1: 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]

2: Eur J Immunol. 2005 Nov;35(11):3268-76.

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Prevention of allergy by a recombinant multi-allergen vaccine with reduced IgE binding and preserved T cell epitopes.

Karamloo F, Schmid-Grendelmeier P, Kussebi F, Akdis M, Salagianni M, von Beust BR, Reimers A, Zumkehr J, Soldatova L, Housley-Markovic Z, Muller U, Kundig T, Kemeny DM, Spangfort MD, Blaser K, Akdis CA.

Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland. karamlooflory@compuserve.de

Novel approaches for the prevention of allergy are required, because of the inevitably increasing prevalence of allergic diseases during the last 30 years. Here, a recombinant chimeric protein, which comprises the whole amino acid sequences of three bee venom major allergens has been engineered and used in prevention of bee venom sensitization in mice. Phospholipase A2 (Api m 1), hyaluronidase (Api m 2) and melittin (Api m 3) fragments with overlapping amino acids were assembled in a different order in the Api m (1/2/3) chimeric protein, which preserved entire T cell epitopes, whereas B cell epitopes of all three allergens were abrogated. Accordingly, IgE cross-linking leading to mast cell and basophil mediator release was profoundly reduced in humans. Supporting these findings, the Api m (1/2/3) induced 100 to 1000 times less type-1 skin test reactivity in allergic patients. Treatment of mice with Api m (1/2/3) led to a significant reduction of specific IgE development towards native allergen, representing a protective vaccine effect in vivo. These results demonstrate a novel prototype of a preventive allergy vaccine, which preserves the entire T cell epitope repertoire, but bypasses induction of IgE against native allergen, and side effects related to mast cell/basophil IgE FcepsilonRI cross-linking in sensitized individuals.

PMID: 16206231 [PubMed - indexed for MEDLINE]

3: In Vivo. 2005 Jul-Aug;19(4):801-5.

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Inhibitory effect of whole bee venom in adjuvant-induced arthritis.

Lee JY, Kang SS, Kim JH, Bae CS, Choi SH.

College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Republic of Korea.

The aim of this study was to assess the inhibitory effect of whole bee venom (BV) on adjuvant-induced arthritis in the rat. Rats were divided into pre-apitherapy, post-apitherapy and control experimental groups. The pre-apitherapy group was subcutaneously stung with a honeybee (Apis mellifera L.) and the control group was subcutaneously injected with 0.1 ml of physiological saline solution one day prior to complete Freund's adjuvant (CFA) injection. The post-apitherapy group was subcutaneously stung with a honeybee on day 14 after CFA injection. When arthritis had developed in the rat, the post-apitherapy group was subcutaneously administered whole BV every other day for a further 14 days. Clinical signs, hematological values and radioglogical features were observed during the entire experimental period. In the pre-apitherapy group, the development of inflammatory edema and polyarthritis was inhibited. Significant differences in lameness score, hind paw edema volume and radiological features were observed between control and pre-apitherapy rats. White blood cell counts indicated that the degree of leucocytosis was significantly different between the pre-apitherapy and control groups (p < 0.01). Inflammatory edema, polyarthritis and bone change into the right hind paw were effectively inhibited in pre-apitherapy rats during the two-week period post-CFA injection. In conclusion, whole BV was found to inhibit arthritic inflammation and bone changes in the rat. This may be an alternative treatment for arthritis in humans.

PMID: 15999553 [PubMed - indexed for MEDLINE]

4: Brain Res. 2005 Jul 12;1049(2):210-6.

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The anti-inflammatory effect of peripheral bee venom stimulation is mediated by central muscarinic type 2 receptors and activation of sympathetic preganglionic neurons.

Yoon SY, Kim HW, Roh DH, Kwon YB, Jeong TO, Han HJ, Lee HJ, Choi SM, Ryu YH, Beitz AJ, Lee JH.

Department of Veterinary Physiology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea.

The anti-inflammatory effect (AI) induced by peripheral injection of diluted bee venom (dBV) involves activation of spinal cord circuits and is mediated by catecholamine release from adrenal medulla, but the precise neuronal mechanisms involved are not fully understood. In a recent study, we demonstrated that an increase in spinal acetylcholine is involved in mediating the anti-inflammatory effect of dBV and that this mediation also involves adrenomedullary activation. The present study utilized the mouse air pouch inflammation model to evaluate the involvement of spinal acetylcholine receptors and sympathetic preganglionic neurons (SPNs) in dBV's anti-inflammatory effect (dBVAI). Intrathecal (IT) pretreatment with atropine (muscarinic cholinergic antagonist) but not hexamethonium (nicotinic cholinergic antagonist) significantly suppressed dBVAI on zymosan-evoked leukocyte migration. Subsequent experiments showed that IT pretreatment with methoctramine (a muscarinic receptor type 2; M(2) antagonist), but not pirenzepine (an M(1) antagonist) or 4-DAMP (an M(3) antagonist), suppressed the dBVAI. In addition, dBV stimulation specifically increased Fos expression in SPNs of the T7-T11, but not the T1-T6 or T12-L2 spinal cord segments, in animals with zymosan-induced inflammation. Moreover, IT methoctramine pretreatment suppressed this dBV-induced Fos expression specifically in SPNs of T7-T11 level. Peripheral sympathetic denervation using 6-hydroxydopamine (6-OHDA) treatment (which spares sympathetic adrenal medullary innervation) did not alter dBVAI. Collectively these results indicate that dBV stimulation leads to spinal cord acetylcholine release that in turn acts on spinal M(2) receptors, which via a hypothesized disinhibition mechanism activates SPNs that project to the adrenal medulla. This activation ultimately leads to the release of adrenal catecholamines that contribute to dBVAI.

