly intervals with minute (0.1 micrograms/injection) or three-times with large (5 micrograms/injection) amounts of the bee venom allergen phospholipase A2 (PLA2). Under both conditions the antibody response is characterized by the production of predominantly IgG1 as well as IgE but very little IgG2a, IgG2b and IgG3 antibodies. Simultaneous application of low doses of IL-12 (1 or 10 ng/day) led to a 2- to 4-fold enhancement of IgE production (PLA2-specific IgE or total IgE). Only a high dose of 1 micrograms IL-12/day resulted in a 3- to 10-fold reduction of the IgE response. This suppression was not stable, however, because the synthesis of IgE antibodies was stimulated to a high level when these mice subsequently received a second course of immunizations in the absence of IL-12. Likewise, the synthesis of IgE was only temporarily suppressed by IL-12 treatment in CBA/J mice immunized with keyhole limpet hemocyanin (KLH) as antigen. However, application of low (10 ng/day) or high (1 microgram/day) doses of IL-12 during the primary course of immunizations of CBA/J mice with KLH suppressed the IgE response slightly or strongly respectively. In striking contrast, the KLH-specific IgE response of BALB/c mice was upregulated even when high doses of IL-12 (1 microgram/day) were injected simultaneously with the immunizations. Thus, these results demonstrate a great variability regarding the influence of IL-12 treatment on ongoing IgE responses in vivo.

PMID: 8562510 [PubMed - indexed for MEDLINE]

31: J Immunol. 1995 Sep 1;155(5):2605-13.

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A link between catalytic activity, IgE-independent mast cell activation, and allergenicity of bee venom phospholipase A2.

Dudler T, Machado DC, Kolbe L, Annand RR, Rhodes N, Gelb MH, Koelsch K, Suter M, Helm BA.

Swiss Institute of Allergy and Asthma Research, Davos, Switzerland.

The molecular and cellular mechanisms controlling Ab isotype selection following encounter of a given Ag are still unclear, although the regulatory role of cytokines is established. In the present study we explored the possibility that the nonimmunologic interaction of an allergen with cells of the innate immune system might result in a release of mediators that promote IgE isotype selection in adaptive responses. Using the bee venom allergen phospholipase A2 (PLA2) and a mutant variant lacking enzymatic function, we show that PLA2, but not its catalytically inactive variant, is able to induce IgE-independent mediator release, including IL-4, from rodent mast cells. Assessing the in vivo relevance of these observations, we find that repeated injections of low doses of active enzyme into mice induce the synthesis of high levels of PLA2-specific IgE, while immunization with the inactive form yields no detectable IgE response. Both Ags were similarly immunogenic when high doses of Ag were used for immunization. These findings suggest that mast cells might be a source of IL-4 at the onset of specific immunity against sources of allergens such as bee venom that contain PLA2 and support the concept that the biologic action of an Ag on cells of the innate immune system can play a role in determining adaptive immune responses.

PMID: 7544378 [PubMed - indexed for MEDLINE]

32: J Immunol. 1995 Apr 15;154(8):4027-31.

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Phospholipase A2-activating protein induces the synthesis of IL-1 and TNF in human monocytes.

Bomalaski JS, Ford T, Hudson AP, Clark MA.

Medical College of Pennsylvania, Philadelphia 19129, USA.

Phospholipase A2-activating protein (PLAP) is an important mediator of eicosanoid generation. PLAP can also be found in high concentrations in synovial fluid from patients with rheumatoid arthritis, and injection of PLAP into animal joints results in an inflammatory, rheumatoid-like lesion. We have demonstrated previously that TNF-alpha and IL-1 beta stimulate formation of PLAP before phospholipase A2 (PLA2) enzyme activation and production of eicosanoids. To further explore the mechanisms found in the inflammatory response, we examined the ability of PLAP to stimulate release of TNF and IL-1 from human peripheral blood monocytes. TNF and IL-1 protein levels were measured by ELISA, and IL-1 and TNF mRNA were determined by Northern blotting. PLAP, PLAP peptide, and melittin, a bee venom PLA2 activator with homology with PLAP, all increased IL-1 and TNF production in a time- and dose-dependent manner. Heat-denatured PLAP and actin (an irrelevant protein) failed to exert this effect. PLAP stimulation of TNF and IL-1 could be enhanced with co-treatment of cells with free fatty acids, such as arachidonic or linoleic acid, but it was not blocked completely by PLA2 inhibitors. These results demonstrate not only that synthesis of PLAP can be stimulated by cytokines, but also that PLAP may regulate cytokine synthesis and thus perpetuate an immune or inflammatory response.

PMID: 7706741 [PubMed - indexed for MEDLINE]

33: J Pept Sci. 1995 Mar-Apr;1(2):140-8.

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Immunogenicity of dinitrocarboxyphenylated melittin: the influence of C-terminal chain shortening, N-terminal substitution and prolin insertion at positions 5 and 10.

Zhao Z, Rolli H, Schneider CH.

Institute of Immunology and Allergology, University of Bern, Inselspital, Switzerland.

Peptides derived from the bee-venom melittin were fitted with the haptenic group dinitrocarboxyphenyl (Dncp) and tested in out-bred guinea pigs for immunogenicity by measuring the IgG anti-Dncp antibody response by ELISA. Dncp-conjugates comprising virtually the entire melittin proved to be strong immunogens producing antibody responses comparable to those of proteins. Weak responses were obtained with considerably shortened sequences. Conjugates with N-terminal Dncp gave markedly reduced antibody responses compared to peptides with C-terminal Dncp. An N-terminal biotinyl substituent abolished the immune response whereas N-terminal lauryl and caprylyl had little effect. Insertion of L-proline into a hexadecapeptide conjugate abolishing the possibility of helix formation gave an immunogen to which individual animals clearly responded on a low level. Oligomerisation, but not the cytolytic activity of melittin peptides, may contribute to the immunogenicities observed.

PMID: 9222991 [PubMed - indexed for MEDLINE]

34: Allergy. 1995 Feb;50(2):119-25.

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Anti-IgE autoantibodies and bee-sting allergy.

Yu Y, de Weck AL, Stadler BM, Muller U.

Medical Division, Zieglerspital, Bern, Switzerland.

Serum anti-IgE autoantibodies (aaIgE) were investigated by dot immunobinding assay in bee-allergic patients in comparison with nonallergic beekeepers, healthy blood donors, and atopic subjects. Elevated serum levels of aaIgE--either free or combined with IgE--were found in both bee-allergic patients and atopic subjects as compared with beekeepers and healthy donors. With regard to a possible significance of aaIgE for the protective mechanism induced by specific allergen immunotherapy, we estimated aaIgE in bee-allergic patients before, during, and after bee-venom immunotherapy (BVIT) in relation to the outcome of a provocation test with a living bee (PT). During the first year of BVIT, there was no significant change in either free or combined aaIgE, but aaIgE decreased during protracted BVIT over 3-7 years. By using two monoclonal anti-IgE antibodies with different epitope specificity (Le27 and BSW17), we were able to detect two kinds of IgE/aaIgE immune complexes. Our data show that during and after BVIT the levels of one kind of IgE-aaIgE immune complex (the non-BSW17 type) tended to decrease in PT-negative patients but stayed elevated in PT-positive patients. The levels of the other kind of immune complex (the non-Le27 type) were similar in treatment failures and successfully treated patients. These data suggest that there are various kinds of aaIgE with different in vivo functions related to their epitope specificity. Some of them (non-BSW17 type) might be associated with BVIT failure.

PMID: 7604933 [PubMed - indexed for MEDLINE]

35: Immunology. 1995 Feb;84(2):285-9.

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Isotype-associated recognition of allergen epitopes and its modulation by antigen dose.

Kolbe L, Heusser CH, Kolsch E.

Institut fur Immunologie, Universitat Munster, Germany.

The existence of competing and blocking IgG antibodies and their interrelationship with IgE antibodies was investigated in the IgE immune response of CBA/J mice to bee venom phospholipase A2 (PLA2) and keyhole limpet haemocyanin (KLH). Minute doses of antigen induced high titres of both IgE and IgG antibodies, whereas large doses elicited high IgG responses but only weak IgE antibody titres. Immunization with minute doses of antigen led to the production of antibodies with class-associated epitope specificities (designated epitopes G for IgG and E for IgE antibodies), and therefore no competition was observed. For IgG antibody production, G epitopes were immunodominant and IgG antibodies directed to E epitopes were produced only after immunization with an increased immunogen dose. Under this condition IgG antibodies recognizing the E epitopes acted as blocking antibodies competing with IgE antibodies for binding to E epitopes, the immunodominant epitopes for the IgE response. The data demonstrate the existance of distinct immunodominant antigenic determinants for IgE and IgG antibodies on PLA2.

PMID: 7538490 [PubMed - indexed for MEDLINE]

36: J Allergy Clin Immunol. 1994 Jul;94(1):61-70.

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Human monoclonal or polyclonal antibodies recognize predominantly discontinuous epitopes on bee venom phospholipase A2.

Schneider T, Lang AB, Carballido JM, Santamaria Babi LF, Dudler T, Kagi MK, Blaser K, Suter M.

Swiss Institute of Allergy and Asthma Research, Davos.

BACKGROUND: Two hybridomas, which secrete human monoclonal antibodies of IgG4 isotype specific for the main bee venom antigen/allergen phospholipase A2, were generated. The antigenic determinants recognized by these antibodies were mapped and compared with the binding sites of murine monoclonal and human polyclonal antibodies raised against the same antigen. METHODS: Two hybridomas were developed by fusing heteromyelomas to Epstein-Barr virus immortalized B cells obtained from beekeepers. The cloned hybridomas were stable and secreted up to 40 mg/L of antibody into the culture supernatant. Phospholipase A2 specificity of the human monoclonal antibodies was confirmed by binding and inhibition ELISA and by Western blot analysis. Epitope mapping on phospholipase A2 was done with the PEPSCAN method and ELISA techniques. RESULTS: The epitopes recognized by the human monoclonal antibodies were shown to be discontinuous and did not contain the sugar residue. Similar results were obtained with polyclonal antibodies of IgG4 isotype (from beekeepers) specific for phospholipase A2, which could also inhibit the binding of the human monoclonal antibodies to phospholipase A2. In contrast, antigen binding of the human monoclonal antibodies could not be inhibited by murine monoclonal antibodies against bee venom phospholipase A2. CONCLUSIONS: The data indicate that the human monoclonal antibodies obtained are representative of a part of the polyclonal immune response to phospholipase A2 from beekeepers and may allow a more precise analysis of the humoral immune response to phospholipase A2 that is associated with protection.

PMID: 7517969 [PubMed - indexed for MEDLINE]

37: J Immunol. 1994 Jun 1;152(11):5514-22.

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Antigenic surface of the bee venom allergen phospholipase A2. Structural functional analysis of human IgG4 antibodies reveals potential role in protection.

Dudler T, Schneider T, Annand RR, Gelb MH, Suter M.

Swiss Institute of Allergy and Asthma Research (SIAF), Davos.

In the human immune response to the main bee venom allergen phospholipase A2 (PLA2), Abs of the IgG4 isotype are associated with protection. The antigenic sites of PLA2 that are recognized by two mAb of this isotype have been analyzed at the molecular level. The interaction of PLA2 with these two Abs has previously been shown to depend on the conformation of the Ag and to be sensitive to lysine modifications. Therefore, genetically engineered Ag displaying single point mutations of lysine residues were generated. Nine out of twelve surface-exposed lysine residues were substituted by glutamate or glutamine, and the effect of individual mutations on mAb binding was analyzed in the context of the folded Ag. Substitution of lysine at position 25 with either glutamate or glutamine completely abrogates binding to both mAb. All other mutants do not show a difference in binding when compared with wild-type Ag. Probing of sera from bee keepers with lysine 25 mutants reveals that a significant proportion of serum Abs of the IgG4 isotype display specificity for this epitope, suggesting that this represents a major B cell-antigenic site in hyperimmune individuals. We further found that both mAbs inhibit the catalytic activity of PLA2, a property that may account for the protective role of IgG4 Abs in hyperimmune individuals.

PMID: 8189069 [PubMed - indexed for MEDLINE]

38: Pneumonol Alergol Pol. 1993;61(7-8):342-5.

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[Skin tests, total IgE and venom specific IgE and IgG4 in serum of bee-keepers]

[Article in Polish]

Nittner-Marszalska M, Malolepszy J, Zak-Nejmark T.

Katedry i Kliniki Chorob Wewnetrznych i Alergologii AM, Wroclawiu.

In 14 occupationally active bee-keepers, who tolerated stinging well, two diagnostic tests have been performed: 1. skin test at the venom concentration of 10(-3) g/l, 2. both total and venom specific IgE and IgG4 assessments. The study group consisted of 13 male and 1 female aged 34-56 (mean age 44.3). 7 out of 14 bee-keepers (50%) had positive skin tests; mean size being mean = 3.57 +/- 4.07 mm. None of the subjects showed increased total IgE levels, but 71.4% showed bee venom specific IgE (mean mean 1.64 +/- 1.34 FAST). Specific bee venom IgG4 occurred in 13 out 14 individuals (92.8%). Their average serum level was mean 21.74 +/- 17.16 IU/ml.

PMID: 8401338 [PubMed - indexed for MEDLINE]

39: Clin Exp Allergy. 1992 Oct;22 Suppl 3:1-44.

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Allergy. Conventional and alternative concepts. A report of the Royal College of Physicians Committee on Clinical Immunology and Allergy.

Kay AB, Lessof MH.

Allergy is an exaggerated response of the immune system to external substances. It plays a role in a wide range of diseases. In some, such as summer hayfever, the symptoms are due entirely to allergy. In other conditions, particularly asthma, eczema and urticaria, allergy plays a part in some patients but not all. In these situations, allergy may have either a major role or provide just one of many triggers. In an individual patient's illness, the importance of allergy may change with time. The most common allergens (substances causing allergy) are grass and tree pollens, the house dust mite, products from pets and other animals, agents encountered in industry, wasp and bee venom, drugs, and certain foods. Food allergy presents a particularly difficult problem. Some individuals who react to food suffer from food allergy in its strict sense but in others there is no evidence of an alteration in the immune system. Here the term 'food intolerance' is preferable. Conventional doctors treat allergy by allergen avoidance--where this is possible--and drugs that relieve symptoms. In a few selected cases, in which other methods have failed, immunotherapy (desensitisation or hyposensitisation) is recommended. Although patients who consult practitioners of alternative allergy may do so by preference, it is often also because they are dissatisfied with the conventional approach to diagnosis and treatment, or because they have conditions which conventional doctors do not accept as having an allergic basis. There is a very wide range of alternative approaches to allergy, including the methods used by clinical ecologists and other treatments such as acupuncture and homoeopathy. Hypnosis may have a small role to play in helping the asthmatic and similar effects have been suggested for acupuncture. Furthermore, it is likely that there are still many active ingredients in medicinal plants used by herbalists but these need to be clearly identified and purified before their usefulness can be evaluated properly. Apart from these situations, we have yet to be convinced by substantial evidence that any of the other alternative methods of diagnosing or treating allergic disease are of proven value. There have, however, been many false and misleading claims and serious harm may be caused by misdiagnosis or delays in appropriate treatment. The public should be warned against costly methods of diagnosis and treatment which have not been validated.(ABSTRACT TRUNCATED AT 400 WORDS)

Publication Types:

·       Review

PMID: 1422946 [PubMed - indexed for MEDLINE]

40: J R Coll Physicians Lond. 1992 Jul;26(3):260-4.