PMID: 15953592 [PubMed - indexed for MEDLINE]

5: Int Immunopharmacol. 2005 Aug;5(9):1406-14. Epub 2005 Apr 20.

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Bee venom modulates murine Th1/Th2 lineage development.

Nam S, Ko E, Park SK, Ko S, Jun CY, Shin MK, Hong MC, Bae H.

College of Oriental Medicine, Kyung Hee University, #1 Hoeki-dong Dongdaemoon-gu, Seoul, 130-701, Korea.

Administration of bee venom (BV) elicits anti-inflammatory, anti-nociceptive and anti-allergic effects in various animal models. This study was designed to evaluate the direct effects of BV on helper T cell activities and on Th1/Th2 lineage development using both in vitro and in vivo conditions. In the Th1 skewed condition, BV increased the expression of IFN-gamma mRNA and enhanced the expression of T-bet on purified CD4(+) T cells from splenocytes of BALB/c mice. On the other hand, BV treatment did not alter the expression of IL-4 or GATA-3 in a Th2 driven environment. To elucidate the effects of BV on Th1/Th2 lineage development under in vivo conditions, BV was given by intraperitonial injection to BALB/c mice. It significantly increased the CD4(+) T cell population and enhanced IFN-gamma expression, while IL-4 transcripts were not altered upon in vivo activation using an anti-CD3 antibody injection. Taken together, these results imply that BV induces Th1 lineage development from CD4(+) T cells by increasing the expression of a Th1-specific cytokine, IFN-gamma. In addition, this result may be mediated by inducing a Th1-specific transcription factor, T-bet.

PMID: 15953567 [PubMed - indexed for MEDLINE]

6: J Allergy Clin Immunol. 2005 May;115(5):1063-7.

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Increased expression of osteopontin is associated with long-term bee venom immunotherapy.

Konno S, Golden DB, Schroeder J, Hamilton RG, Lichtenstein LM, Huang SK.

Johns Hopkins Asthma and Allergy Center,Baltimore, MD 21224, USA.

BACKGROUND: Venom allergen immunotherapy (VIT) is proven to be highly effective for insect allergy, but the mechanisms and the biomarkers associated with clinical efficacy remain elusive. OBJECTIVE: The aim of this study was to identify candidate biomarkers associated with successful VIT. METHODS: Gene chip array and clustering analyses of PBMCs from subjects with or without VIT were performed. RESULTS: From gene chip array and clustering analyses, an increased expression of osteopontin was found in patients who completed 5 to 6 years of VIT and discontinued therapy for 3 to 6 years (completed treatment group) compared with the untreated group. A significantly higher level of serum osteopontin was found in the completed treatment group compared with the untreated group (n = 16 in each group; P < .001). CONCLUSION: The upregulation of osteopontin after VIT suggests a role of osteopontin as a candidate biomarker for VIT.

PMID: 15867867 [PubMed - indexed for MEDLINE]

7: J Ethnopharmacol. 2005 May 13;99(1):157-60.

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Effects of bee venom on the pro-inflammatory responses in RAW264.7 macrophage cell line.

Jang HS, Kim SK, Han JB, Ahn HJ, Bae H, Min BI.

Department of East-West Medicine, Graduate School, Kyung-Hee University, Seoul 130-701, South Korea.

The purpose of this study is to elucidate the molecular mechanism of anti-inflammatory effect of bee venom (BV), which has been used for the treatment of various inflammatory diseases in oriental medicine. With this aim, we examined the effects of BV on the nitric oxide (NO) production by lipopolysaccharide (LPS) or sodium nitroprusside in RAW264.7 macrophages. We further investigated the effects of BV on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) with RT-PCR in LPS-stimulated RAW264.7 cells. BV suppressed the NO production and decreased the levels of iNOS, COX-2, NF-kappaB and MAPK mRNA in a dose-dependent manner. These results suggest that BV has an anti-inflammatory effect by inhibiting iNOS and COX-2 expression, possibly through suppression of NF-kappaB and MAPK expression.

PMID: 15848037 [PubMed - indexed for MEDLINE]

8: Clin Exp Allergy. 2005 Mar;35(3):367-73.