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Allergy: conventional and alternative concepts. Summary of a report of the Royal College of Physicians Committee on Clinical Immunology and Allergy.

[No authors listed]

Allergy is an exaggerated response of the immune system to external substances. It plays a role in a wide range of diseases. In some, such as summer hayfever, the symptoms are entirely due to allergy. In other conditions, particularly asthma, eczema and urticaria, allergy plays a part in some patients but not all. In these situations, allergy may either have a major role or provide just one of many triggers. In an individual patient's illness, the importance of allergy may change with time. The most common allergens (substances causing allergy) are grass and tree pollens, the house dust mite, products from pets and other animals, agents encountered in industry, wasp and bee venom, drugs, and certain foods. Food allergy presents a particularly difficult problem. Some individuals who react to food suffer from true food allergy but in others there is no evidence of an alteration in the immune system. Here the term 'food intolerance' is preferable. Conventional doctors treat allergy by allergen avoidance--where this is possible--and drugs that relieve symptoms. In a few selected cases, in which other methods have failed, immunotherapy (desensitisation or hyposensitisation) is recommended. Patients who consult practitioners of alternative allergy often do so because they are dissatisfied with the conventional approach to diagnosis and treatment, and sometimes because they have conditions which conventional doctors do not accept as having an allergic basis. There is a very wide range of alternative approaches to allergy, including the methods used by clinical ecologists, acupuncturists and homoeopathists. Hypnosis may have a small role to play in asthma, and similar claims for acupuncture need to be evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication Types:

·       Guideline

·       Practice Guideline

PMID: 1404018 [PubMed - indexed for MEDLINE]

41: Eur J Immunol. 1992 Jun;22(6):1357-63.

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Bee venom phospholipase A2-specific T cell clones from human allergic and non-allergic individuals: cytokine patterns change in response to the antigen concentration.

Carballido JM, Carballido-Perrig N, Terres G, Heusser CH, Blaser K.

Swiss Institute of Allergy and Asthma Research, Davos.

Protein antigens with both allergenic and immunoprotective properties represent appropriate molecules to study IgE and IgG regulation. We have established a panel of T cell clones specific to bee venom phospholipase A2 (PLA) from human individuals allergic, hyposensitized or immune (protected) to bee sting. All clones obtained were CD3+, CD4+ and expressed alpha, beta T cell receptor. Depending on the T cell clone, maximal stimulation required 1 to 100 micrograms/ml of PLA, and the addition of interleukin (IL)-2 and/or IL-4 increased their antigen-dependent proliferation. Following antigen stimulation, the clones produced IL-4, interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor. Most clones also produced tumor necrosis factor alpha (TNF-alpha) and tumor necrosis factor beta (TNF-beta), and some produced IL-5 and/or IL-2. Both absolute and relative amounts of secreted cytokines depended on the antigen concentration. At low antigen doses, IL-4 was produced but little or not IFN-gamma, whereas at higher PLA concentrations significant amounts of both IL-4 and IFN-gamma were obtained. Thus, these PLA-specific T cell clones could be classified according to the changes in the ratio of IL-4/IFN-gamma production in response to increasing antigen concentrations. Clones derived from allergic and hyposensitized individuals required higher critical amounts of antigen for IFN-gamma induction, and expressed increasing IL-4/IFN-gamma ratios with increasing concentrations of PLA. Modulation of cytokine patterns by the dose of the antigen may be a driving force for IgE or IgG formation resulting in allergy or immunoprotection.

PMID: 1601030 [PubMed - indexed for MEDLINE]

42: Asian Pac J Allergy Immunol. 1991 Dec;9(2):131-6.

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Immunoblot analysis of IgE and IgG antibodies to honey bee venom: cross sectional and sequential studies in bee sensitive subjects.

Roberts-Thomson PJ, Koh S, Shepherd K, Kupa A, Heddle RJ.

Department of Clinical Immunology, Flinders Medical Centre, Bedford Park.

To investigate the specific IgE and IgG immune response to honey bee venom (bv), we performed immunoblot analysis of sera from 47 bee sensitive subjects and followed the response during and after venom immunotherapy in 15 of these subjects. Fifteen venom proteins varying in molecular size from 20 to 105 kDa were identified as being antigenic and consisted of a high molecular weight (HMW) group (5 to 105 kDa, containing the previously identified allergens B and C) and a low molecular weight group (LMW) containing hyaluronidase and phospholipase A. In general for a given individual the anti-venom IgE and IgG response was qualitatively similar although some variation between individuals was apparent. Reactivity with hyaluronidase and phospholipase A appeared only in those subjects showing reactivity with HMW components. During immunotherapy specific anti-venom IgG and IgE responses tended to be linked. Increased responses being seen against all components in 4 of 12 subjects, reductions in 3 and unchanged responses in the remainder. Following immunotherapy (mean 4.0 years), spontaneous reduction of IgE and IgG was seen in 5 of 5 subjects.
Loss of reactivity with the LMW components was prominent in these sera.

PMID: 1807261 [PubMed - indexed for MEDLINE]

43: Patol Fiziol Eksp Ter. 1991 Jul-Aug;(4):22-4.

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[The effect of zootoxins on the formation of delayed hypersensitivity]

[Article in Russian]

Romanova EB, Podoplelov II.

The effect of bee venom (0.05 and 0.5 mg/kg) and the venom of Vipera lebetina (0.05 and 0.5 mg/kg) on the reaction of delayed hypersensitivity was studied in experiments on CBA mice. The character and trend of the effect was determined by the zootoxin species. The venom of Vipera lebetina completely suppressed the cell immune reaction in a dose of 0.5 mg/kg and produced an adjuvant effect in a dose of 0.05 mg/kg. Bee venom (0.5 mg/kg) failed to cause a suppressive effect and in a small dose (0.05 mg/kg) stimulated cellular immunity. The adjuvant properties of the venoms were comparable with the effect of Evans' blue dye.

PMID: 1798644 [PubMed - indexed for MEDLINE]

44: Q Rev Biol. 1991 Mar;66(1):23-62.

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The function of allergy: immunological defense against toxins.

Profet M.

Division of Biochemistry & Molecular Biology, University of California, Berkely 94720.

This paper proposes that the mammalian immune response known as "allergy" evolved as a last line of defense against the extensive array of toxic substances that exist in the environment in the form of secondary plant compounds and venoms. Whereas nonimmunological defenses typically can target only classes of toxins, the immune system is uniquely capable of the fine-tuning required to target selectively the specific molecular configurations of individual toxins. Toxic substances are commonly allergenic. The pharmacological chemicals released by the body's mast cells during an IgE antibody-mediated allergic response typically cause vomiting diarrhea, coughing, tearing, sneezing, or scratching, which help to expel from the body the toxic substance that triggered the response; individuals frequently develop aversions to substances that have triggered such responses. A strong allergic response often includes a decrease in blood pressure, which slows the rate at which toxins circulate to target organs. The immune system identifies as toxic the following kinds of substances: (1) those low-molecular-weight substances that bind covalently to serum proteins (e.g., many plant toxins); (2) nontoxic proteins that act as carriers of toxins with low molecular weights (e.g., plant proteins associated with plant toxins); (3) specific substances of high molecular weight that harmed individuals in ancestral mammalian populations for a span of time that was significant from the standpoint of natural selection (e.g., the toxic proteins of bee venom. Substances that bind covalently to serum proteins generally are acutely toxic, and because many of these substances also bind covalently to the DNA of target cells, they are potentially mutagenic and carcinogenic as well. Thus, by protecting against acute toxicity, allergy may also defend against mutagens and carcinogens. The toxic hypothesis explains the main phenomena of allergy; why IgE-mediated allergies usually occur within minutes of exposure to an allergen and why they are often so severe; why the manifestations of allergy include vomiting, diarrhea, coughing, sneezing, scratching, tearing, and a drop in blood pressure; why covalent binding of low-molecular-weight substances to serum proteins frequently causes allergy; why allergies occur to many foods, pollens, venoms, metals, and drugs; why allergic cross-reactivity occurs to foods and pollen from unrelated botanical families; why allergy appears to be so capricious and variable; and why allergy is more prevalent in industrial societies than it is in foraging societies. This hypothesis also has implications for the diagnosis, prevention, and treatment of allergy.

Publication Types:

·       Review

PMID: 2052671 [PubMed - indexed for MEDLINE]

45: Biochim Biophys Acta. 1990 Sep 18;1046(2):111-9.

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Acyl carrier protein interacts with melittin.

Ernst-Fonberg ML, Williams SG, Worsham LM.

Department of Biochemistry, James H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614-0002.

Acyl carrier protein (ACP) from Escherichia coli has been shown to form complexes with melittin, a cationic peptide from bee venom. ACP is a small (Mr 8847), acidic, Ca2(+)-binding protein, which possesses some characteristics resembling those of regulatory Ca2(+)-binding proteins including interaction with melittin. Complexing between melittin and ACP which occurred both in the presence and absence of Ca2+ was evident by chemical cross-linking the two peptides, fluorescence changes (including anisotropy measurements), and inhibition by melittin of the activity of a nonaggregated fatty acid synthetase from Euglena. Also, anti-Apis mellifera antibodies which contained antibodies against melittin specifically inhibited the same enzyme system activity relative to non-immune IgG.

PMID: 2223852 [PubMed - indexed for MEDLINE]

46: Proc Natl Acad Sci U S A. 1990 Aug;87(15):5643-7.

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Folding and activity of hybrid sequence, disulfide-stabilized peptides.

Pease JH, Storrs RW, Wemmer DE.

Department of Chemistry, University of California, Berkeley 94720.

Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a beta-turn and an alpha-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. We show that in S peptide the alpha-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure-function relationships for many biologically active sequences.

PMID: 2377603 [PubMed - indexed for MEDLINE]

47: J Allergy Clin Immunol. 1990 Aug;86(2):160-70.

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An HLA-associated nonresponsiveness to mellitin: a component of bee venom.

Lympany P, Kemeny DM, Welsh KI, Lee TH.

Department of Allergy and Allied Respiratory Disorders, United Medical and Dental Schools, Guy's Hospital, London, England.

Previous work has demonstrated a close association between certain histocompatibility antigens and the gene that controls the IgE response to certain ragweed allergens. For example, there is a 90% association between IgE production to the short ragweed allergen, Amb a V, and an HLA class II allele. To assess whether these HLA linkages are specific for ragweed, we have investigated the association between HLA antigens and the capacity of individuals to mount a specific IgE response to melittin in patients with bee-venom allergy. Twenty-two subjects with bee-venom sensitivity, 22 healthy beekeepers without bee-venom allergy, and a normal population of 149 unselected individuals were studied. With serologic tissue typing and restriction fragment length polymorphism analysis, we have demonstrated a significant decrease in the HLA-DR4 and DQw3 alleles in subjects who are allergic to melittin compared to the control populations. There was also a negative association between the presence of HLA-DR4 and DQw3 alleles with the capacity of the individuals to mount an IgE response to phospholipase A2 (PLA2). The bee-venom sensitive subjects had a slightly lower titer of anti-PLA2 IgG when these subjects were compared to the bee-venom insensitive beekeepers. These results support the view that either HLA-DR or HLA-DQ has a protective role in controlling the IgE immune response. Lack of an IgE response to melittin or PLA2 is unlikely to be due to a failure to recognize allergen.

PMID: 1974559 [PubMed - indexed for MEDLINE]

48: Cryobiology. 1989 Jun;26(3):277-84.

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The gravimetric method for the determination of residual moisture in freeze-dried biological products.

May JC, Wheeler RM, Grim E.

Department of Health and Human Services, Food and Drug Administration, Bethesda, Maryland 20892.

The gravimetric test for the determination of residual moisture in freeze-dried biological products performed in a humidity- and temperature-controlled room with the use of scrupulous gravimetric analytical technique can be used to accurately determine residual moisture in freeze-dried biological products such as antihemophilic factor (human) or honey bee venom allergenic extract. This method determines the first water of hydration of sodium tartrate dihydrate (7.93%) to within 1.3% of the calculated value with a relative standard deviation of 0.3% for 10 replicates. For this gravimetric procedure, freeze-dried samples containing from 1.12 to 4.4% residual moisture had relative standard deviations ranging from 3.6 to 9.1%. Samples containing less than 1.0% residual moisture by the gravimetric method such as intravenous immune globulin and antihemophilic factor (human) had relative standard deviations ranging from 16.7 to 47.0%. Relative standard deviations for residual moisture tests performed on comparable samples by the Karl Fischer and thermogravimetric methods showed similar variability.

PMID: 2743789 [PubMed - indexed for MEDLINE]

49: J Immunol. 1989 Jun 1;142(11):3957-62.

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A phospholipase A2-activating protein (PLAP) stimulates human neutrophil aggregation and release of lysosomal enzymes, superoxide, and eicosanoids.

Bomalaski JS, Baker DG, Brophy L, Resurreccion NV, Spilberg I, Muniain M, Clark MA.

Veterans Administration Medical Center, Medical College of Pennsylvania, Philadelphia 19104.

We have recently isolated a human phospholipase A2-activating protein (PLAP) that shares antigenic and biochemical similarities with melittin, a well characterized bee venom phospholipase-stimulatory peptide. To explore the potential mechanisms of action of PLAP that extend beyond its effects on eicosanoid synthesis, we examined its effects on the release of human neutrophil lysosomal enzymes and superoxide, and on RBC hemolysis. These results were compared to the effects of melittin, which has been reported to induce enzyme release and hemolysis. We also examined the effects of PLAP on neutrophil aggregation and chemotaxis. PLAP induced neutrophils to release beta-glucuronidase and metalloproteinase enzyme activities as well as produce superoxide ion in both a dose- and time-dependent manner. Eicosanoid synthesis inhibitors did not abrogate these responses. PLAP induced release of arachidonic acid metabolites, but this response could be abrogated by eicosanoid synthesis inhibitors. PLAP also induced neutrophil aggregation and chemokinesis, but not chemotaxis. Concentrations of PLAP that induced these responses did not induce cellular toxicity as determined by light and electron microscopy, lactic dehydrogenase release, trypan blue dye exclusion, and RBC hemolysis. In contrast, prolonged incubation with higher concentrations of PLAP induced cell death that was similar to that observed with melittin. These findings suggest that the mechanisms of action of PLAP extend beyond the eicosanoid synthetic pathway, and that disordered regulation of PLAP may be responsible, at least in part, for chronic immune and inflammatory states.

PMID: 2541202 [PubMed - indexed for MEDLINE]

50: J Immunol Methods. 1989 Apr 21;119(1):1-8.

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Quantification of IgG and IgG4 antibodies to bee venom phospholipase A2 by competitive inhibition in ELISA.

Rieben R, Blaser K.

Laboratory of Molecular Immunology, University of Bern, Switzerland.