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Characterization of the human T cell response to antigen 5 from Vespula vulgaris (Ves v 5).

Bohle B, Zwolfer B, Fischer GF, Seppala U, Kinaciyan T, Bolwig C, Spangfort MD, Ebner C.

Department of Pathophysiology, Medical University of Vienna, VA-1090 Vienna, Austria. barbara.bohle@meduniwien.ac.at

BACKGROUND: The T cell reactivity to the major allergen of bee venom, phospholipase A2, has been thoroughly characterized. In contrast, only little is known about the human cellular response to major allergens from wasp venom. OBJECTIVE: To characterize the human T cell response to antigen 5 from Vespula vulgaris, Ves v 5. METHODS: Recombinant Ves v 5 was used to establish allergen-specific T cell lines (TCL) and T cell clones (TCC) from the peripheral blood of vespid-allergic and non-allergic individuals. Ves v 5-specific TCL were mapped for T cell epitopes using overlapping synthetic peptides representing the complete amino acid sequence of Ves v 5. Ves v 5-specific TCC were analysed for antigen-induced secretion of IL-4, IFN-gamma and IL-10. RESULTS: Seventeen distinct T cell epitopes were recognized by allergic individuals among which Ves v 5(181-192) was identified as a dominant T cell epitope. Partially different epitopes were observed in TCL from non-allergic subjects and the dominant epitope Ves v 5(181-192) was not prevalent in these cultures. Ves v 5-specific TCC isolated from allergic individuals did not show the typical T helper type 2 (Th2)-like cytokine profile in response to specific stimulation, i.e. high amounts of IL-4 and low IFN-gamma. TCC from non-allergic individuals showed a Th1-like cytokine pattern. CONCLUSIONS: Our findings provide evidence that the allergic T cell response to Ves v 5 is not Th2-dominated and that different immunogenic sites on this major wasp venom allergen are recognized by allergic and non-allergic individuals.

PMID: 15784117 [PubMed - indexed for MEDLINE]

9: FEBS Lett. 2005 Mar 14;579(7):1658-64.

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Cross-presentation of a CMV pp65 epitope by human dendritic cells using bee venom PLA2 as a membrane-binding vector.

Babon A, Almunia C, Boccaccio C, Beaumelle B, Gelb MH, Menez A, Maillere B, Abastado JP, Salcedo M, Gillet D.

Protein Engineering and Research Department (DIEP), bat 152, CEA-Saclay, 91191 Gif sur Yvette cedex, France.

We have used bee venom phospholipase A2 as a vector to load human dendritic cells ex vivo with a major histocompatibility complex (MHC) class I-restricted epitope fused to its C-terminus. The fusion protein bound to human monocyte-derived dendritic cells and was internalized into early endosomes. In vitro immunization experiments showed that these dendritic cells were able to generate specific CD8 T cell lines against the epitope carried by the fusion protein. Cross-presentation did not require proteasome, transporter associated with antigen processing, or endosome proteases, but required newly synthesized MHC molecules. Comparison of the antigen presentation pathway observed in this study to that followed by other toxins used as vectors is discussed.

PMID: 15757657 [PubMed - indexed for MEDLINE]

10: J Allergy Clin Immunol. 2005 Feb;115(2):323-9.

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A major allergen gene-fusion protein for potential usage in allergen-specific immunotherapy.

Kussebi F, Karamloo F, Rhyner C, Schmid-Grendelmeier P, Salagianni M, Mannhart C, Akdis M, Soldatova L, Markovic-Housley Z, Von Beust BR, Kundig T, Kemeny DM, Blaser K, Crameri R, Akdis CA.

Swiss Institute of Allergy and Asthma Research, Davos, Switzerland. fatimah.kussebi@charite.de

BACKGROUND: Specific immunotherapy is a common treatment of allergic diseases and could potentially be applied to other immunologic disorders. Despite its use in clinical practice, more defined and safer allergy vaccine preparations are required. Differences between epitopes of IgE that recognize the 3-dimensional structure of allergens and T cells that recognize linear amino acid sequences provide a suitable tool for novel vaccine development for specific immunotherapy. OBJECTIVE: The aim of the study was to delete B-cell epitopes and prevent IgE crosslinking, but to preserve T-cell epitopes by fusion of 2 major allergens of bee venom because of a change in the conformation. METHODS: By genetic engineering, we produced a fusion protein composed of the 2 major bee venom allergens: phospholipase A 2 (Api m 1) and hyaluronidase (Api m 2). RESULTS: The Api m [1/2] fusion protein induced T-cell proliferation and both T H 1-type and T H 2-type cytokine responses. In contrast, IgE reactivity was abolished, and profoundly reduced basophil degranulation and type 1 skin test reactivity was observed. Pretreatment of mice with Api m [1/2] fusion protein significantly suppressed the development of specific IgE as well as other antibody isotypes after immunization with the native allergen. CONCLUSION: The novel fusion protein of 2 major allergens bypasses IgE binding and mast cell/basophil IgE FcepsilonRI crosslinking and protects from IgE development.