Phospholipase A2 (PLA) is the major antigen of bee venom. Individuals often stung by bees, such as bee keepers, show a restricted immune response mainly of anti-PLA IgG4 antibodies. In contrast, patients allergic to bee venom produce high levels of PLA-specific IgE. This isotype restriction, the clinical relevance and the well defined structure of the PLA antigen, provide a useful model for the study of the principles regulating isotype expression in the human antibody response. A fundamental requirement for such studies is the availability of quantitative and sensitive assays to measure PLA-specific antibodies. Here we describe an ELISA method for direct isotype-specific quantification of anti-PLA IgG and IgG4 antibodies. Serum containing anti-PLA IgG antibodies was added at a predetermined dilution to PLA coated microtiter plates. Then mouse monoclonal antibodies to human IgG or IgG4 and different concentrations of purified human IgG and IgG4, respectively, were added simultaneously. The concentration of anti-PLA IgG and IgG4 antibodies in the serum was calculated from the resulting inhibition curve. Additionally, an analytical method to compare unknown antibody samples with a standard in ELISA - avoiding problems of different affinities - is described. Using the technique described here, isotype-specific quantification of anti-PLA antibodies can be performed at a sensitivity of approximately 70 pg/ml with a reproducibility range of 10-15%.

PMID: 2708823 [PubMed - indexed for MEDLINE]

51: Int Arch Allergy Appl Immunol. 1989;89(1):43-8.

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Histamine release from peripheral blood leukocytes with purified bee venom allergens: effect of hyperimmune beekeeper plasma.

Clinton PM, Kemeny DM, Youlten LJ, Lessof MH.

Department of Allergy and Allied Respiratory Disorders, United Medical School of Guy's Hospital, London, UK.

The response of 15 strongly bee-venom-allergic patients to highly purified venom allergens was compared using skin prick test titration, peripheral blood leukocyte (PBL) histamine release and radioallergosorbent test with three highly purified bee venom allergens: phospholipase (PLA2), hyaluronidase (HYAL) and acid phosphatase (ACID P). Sensitivity to the three allergens ranked in the same order for all three tests and in each case PLA2 was found to the most potent allergen. In the presence of hyperimmune beekeeper plasma, maximum histamine release was reduced significantly for all three allergens (p less than 0.001). Furthermore, hyperimmune beekeeper plasma increased the amount of allergen required for a comparable release of histamine (mean shift in dilution curve PLA2 917-fold; HYAL, 492-fold; ACID P, 61-fold). The release of histamine from whole blood was also compared with PBL + 10% normal human serum (NHS). For all three allergens maximum release was much lower from whole blood compared with washed cells + 10% NHS (p less than 0.001). These data confirm PLA2 as the major bee venom allergen by all three tests. Hyperimmune beekeeper plasma reduces maximum histamine release and increases its threshold. Histamine release in response to ACID P appears harder to block with hyperimmune beekeeper plasma than that provoked by PLA2 or HYAL (p less than 0.01). Whole blood releases less histamine and requires more allergen than washed cells, indicating that sensitivity of PBL in vivo is unlikely to be as great as washed PBL in vitro.

PMID: 2471697 [PubMed - indexed for MEDLINE]

52: Boll Ist Sieroter Milan. 1988;67(5-6):386-92.

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[Epidemiological and clinical study on bee venom allergy among beekeepers]

[Article in Italian]

Pastorello EA, Incorvaia C, Sarassi A, Qualizza R, Bigi A, Farioli L.

Istituto di Clinica Medica I, Universita degli Studi di Milano.

A randomized population of 222 beekeepers from Lombardy (203 males, 19 females, of mean age 42.5 years) was studied to determine the frequency of allergic reactions to bee sting. The type of reactions, the clinical evolution at the subsequent stings and the risk factors concerning the development of allergy (presence of venom specific IgE, number of stings in a year, atopy, age) were evaluated. It was found that 170 beekeepers never presented reactions to stings while 52 (23.4%) showed allergic reactions consisting in 31 large local reactions and 21 systemic reactions; of these, 3 (5.7%) were life-threatening. In the group of beekeepers with allergic reactions at the subsequent stings, 26 (50%) showed a spontaneous loss of reactivity, 16 (30.8%) presented persistent, but unchanged in severity, reactions and 10 (19.2%) had a worsening of symptoms. Specific honey bee venom IgE levels (measured by means of RAST) were significantly lower in immune beekeepers when compared with the group with allergic reactions (p less than 0.01) and in beekeepers with previous allergy when compared to the ones with persistent reactions (p less than 0.05). We also found significant differences about the number of stings received in a year by beekeepers with persistent allergic reactions (17.5 stings), beekeepers with previous allergy (89.8 stings) and immune subjects (126.9 stings). On the contrary, no significant differences were detected about the age and the presence of atopy. These results suggest that practice of bee-keeping induces a relatively high incidence of allergic reactions but with a trend to the spontaneous improvement of symptoms and a low incidence of severe reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 3154984 [PubMed - indexed for MEDLINE]

53: Int Arch Allergy Appl Immunol. 1988;87(4):361-6.

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Regulation of the immune response to allergens by immunosuppressive allergenic fragments. 1. Peptic fragments of honey bee venom phospholipase A2.

Litwin A, Pesce AJ, Michael JG.

Department of Microbiology and Molecular Genetics, University of Cincinnati College of Medicine, Ohio.

Nine patients with anaphylactic sensitivity to honey bee venom (HBV) were treated with P-1, a pepsin derived fragment of HBV phospholipase A2 (PLA2). P-1 caused only rare reactions with doses of 100 micrograms/injection. Treatment resulted in a substantial decrease in specific anti-PLA2 IgE and IgG antibodies as well as a decline in skin test sensitivity to PLA2. Another group of HBV-sensitive patients was treated with unaltered PLA2. Doses greater than 20 micrograms/injection were not tolerated. PLA2 injections caused an increase in anti-PLA2 IgG and IgE antibodies as well as increase in skin test sensitivity. This study demonstrates that a nonimmunogenic fragment derived from an allergen can downregulate immune responses and thus offer a new modality for therapy of allergic diseases.

PMID: 3147947 [PubMed - indexed for MEDLINE]

54: J Immunol Methods. 1987 Nov 23;104(1-2):223-9.

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An indirect enzymoimmunological assay for hyaluronidase.

Delpech B, Bertrand P, Chauzy C.

Laboratory of Immunochemistry, Centre Henri-Becquerel, Rouen, France.

The use of a hyaluronic acid-binding proteoglycan (hyaluronectin) as a probe for the detection of hyaluronic acid has facilitated the development of an indirect enzymo-immunological assay for hyaluronidase. Plastic microtest ELISA plates were coated with hyaluronic acid. Incubation with hyaluronidase led to the destruction of insolubilized hyaluronic acid in proportion to the hyaluronidase concentration of samples. Residual hyaluronic acid was assayed by its capacity to bind immune complexes made up of hyaluronectin supplemented with alkaline phosphatase-conjugated anti-hyaluronectin antibodies. The technique was very sensitive and permitted the detection of as little as 10(-10) NFU of bovine testicular hyaluronidase. Hyaluronidase was detected by this technique in human sera, bee venom and culture medium of human hepatoma cell lines.

PMID: 3316396 [PubMed - indexed for MEDLINE]

55: Int Arch Allergy Appl Immunol. 1987;83(2):113-20.

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Immune response to bee venom. II. Quantitation of the absolute amounts of IgE and IgG antibodies by saturation analysis.

Kemeny DM, Lessof MH.