PMID: 15696088 [PubMed - indexed for MEDLINE]

11: Arthritis Rheum. 2004 Nov;50(11):3504-15.

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Antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of NF-kappaB through interaction with the p50 subunit.

Park HJ, Lee SH, Son DJ, Oh KW, Kim KH, Song HS, Kim GJ, Oh GT, Yoon do Y, Hong JT.

College of Pharmacy, Chungbuk National University, 48 Gaesin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, South Korea.

OBJECTIVE: To investigate the molecular mechanisms of the antiarthritic effects of bee venom (BV) and melittin (a major component of BV) in a murine macrophage cell line (Raw 264.7) and in synoviocytes obtained from patients with rheumatoid arthritis. METHODS: We evaluated the antiarthritic effects of BV in a rat model of carrageenan-induced acute edema in the paw and in a rat model of chronic adjuvant-induced arthritis. The inhibitory effects of BV and melittin on inflammatory gene expression were measured by Western blotting, and the generation of prostaglandin E(2) (PGE(2)) and nitric oxide (NO) and the intracellular calcium level were assayed. NF-kappaB DNA binding and transcriptional activity were determined by gel mobility shift assay or by luciferase assay. Direct binding of BV and melittin to the p50 subunit of NF-kappaB was determined with a surface plasmon resonance analyzer. RESULTS: BV (0.8 and 1.6 mug/kg) reduced the effects of carrageenan- and adjuvant-induced arthritis. This reducing effect was consistent with the inhibitory effects of BV (0.5, 1, and 5 mug/ml) and melittin (5 and 10 mug/ml) on lipopolysaccharide (LPS; 1 mug/ml)-induced expression of cyclooxygenase 2, cytosolic phospholipase A(2), inducible NO synthase, generation of PGE(2) and NO, and the intracellular calcium level. BV and melittin prevented LPS-induced transcriptional and DNA binding activity of NF-kappaB via the inhibition of IkappaB release and p50 translocation. BV (affinity [K(d)] = 4.6 x 10(-6)M) and melittin (K(d) = 1.2 x 10(-8)M) bound directly to p50. CONCLUSION: Target inactivation of NF-kappaB by directly binding to the p50 subunit is an important mechanism of the antiarthritic effects of BV.

PMID: 15529353 [PubMed - indexed for MEDLINE]

12: 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]

13: Protein Sci. 2004 Nov;13(11):2970-8. Epub 2004 Sep 30.

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Alteration of the tertiary structure of the major bee venom allergen Api m 1 by multiple mutations is concomitant with low IgE reactivity.

Buhot C, Chenal A, Sanson A, Pouvelle-Moratille S, Gelb MH, Menez A, Gillet D, Maillere B.

Protein Engineering and Research Department, batiment 152, CEA-Saclay, 91191 Gif sur Yvette, France.

We have engineered a recombinant form of the major bee venom allergen (Api m 1) with the final goal of reducing its IgE reactivity. This molecule (Api mut) contains 24 mutations and one deletion of 10 amino acids. The successive introduction of these sequence modifications led to a progressive loss of specific IgE and IgG reactivity and did not reveal any immunodominant epitopes. However, Api mut exhibited a clear loss of reactivity for Api m 1-specific IgE and IgG. Injection of Api mut into mice induced specific antibody production. This humoral response was as high as that induced by the Api m 1 but the cross-reactivity of the antibodies was weak. As inferred by far UV circular dichroism, this mutant was correctly folded. However, near UV circular dichroism and denaturation curves of Api mut showed that it exhibits a dynamic tertiary structure and that it is a highly flexible molecule. Finally, as all the sequence modifications have been introduced outside the human and murine T cell epitope regions, we investigated its T cell properties in mice. We showed that Api mut-specific T lymphocytes induced in vivo were stimulated in vitro by both proteins. These data provide new insights in the design of hypoallergenic molecules.

PMID: 15459335 [PubMed - indexed for MEDLINE]

14: Allergy. 2004 Oct;59(10):1110-7.

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The CD63 basophil activation test in Hymenoptera venom allergy: a prospective study.

Sturm GJ, Bohm E, Trummer M, Weiglhofer I, Heinemann A, Aberer W.

Department of Experimental and Clinical Pharmacology, University of Graz, Graz, Austria.

BACKGROUND: The basophil activation test (BAT), which relies on flow cytometric quantitation of the allergen-induced up-regulation of the granule-associated marker CD63 in peripheral blood basophils, has been suggested to be a useful approach in detecting responsiveness to allergens. The purpose of this study was to establish the usefulness of the BAT with regard to the clinical history and current diagnostic tools in Hymenoptera venom allergy using a prospective study design. METHODS: Fifty-seven consecutive patients allergic to Hymenoptera venom as defined by a systemic reaction after an insect sting, and 30 age- and sex-matched control subjects with a negative history were included. The degree and nature of sensitization was confirmed by skin testing, specific immunoglobulin E (IgE), serum tryptase levels and BAT. In the nonallergic control group only analysis of specific IgE and BAT were performed. Correlation of BAT, skin test and specific IgE, respectively, with the clinical history in the allergic group was termed as sensitivity and in the control group as specificity. RESULTS: Twenty one of 23 (91.3%) bee venom allergic patients and 29 of 34 (85.3%) patients allergic to wasp and hornet venom tested positive in BAT. The overall sensitivity of BAT, specific IgE and skin tests were 87.7, 91.2 and 93.0%, respectively. The overall specificities were 86.7% for BAT and 66.7% for specific IgE. No correlation between the severity of clinical symptoms and the magnitude of basophil activation was observed. CONCLUSION: The BAT seems to be an appropriate method to identify patients allergic to bee or wasp venom with a comparable sensitivity to standard diagnostic regimens. The higher specificity of BAT as compared with specific IgE makes this test a useful tool in the diagnosis of Hymenoptera venom allergy.

Publication Types:

·       Evaluation Studies

PMID: 15355471 [PubMed - indexed for MEDLINE]

15: Allergy. 2004 Oct;59(10):1102-9.

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The basophil activation test in wasp venom allergy: sensitivity, specificity and monitoring specific immunotherapy.

Erdmann SM, Sachs B, Kwiecien R, Moll-Slodowy S, Sauer I, Merk HF.

Department of Dermatology and Allergology, University Hospital of Aachen, Aachen, Germany.

BACKGROUND: As in vitro diagnosis of wasp venom sensitization by specific serum IgE has a sensitivity of only 60-80%, additional in vitro tests are desirable. Basophil activation is associated with the expression of CD63 and its measurement has been proposed as a novel in vitro test for immediate-type allergy. Furthermore, to date, no in vitro test exists to monitor successful specific immunotherapy (SIT) with wasp venom. Therefore, the potentially harmful sting challenge is still recommended. OBJECTIVE: We compared the CD63-based basophil activation test (BAT) in the diagnosis of wasp venom allergy with skin tests and measurement of specific IgE. Furthermore, we investigated whether BAT can predict the outcome of the sting challenge in patients on SIT. METHODS: Fifty patients with a systemic reaction caused by a wasp sting and 20 controls were studied. Intracutaneous tests were performed with wasp and bee venom in the suspected allergics. Specific IgE was determined by the CAP-FEIA method and basophil activation by flow cytometry upon double staining with anti-IgE/anti-CD63 mAb. Twenty-five patients were sting challenged 6 months after starting SIT and the BAT was repeated before challenge. RESULTS: Sensitivity of the intracutaneous tests, specific IgE and BAT was 100, 76, and 92%, respectively. Specificity of specific IgE and the BAT was 85 and 80%, respectively. The cut-off for a positive BAT was 15% CD63+ basophils. There was a positive correlation between IgE reactivity to wasp venom and the number of CD63+ basophils (r = 0.65). Although no patient had a systemic reaction upon sting challenge, in most subjects basophil activation did not decrease when compared with the BAT before SIT. CONCLUSIONS: Quantitation of basophil activation by CD63 expression is a valuable new in vitro method for diagnosis of allergy to hymenopteran venoms. The CD63-based BAT is a helpful tool for the complementation of routine diagnostic tests such as specific IgE as it increases sensitivity of in vitro detection of sensitization. However, this in vitro method does not offer an alternative to the sting challenge in monitoring successful SIT.

PMID: 15355470 [PubMed - indexed for MEDLINE]

16: 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]

17: Clin Exp Allergy. 2003 Sep;33(9):1209-15.

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Impaired secretion of interleukin-4 and interleukin-13 by allergen-specific T cells correlates with defective nuclear expression of NF-AT2 and jun B: relevance to immunotherapy.

Faith A, Richards DF, Verhoef A, Lamb JR, Lee TH, Hawrylowicz CM.

Department of Respiratory Medicine and Allergy, The Guy's, King's College and St Thomas' Hospitals School of Medicine, London, UK. alex.faith@kcl.ac.uk