We have measured in allergic and non-allergic beekeepers IgE and IgG antibodies to the bee venom allergens, phospholipase A2 (PLA2) and hyaluronidase (HYAL), by the radioallergosorbent test (RAST) and a 125I-radiolabelled antigen-binding assay. The absolute amount of IgG antibody in a reference serum was determined by saturation analysis using 125I-radiolabelled PLA2 and HYAL. Using monoclonal anti-IgE coated microtitre plates, the absolute amount of IgE antibody to the same antigen was also determined by saturation analysis. Regardless of the IgE response to the different allergens, IgG antibody concentrations to PLA2 were invariably higher than those to HYAL. In addition, the ratio of IgG to IgE antibody was higher for PLA2 (220:1) than for HYAL (10:1). Higher levels of IgG antibody to both allergens (especially HYAL) were found in those who had had prolonged exposure to bee stings. These data suggest that the level of IgG antibody produced is related to the dose administered, while the amount of IgE antibody may be regulated by other factors.

PMID: 3583409 [PubMed - indexed for MEDLINE]

56: J Biol Stand. 1986 Oct;14(4):363-75.

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A survey of the concentrations of eleven metals in vaccines, allergenic extracts, toxoids, blood, blood derivatives and other biological products.

May JC, Rains TC, Maienthal FJ, Biddle GN, Progar JJ.

Approximately 85 samples of injectable biological products regulated by the Center for Drugs and Biologics of the United States Food and Drug Administration were surveyed for the presence of 11 elements, namely aluminum, arsenic, barium, cadmium, chromium, lead, mercury, selenium, thallium and zinc, by flame and flameless methods of atomic absorption spectrometry and flame emission spectrometry. The range of products tested included whole blood, red cells, plasma, normal serum albumin, antihemophilic factor, and other products derived from blood; allergenic extracts including honey bee venom and house dust allergenic extracts; vaccines such as measles virus vaccine and typhoid vaccine; and tetanus toxoid. The metal concentrations found in the majority of these products were low or undetectable. The metal levels varied from manufacturer to manufacturer, product and lot-to-lot of the same manufacturer's products. House dust allergenic extracts had the highest concentrations of arsenic (2.4 ppm), cadmium (0.28 ppm), chromium (0.6 ppm) and lead (1.5 ppm) found in the study. A high zinc concentration (24 ppm) in an immune serum globulin was attributed to the zinc-containing rubber stopper in contact with the product. A range of 0.36-3.30 ppm aluminum was found for seven 25% normal serum albumin samples from seven manufacturers. Values of 8.2, 17 and 18 ppm aluminum were found in one manufacturer's 25% normal serum albumin. These aluminum values appeared to be the result of an anomaly in this manufacturer's production that has not been repeated to date.

PMID: 3558419 [PubMed - indexed for MEDLINE]

57: J Allergy Clin Immunol. 1986 Jul;78(1 Pt 1):25-30.

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Lack of responsiveness of beekeeper mononuclear cells to in vitro stimulation with pure bee venom.

Lomnitzer R, Rabson AR.

Lymphocyte proliferation activity after in vitro bee venom (BV) stimulation was examined in a group of patients allergic to bee stings and in a group of beekeepers. Although the allergic patients responded strongly to increasing doses of BV, the beekeepers demonstrated no proliferative activity and an inability to produce interleukin-2 after BV stimulation. Removal of adherent cells or various populations of suppressor cells, including T gamma cells and OKT8 positive cells, did not influence the cellular unresponsiveness of cells of beekeepers after BV stimulation. Furthermore, cells of beekeepers, when they were trypsinized or when they were preincubated for 72 hours, did not proliferate after BV challenge. It is concluded that the lack of proliferation of lymphocytes of beekeepers and the inability to produce interleukin-2 is not due to a suppressor mechanism or to the presence of anti-idiotype antibodies coating the surface of lymphocytes of beekeepers. The mechanism behind the failure of cells of beekeepers to proliferate remains unclear.

PMID: 3487563 [PubMed - indexed for MEDLINE]

58: Clin Exp Immunol. 1986 May;64(2):415-22.

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Inhibition of complement activation by IgG4 antibodies.

van der Zee JS, van Swieten P, Aalberse RC.

Prolonged exposure to antigens may result in high IgG4 antibody titres as was shown in a previous paper (Aalberse et al., 1983b). In novice bee keepers, a shift in the IgG1/IgG4 ratio of the response against phospholipase-A (PLA; a major component of bee venom) occurred. This resulted in an IgG4-dominated response after approximately 2 years of bee-keeping experience. Subject of the present study was the influence of relatively high concentrations of IgG4 antibodies on the biological activity of immune complexes. In the PLA antigen model, it was demonstrated that IgG4-containing immune complexes do not activate complement and that IgG4 antibodies effectively inhibit immune precipitation and complement activation by IgG1 antibodies. Evidence is provided that IgG4 antibodies inhibit binding of C1q to IgG1 in mixed, IgG1- and IgG4-containing complexes. It is proposed that IgG4 antibodies protect against the biological effects of the complement-fixing IgG subclasses. For this reason, determination of the total IgG response or just determination of antibody activity in the complement-fixing isotypes is insufficient in immune-complex diseases. The modulating effect of the non-complement-fixing isotypes should be taken into account to predict the biological activity of the immune complexes.

PMID: 3488859 [PubMed - indexed for MEDLINE]

59: Toxicon. 1986;24(5):435-40.

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Immunological effects of honey bee (Apis mellifera) venom using BALB/c mice.

Hyre HM, Smith RA.

Honey bee venom at concentrations up to 50 micrograms/ml was not toxic in vitro for unstimulated murine splenic lymphocytes, as assayed by tritiated thymidine incorporation. However, venom concentrations greater than 6 micrograms/ml significantly (P less than 0.01) inhibited concanavalin A (con A) simulation of murine lymphocytes. In contrast to this finding, the splenic lymphocytes from mice receiving 4 s.c. injections of honey been venom on alternate days were significantly (P less than 0.01) enhanced in their proliferative response to con A compared to a sham-injected group of mice. Mice injected with the venom prior to and following an injection of sheep red blood cells produced significantly more direct IgM plaques (P less than 0.02) than the sham-injected group. In this study both suppression and enhancement of immune reactivity was seen using honey bee venom when assayed on the splenic lymphocytes of BALB/c mice.

PMID: 3520958 [PubMed - indexed for MEDLINE]

60: Immunogenetics. 1986;23(6):417-20.

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Immune response gene control of the murine antibody response to the phospholipase A2 of honey bee venom.

Lucas A, Hamburger RN.

PMID: 3087870 [PubMed - indexed for MEDLINE]

61: Clin Exp Immunol. 1984 Aug;57(2):449-53.

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Humoral and cellular immune response of the rat to immunization with bee venom.

Schneider H, Urbanek R.

Lewis rats were immunized with bee venom allergen in Freund's complete adjuvant (FCA) or with FCA only. Animals immunized with bee venom developed specific IgG antibodies but no specific IgE antibodies were detected. Lymphocytes from lymph nodes when cultured with antigen in vitro showed an increased stimulation index from day 17 onwards. A concomitant augmentation of T suppressor cells was observed; the T helper/T suppressor cell ratio declined from 4.5:1 before immunization to 1:1 from day 5 onwards.

PMID: 6235990 [PubMed - indexed for MEDLINE]

62: Clin Allergy. 1984 Jul;14(4):341-50.

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Bee keepers' IgG and IgE antibody responses to bee venom studied by means of crossed radioimmunoelectrophoresis.

Nordvall SL, Uhlin T, Einarsson R, Johansson SG, Ohman S.