BACKGROUND: Allergen immunotherapy (IT) is a successful treatment associated with decreased Th2 cytokine production by allergen-specific T cells. We have previously demonstrated (Faith et al., J Immunol 1997; 159:53-57) that inhibition of Th2 cytokine production in vitro correlates with impaired tyrosine kinase activity through the TCR. The transcription factor complex, nuclear factor of activated T cells (NF-AT), which regulates Th2 cytokine production is controlled by the activity of tyrosine kinases. OBJECTIVE: To address whether decreased Th2 cytokine production by allergen-specific CD4+ T cells following IT is correlated with altered translocation and nuclear expression of the NF-AT family member, NF-AT2, and the activator protein 1 (AP1) component of NF-AT, jun B. METHODS: T cell lines specific for insect venom phospholipase A2 (PLA) were derived from patients prior to and during conventional venom IT. Nuclear expressions of NF-AT and jun B were assessed following stimulation through the TCR. Th1 and Th2 cytokine and IL-10 production by insect venom-specific T cells were also determined. Results were compared with a well-established model system in which anergy was induced in cloned, allergen-specific Th2 cells. RESULTS: Impaired translocation and decreased expression of NF-AT2 and jun B were detected in PLA-specific T cell lines derived from bee venom-allergic individuals following 16 weeks treatment compared to pre-treatment. These results correlated with significantly reduced production of IL-4 and IL-13 and significantly increased production of IFN-gamma and IL-10 by PLA-specific T cells. Impaired IL-4 and IL-13 production also correlated with defective nuclear expression of NF-AT2/jun B in cloned, anergic allergen-specific Th2 cells. CONCLUSION: These results suggested that optimal production of IL-4 and IL-13 by allergen-specific T cells is dependent on the nuclear expression of NF-AT2 and jun B. Thus, specific inhibition of NF-AT2/jun B might be an option in novel and improved forms of allergen IT.

PMID: 12956740 [PubMed - indexed for MEDLINE]

18: 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]

19: 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]

20: 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]

21: 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]

22: J Allergy Clin Immunol. 2003 Feb;111(2):426-8.

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Structural similarity between the bee venom peptides and the immunodominant human myelin basic proteins: role for pathogenesis of acute disseminated encephalomyelitis.

Dharmasaroja P.

Publication Types:

·       Letter

PMID: 12589368 [PubMed - indexed for MEDLINE]

23: J Neuroendocrinol. 2003 Jan;15(1):93-6.

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The anti-inflammatory effect of bee venom stimulation in a mouse air pouch model is mediated by adrenal medullary activity.

Kwon YB, Kim HW, Ham TW, Yoon SY, Roh DH, Han HJ, Beitz AJ, Yang IS, Lee JH.

Department of Veterinary Physiology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon, South Korea.

Cutaneous electrical or chemical stimulation can produce an anti-inflammatory effect, which is dependent on adrenal medullary-sympathetic activation. We have previously shown that peripheral injection of bee venom (BV) also produces a significant anti-inflammatory effect that is neurally mediated. In the present study, we examined whether this anti-inflammatory effect is also dependent on the adrenal gland using the mouse inflammatory air pouch model. Subcutaneous (s.c.) BV injection produced a marked suppression of leucocyte migration and tumour necrosis factor (TNF)-alpha concentration induced by zymosan injection into the air pouch. The role of the adrenal gland in this suppression was evaluated in adrenalectomized mice. Adrenalectomy significantly reversed the suppression of leucocyte migration and TNF-alpha elevation caused by BV. Serum concentrations of corticosteroid were increased in mice with zymosan-induced air-pouch inflammation and this increase was reduced by BV administration, suggesting that adrenal corticosteroid release is not involved in mediating the anti-inflammatory effects of BV. To test this hypothesis, the corticosteroid receptor antagonist (RU486) was administered and found not to affect the BV-induced inhibition of leucocyte migration. By contrast, pretreatment with the beta-adrenergic antagonist propranolol reversed the BV-induced inhibitory effect on leucocyte migration. These results suggest that the anti-inflammatory effect of s.c. BV administration is mediated in part by the release of catecholamines from the adrenal medulla.

PMID: 12535175 [PubMed - indexed for MEDLINE]

24: Eur J Immunol. 2002 Dec;32(12):3699-707.

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Emerging principles for the design of promiscuous HLA-DR-restricted peptides: an example from the major bee venom allergen.

Texier C, Pouvelle-Moratille S, Buhot C, Castelli FA, Pecquet C, Menez A, Leynadier F, Maillere B.

Protein Engineering and Research Department, CEA-Saclay, Gif sur Yvette, France.

Mechanisms underlying successful immunotherapy of allergic patients operate at the level of CD4+ helper T cells. T cell epitopes from allergens may thus constitute interesting molecules for immunotherapy, provided they are efficient for all patients and are not recognized by IgE. In an attempt to define such peptides for allergy to bee venom, we have investigated the capacity of peptides encompassing the sequence of the major bee venom allergen to stimulate PBMC from allergic patients and to react specifically with their IgE. The region 77-110 emerged as the most frequently T cell stimulating. We then analyzed the binding modes of the sequence 81-97 for ten different HLA-DR molecules and introduced punctual mutations to enhance the peptide affinity for these molecules. Six different modes have been identified on the sequence 81-97, one mode being common to eight HLA-DR molecules. Four HLA-DR molecules can bind the P85-97 peptide by two different modes with an equivalent affinity. The peptide N89L has a higher affinity for DRB1*0301 and DRB3*0101 and remains as active as the native peptide towards the other HLA-DR molecules.

PMID: 12516563 [PubMed - indexed for MEDLINE]

25: Int Arch Allergy Immunol. 2002 Oct;129(2):160-8.

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Individual hymenoptera venom compounds induce upregulation of the basophil activation marker ectonucleotide pyrophosphatase/phosphodiesterase 3 (CD203c) in sensitized patients.

Binder M, Fierlbeck G, King T, Valent P, Buhring HJ.

Department of Internal Medicine II, Division of Hematology and Immunology, University of Tubingen, Germany.

BACKGROUND: Bee and wasp venom extracts contain potent allergens capable of inducing severe clinical reactions. To analyze immediate-type hypersensitivity to defined hymenoptera venom components, a recently developed in vitro test was applied that is based on the upregulation of CD203c expression on basophils. METHODS: CD203c expression on blood basophils of 9 healthy donors and 39 patients allergic to bee and/or wasp venom was analyzed by flow cytometry before and after activation with the purified bee venom allergens phospholipase A2 (Api m 1), hyaluronidase (Api m 2) and melittin (Api m 4), or the purified wasp venom allergens phospholipase A1 (Ves v 1), hyaluronidase (Ves v 2) and the recombinant antigen 5 (Ves v 5). Venom-induced CD203c upregulation on basophils was compared with skin tests and assessment of specific IgE. Basophils of nonresponders were preincubated with 10 ng/ml interleukin-3 (IL-3) prior to allergen stimulation. RESULTS: CD203c upregulation on basophils was induced by defined hymenoptera venom components in 35/39 patients with a diagnosed allergy to wasp and/or bee venom. Twenty-seven of the 34 tested patients with wasp allergy showed CD203c upregulation in response to Ves v 5, 26 of these patients also reacted with Ves v 2 and 17 with Ves v 1. Nine of 13 patients with bee allergy reacted with Api m 1, 13 individuals with Api m 2 and none of these patients with the minor allergen Api m 4. A diagnosed wasp allergy could also be confirmed in the prestimulated basophils (IL-3) of 2 nonresponder individuals who failed to upregulate CD203c in response to IgE receptor cross-linking prior to culture with IL-3. CONCLUSIONS: Flow-cytometric determination of CD203c upregulation on basophils activated by molecularly defined allergens is a powerful method to identify the precise allergen reactivity in sensitized individuals. Copyright 2002 S. Karger AG, Basel

PMID: 12403934 [PubMed - indexed for MEDLINE]

26: Biol Pharm Bull. 2002 Jun;25(6):710-7.

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Participation of the arachidonic acid cascade pathway in macrophage binding/uptake of oxidized low density lipoprotein.

Beppu M, Watanabe M, Sunohara M, Ohishi K, Mishima E, Kawachi H, Fujii M, Kikugawa K.

School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.

Arachidonic acid cascade inhibitors, including phospholipase A2 inhibitors, dexamethasone and quinacrine (mepacrine), cyclooxygenase inhibitors, indomethacin and aspirin, and lipoxygenase inhibitor AA861, prevented foam cell formation and cholesterol accumulation in the incubation of thioglycollate-induced mouse peritoneal macrophages with oxidized low density lipoprotein (LDL) at 37 degrees C for 24 h. These inhibitors similarly prevented foam cell formation of fibronectin- and Ca ionophore A23187-stimulated macrophages. Binding and/or uptake of Dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine)-acetyl LDL by macrophages at 37 degrees C for 3h and arachidonic acid release from macrophages at 37 degrees C for 4h were inhibited by dexamethasone. Exogenously added phospholipase A2 of bee venom and Crotalus adamanteous venom increased arachidonic acid release during incubation for 2 h, and increased macrophage binding and/or uptake of Dil-acetyl LDL at 37 degrees C for 3 h, and foam cell formation at 37 degrees C for 24 h. Protein kinase inhibitors, ML-9 and staurosporine, that inhibited macrophage binding and/or uptake of Dil-acetyl LDL did not inhibit arachidonic acid release, indicating that protein phosphorylation was not involved in the arachidonic acid pathway in the macrophage scavenger receptor activation. Nordihydroguaiaretic acid that inhibited arachidonic acid release prevented binding and/or uptake of Dil-acetyl LDL. The release of arachidonic acid was not enhanced by fibronectin-stimulation, indicating that Ca influx-dependent stimulation of macrophage activity was not through the activation of phospholipase A2. These results indicate that, as well as the fibronectin-stimulated Ca influx pathway and protein phosphorylation pathway, the arachidonic acid pathway participated in the activation of macrophages to bind and take up oxidized LDL.

PMID: 12081134 [PubMed - indexed for MEDLINE]

27: Toxicon. 2002 May;40(5):519-26.

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Role of melittin-like region within phospholipase A(2)-activating protein in biological function.

Ribardo DA, Kuhl KR, Peterson JW, Chopra AK.

Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1070, USA.