The immune response to honey bee venom in thirty-seven bee keepers' sera was studied by several methods. Specific IgE antibody levels studied by RAST were generally low, whereas specific IgG antibody levels studied by a Sepharose protein A technique were high. Crossed radioimmunoelectrophoresis was applied for a detailed analysis of the antibody specificities towards the different components of venom in seventeen of the bee keepers' sera. Significant amounts of IgG antibodies were found towards most bee-venom components. The highest IgG response was directed towards phospholipase A. Hyaluronidase, acid phosphatase and two uncharacterized antigens also showed distinct IgG binding. The IgG binding to melittin was low. The IgE binding to the bee venom components was low and primarily directed to the phospholipase. IgE binding to hyaluronidase and acid phosphatase occurred, but was also in very small amounts. One bee-keeper serum caused heavy radiostaining to melittin but the others did not show IgE binding to this component. Thus a low IgE but a high IgG response was demonstrated in bee keepers. The major immunogen was phospholipase A, which is known to be the major allergen in bee venom. Generally, the strongest IgG responses were found to the components capable of inducing the strongest IgE responses.

PMID: 6205790 [PubMed - indexed for MEDLINE]

63: Agents Actions. 1984 Jun;14(5-6):662-6.

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Inhibitory effect of honey bee venom on immune complex mediated leukocyte migration into rabbit knee-joints.

Thomsen P, Bjursten LM, Ahlstedt S, Bagge U, Bjorksten B.

The anti-inflammatory effect of purified honey bee venom (HBV) was studied using a recently described animal model in which preformed immune complexes were injected into rabbit knee-joints. As little as a single injection of 1.2 micrograms HBV/kg body weight subcutaneously significantly reduced the immune complex induced joint inflammation as measured by reduction in leukocyte counts in the joint fluid. This decrease was obvious 3 and 6 but not 9 hours after induction of the inflammation. There was no significant effect on leukocyte random migration, chemotactic responsiveness or phagocytosis, indicating that HBV did not interfere with normal phagocyte motility and ingestion. The modifying effects by HBV on the inflammatory response to immune complexes in vivo is most likely due to interference with other components of the inflammatory response.

PMID: 6475661 [PubMed - indexed for MEDLINE]

64: Dev Comp Immunol. 1984 Fall;8(4):791-801.

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The humoral immune response in the American cockroach, Periplaneta americana: reactivity to a defined antigen from honeybee venom, phospholipase A2.

Rheins LA, Karp RD.

Our previous experiments demonstrated that honeybee venom could induce a specific, adaptive humoral immune response in the American cockroach. Since honeybee venom is a complex substance made up of several proteins, a more defined antigen is needed for future characterization studies. One of the components of bee venom, phospholipase A2 (PA2) was found to be highly lethal and immunogenic in the roach. Roaches injected with PA2 generated a specific primary response that developed over a period of time, peaking within 10 days, and then gradually subsiding by the fifth week. Specificity of this response was demonstrated by the fact that immunized animals were protected against the original immunizing PA2, but not to PA2 from a heterologous source. In addition, a secondary response could be induced with PA2, demonstrating the existence of immunologic memory. Thus, we established that PA2 could induce as good, if not better, humoral responsiveness as whole bee venom, and therefore could be utilized as a more defined antigen in studies designed to characterize the inducible humoral factor in the roach.

PMID: 6519337 [PubMed - indexed for MEDLINE]

65: Biochem Pharmacol. 1982 Mar 15;31(6):1139-46.

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Effect of honeybee (Apis mellifera) venom on the course of adjuvant-induced arthritis and depression of drug metabolism in the rat.

Eiseman JL, von Bredow J, Alvares AP.

The ability of honeybee venom to suppress Mycobacterium butyricum-induced arthritis was studied in Lewis rats. Bee venom, 2 mg.kg-1.day-1 for 24 days, suppressed but did not abolish the primary and secondary inflammatory responses to the adjuvant as monitored by decreases in the swelling of the left and right hind paws and adjuvant-induced arthritis on heme metabolism were also examined. Bee venom or adjuvant had no effect on hepatic delta-aminolevulinic acid synthase, porphyrin content, or ferrochelatase activity. However, with both treatments cytochrome P-450 and the associated enzymic activities of ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase were depressed markedly. In contrast, both treatments caused several-fold enhancement of hepatic microsomal heme oxygenase activity. Adjuvant-treated rats receiving bee venom showed changes in heme metabolism which were of a magnitude similar to those observed when either agent was administered to the experimental animals. Although the bee venom appears to suppress adjuvant-induced arthritis to a greater extent in female than in male rats, the alterations in heme metabolism were similar in bee venom-treated male and female rats. The observed changes in heme metabolism elicited by the venom or by the adjuvant are strongly suggestive of perturbations of the immune system causing alterations in hepatic microsomal enzymes.

PMID: 6177321 [PubMed - indexed for MEDLINE]

66: Int Arch Allergy Appl Immunol. 1982;68(3):268-74.

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The immune response to bee venom. Comparison of the antibody response to phospholipase A2 with the response to inhalant antigens.

Kemeny DM, Miyachi S, Platts-Mills TA, Wilkins S, Lessof MH.

The antibody response to phospholipase A2 (PLA2) was studied by using radiolabelled antigen-binding techniques for both IgG and IgE antibodies. The results are compared with results for antigen P1 from Dermatophagoides pteronyssinus and for rye I from rye grass pollen. The response to PLA2 differs from the response to the inhalant allergens in several ways: almost all exposed bee keepers produce IgG antibodies to PLA2; among the allergic bee keepers there was no quantitative correlation between IgG and IgE antibodies; and the antibodies to PLA2 are not produced locally in the nose. Bee keepers with hay fever did not appear to have an increased incidence of allergic reactions to bee venom. In addition, the bee keepers with hay fever and the same levels of IgG and IgE antibodies to rye I as other individuals with hay fever. However, the bee keepers who had no history of hay fever were found to have high levels of IgG antibodies to rye I. These antibodies did not cross-react with PLA2 or bee venom, and the results suggest that bee stings may act as an adjuvant for IgG antibodies to pollen antigens.

PMID: 7085123 [PubMed - indexed for MEDLINE]

67: Schweiz Med Wochenschr. 1981 Nov 14;111(46):1756-65.

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[Antibody response pattern (specific IgE and IgG) of insect sting allergic patients in immunotherapy with venom preparations]

[Article in German]

Wuthrich B, Arrendal H, Lanner A.

The concentrations of venom specific IgE (Phadebas-RAST) and IgG (Phadebas IgG-RAST) were monitored in sera of 22 patients with histories of systemic anaphylactic reactions following insect stings who underwent immunotherapy with venom extracts (bee venom and/or yellow jacket venom). Analysis of the immunological parameters during immunotherapy revealed great individual variation in the degree of response concerning both specific IgE and IgG antibodies. Nevertheless, four typical patterns of immune response could be found. The majority of the patients were able to produce high titers of IgG "blocking" antibodies without an IgE increase. Another group of patients had a marked rise in both specific IgG and IgE following the initial phase of hyposensitization. In contrast, one patient received venom injections without a significant IgG or IgE response. Finally, a small group of patients has a marked increase in specific IgE while synthesis of IgG was not observed. This IgE rise was the cause of systemic reactions in this group after the venom injections. Regular monitoring of venom specific IgE and IgG is useful in evaluating the degree of protection for the patient. Since an increasing IgG/IgE ratio must be obtained during immunotherapy, a knowledge of this relationship serves to adapt the individual treatment schedule.

Publication Types:

·       Case Reports

PMID: 7313646 [PubMed - indexed for MEDLINE]

68: Nature. 1981 Jul 16;292(5820):246-8.

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Sequence and specificity of two antibacterial proteins involved in insect immunity.

Steiner H, Hultmark D, Engstrom A, Bennich H, Boman HG.

Immune responses have been described for many different insect species. However, it is generally acknowledged that immune systems must therefore differ from those of vertebrates. An effective humoral immune response has been found in pupae of the cecropia moth, Hyalophora cecropia. The expression of this multicomponent system requires de novo synthesis of RNA and proteins and its broad antibacterial activity is due to at least three independent mechanisms, the most well known of which is the insect lysozyme. However, this enzyme is bactericidal for only a limited number of Gram-positive bacteria. WE recently purified and characterized P9A and P9B, which are two small, basic proteins with potent antibacterial activity against Escherichia coli and several other Gram-negative bacteria. We believe that P9A and P9B plays an important part in the humoral immune responses described previously and that the P9 proteins represent a new class of antibacterial agents for which we propose the name cecropins. We describe here the primary structures of cecropins A and B. We also show that cecropin A is specific for bacteria in contrast to melittin, the main lytic component in bee venom which lyses both bacteria and eukaryotic cells.

PMID: 7019715 [PubMed - indexed for MEDLINE]

69: Agents Actions. 1979 Jun;9(2):205-11.

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Anti-arthritic effect of bee venom.

Chang YH, Bliven ML.

Bee venom, administered subcutaneously, suppressed the development of carrageenan-induced paw edema and adjuvant arthritis in the rat in a dose-related manner. A single dose of bee venom administered subcutaneously the day before or on the day of injection of complete Freund's adjuvant (CFA) effectively suppressed the development of polyarthritis. This suppressive effect decreased progressively as dosing was delayed. Bee venom was found to be most effective when mixed and injected (sub-plantar) together with CFA, the disease-inducing agent. Similarly, antigens such as egg albumin, when incorporated into CFA, and injected into the hind paw, prevented the development of arthritis. These results suggest that at least two mechanisms are involved in the anti-arthritic action of bee venom: (1) alteration of the immune response, probably via antigen competition, and (2) an anti-inflammatory action via corticosteroids or through an as yet undetermined mechanism.

PMID: 474306 [PubMed - indexed for MEDLINE]

70: Johns Hopkins Med J. 1978 Jan;142(1):1-7.

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Effects of passive antibody in bee venom anaphylaxis.

Lessof MH, Sobotka AK, Lichtenstein LM.

Twelve patients allergic to honeybees were challenged by injections of bee venom; five responded systemically to the venom, with symptoms ranging from angioedema to respiratory distress. These patients were given intramuscular or intravenous infusions of gamma-globulin obtained from the plasma of hyperimmune beekeepers who had high levels of antibody to an allergen (phospholipase A) in the venom. Post-infusion, all five patients tolerated 1.5 to 5 times the venom dose that previously elicited adverse reactions. The quantity of passive IgG antibody infused did not impair the patient's own immune response to venom. These results represent the best available evidence for a direct role for IgG blocking antibodies in clinical protection against anaphylaxis occurring as a result of parenteral antigenic challenge as may be observed in penicillin and insect hypersensitivity.

PMID: 75280 [PubMed - indexed for MEDLINE]

71: J Allergy Clin Immunol. 1977 Mar;59(3):247-53.

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Clinical application of measurements of serum level of bee venom-specific IgE and IgG.

Light WC, Reisman RE, Shimizu M, Arbesman CE.

Bee venom--specific IgE and IgG antibodies were measured in the serum of beekeeepers, bee-allergic persons, and normal persons infrequently stung without adverse effects. Beekeepers, who are stung frequently and relatively "immune" to bee stings, are characterized by high serum levels of IgG- and low serum levels of IgE-specific antibodies. Bee-allergic individuals have high titers of bee venom--specific IgG. Following a bee sting, allergic individuals develop a rising titer of IgE antibodies, accompanied occasionally by a rise in IgG antibodies. Therapy with whole bee body extracts fail to effect IgE or IgG antibody titers. During the course of venom immunotherapy IgG-specific antibodies are stimulated and IgE-specific antibodies continue to decline. These observations suggest that: (1) bee sting allergy is a function of bee venom--specific IgE; (2) bee sting immunity is a function of bee venom--specific IgG; and (3) bee venom is an appropriate therapeutic antigen.

PMID: 838994 [PubMed - indexed for MEDLINE]

72: J Allergy Clin Immunol. 1976 Jul;58(1 PT 1):101-9.

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Immunologic comparison of phospholipases A present in Hymenoptera insect venoms.

Nair BC, Nair C, Denne S, Wypych J, Arbesman CE, Elliott WB.

A comparative study was made of the effect of anti-bee venom phospholipase A2 serum and anti-bald face hornet venom serum on the activity of phospholipase A2 present in yellow hornet, yellow jacket, bald face hornet, and honeybee venoms. Activity of phospholipase A2 present in the venoms was measured titrimetrically using egg yolk dispersion as the substrate. Antiserum against pure bee venom phospholipase A2 activity completely suppressed bee venom phospholipase A2 activity but failed to inhibit phospholipase A2 activity in yellow jacket, yellow hornet, and bald face hornet venoms. Anti-bald face hornet venom serum suppressed the activity of phospholipase A2 present in the four insect venoms with the inhibitory effect decreasing in the order: bald face hornet greater than yellow hornet greater than yellow jacket greater than honeybee. A combination of immunoelectrophoresis and immunodiffusion techniques gave indication of common antigenic sites in a component of yellow hornet venom and a component of bald face hornet venom. The results suggest that IgG antibodies against pure bee venom phospholipase A2 may not give any protection against the reaction(s) due to the phospholipase A2 in yellow hornet, yellow jacket, and bald face hornet venoms, while antibodies against bald face hornet venom phospholipase A2 may give some cross-protection.

PMID: 947973 [PubMed - indexed for MEDLINE]

73: Dokl Bulg Acad Nauk. 1976;29(11):1685-7.

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Effect of bee venom components on immuno-competent cell proliferation at a primary humoral immune response.

Shkenderov S, Gencheva G.

PMID: 1035856 [PubMed - indexed for MEDLINE]

74: J Allergy Clin Immunol. 1975 May;55(5):285-98.

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Click here to read  
Comparison of honeybee venoms and their components from various sources.

Franklin R, Baer H.

The use of honeybee venoms and their components may assist in the elucidation of the pathophysiology of reactions to honeybee stings. This initial study compared venoms from various sources by chemical and biological assays, and significant variations were observed. Ten different bee venoms were compared by nitrogen analysis, mouse toxicity, hyaluronidase content, and antigenicity. Based on mouse toxicity, hyaluronidase content, and gel diffusion analysis, two groups of bee venoms could be differentiated.
Venoms in one group, Group A, were more toxic, contained hyaluronidase, and showed an additional precipitin band. All venoms contained mellitin as a major fraction, which formed nonimmune precipitin bands during gel diffusion analysis. Gel filtration chromatography and dialysis separated the venoms into components that were then identified by enzyme assays, rat mast cell degranulation, hemolytic activity, and gel diffusion analysis. The venoms within Group A showed similar components, some of which, most noticeably hyaluronidase, were not present in Group B. Dialysis showed that a large portion of the venom could pass through a cellophane membrane including a portion of the phospholipase A. Heterogeneous molecular weights were found for phospholipase A by both gel filtration and dialysis, and may reflect variation in carbohydrate content. It appears that bee venom variability for whatever reason, a heterogeneous MW antigen, and a non-immune precipitable component require careful consideration in any study involving this venomm. These studies have yielded relatively pure, identified bee venom components which can be employed in further studies investigating reactions to honeybee stings.

PMID: 804500 [PubMed - indexed for MEDLINE]

75: Proc Soc Exp Biol Med. 1974 Nov;147(2):585-8.

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Immune and nonimmune gel precipitates produced by honey bee venom and its components.

Franklin RM, Baer H.

PMID: 4216035 [PubMed - indexed for MEDLINE]

76: Clin Chim Acta. 1967 Jul;17(1):119-23.

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The specificity of the anti-hyaluronidase developed in beekeepers serum against bee venom hyaluronidase.

Barker SA, Walton KW, Weston PD.

PMID: 4166652 [PubMed - indexed for MEDLINE]