Phospholipase A(2)-activating protein (PLAA) has been implicated in the production of prostaglandins (e.g. PGE(2)) via activation of phospholipases in various stimulated cell types. Human PLAA, with 738 amino acid (aa) residues, contains a region of 38% homology (aa 503-538) with the 26-aa long melittin peptide, a major component of bee venom and a reported regulator of phospholipase A(2) and phospholipase D activity. To learn more about the role of PLAA in the production of eicosanoids and other inflammatory mediators, we synthesized a murine PLAA peptide (36-aa long) having homology to melittin, as well as to human and rat PLAA. The PLAA peptide and melittin increased the expression of genes encoding the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) and cyclooxygenase-2 (COX-2), which is involved in PGE(2) production. We determined that the C-terminal region of the PLAA peptide (aa 515-538) was essential, since truncation of the C-terminal end of the PLAA peptide significantly reduced expression of genes encoding TNFalpha and COX-2 in macrophages. We concluded that PLAA could be important in the regulation of the inflammatory response because of its stimulatory effects on eicosanoid and cytokine synthesis. Consequently, control of plaa gene expression could be a target for the development of new drugs to control the inflammatory response.

PMID: 11821123 [PubMed - indexed for MEDLINE]

28: Int Arch Allergy Immunol. 2001 Dec;126(4):335-42.

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Erratum in:

·       Int Arch Allergy Immunol 2002 Apr;127(4):293.

Hymenoptera-venom-induced upregulation of the basophil activation marker ecto-nucleotide pyrophosphatase/phosphodiesterase 3 in sensitized individuals.

Platz IJ, Binder M, Marxer A, Lischka G, Valent P, Buhring HJ.

University of Tubingen, Department of Internal Medicine II, Division of Hematology and Immunology, Tubingen, Germany.

BACKGROUND: Bee and wasp venoms are potent allergens capable of inducing severe clinical reactions. To detect immediate-type hypersensitivity to these allergens, a rapid in vitro test was developed that relies on the upregulation of ecto-nucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3) on activated basophils. METHODS: Blood basophils of 13 healthy donors and 22 patients allergic to bee or wasp venom were analyzed for E-NPP3 (CD203c) expression using monoclonal antibody 97A6. Basophils were analyzed by flow cytometry after activation with anti-IgE antibody or allergen. Venom-induced E-NPP3 upregulation on basophils was compared with diagnostic parameters, including skin tests and assessment of specific IgE. In selected samples, the increase in E-NPP3 expression on activated basophils was compared with histamine release and CD63 upregulation. RESULTS: In 20/22 patients sensitized to wasp or bee venom, E-NPP3 expression on basophils was upregulated in response to activation by allergen or anti-IgE. The maximum increase in E-NPP3 expression (above ten times of baseline) was achieved after 15 min of stimulation with 1 microg/ml of allergen or anti-IgE antibody. Sensitized individuals who failed to upregulate E-NPP3 in response to IgE receptor cross-linking also failed to induce histamine release and CD63 upregulation. CONCLUSIONS: Flow cytometric determination of hymenoptera-venom-induced upregulation of E-NPP3 is a novel in vitro test to identify sensitized individuals.
Copyright 2002 S. Karger AG, Basel

Publication Types:

·       Evaluation Studies

PMID: 11815741 [PubMed - indexed for MEDLINE]

29: Mol Pharmacol. 2001 Aug;60(2):341-7.

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A peptide derived from bee venom-secreted phospholipase A2 inhibits replication of T-cell tropic HIV-1 strains via interaction with the CXCR4 chemokine receptor.

Fenard D, Lambeau G, Maurin T, Lefebvre JC, Doglio A.

Laboratoire de Virologie, Institut National de la Sante et de la Recherche Medicale U526, Faculte de Medecine, Nice, France.

We have previously shown that secreted phospholipases A2 (sPLA2) from bee and snake venoms have potent anti-human immunodeficiency virus (HIV) activity. These sPLA2s block HIV-1 entry into host cells through a mechanism linked to sPLA2 binding to cells. In this study, 12 synthetic peptides derived from bee venom sPLA2 (bvPLA2) have been tested for inhibition of HIV-1 infection. The p3bv peptide (amino acids 21 to 35 of bvPLA2) was found to inhibit the replication of T-lymphotropic (T-tropic) HIV-1 isolates (ID(50) = 2 microM) but was without effect on monocytotropic (M-tropic) HIV-1 isolates. p3bv was also found capable of preventing the cell-cell fusion process mediated by T-tropic HIV-1 envelope. Finally, p3bv can inhibit the binding of radiolabeled stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and the binding of 12G5, an anti-CXCR4 monoclonal antibody. Taken together, these results indicate that p3bv blocks the replication of T-tropic HIV-1 strains by interacting with CXCR4. Its mechanism of action however appears distinct from that of bvPLA2 because the latter inhibits replication of both T-tropic and M-tropic isolates and does not compete with SDF-1alpha and 12G5 binding to CXCR4.

PMID: 11455021 [PubMed - indexed for MEDLINE]

30: 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]

31: J Allergy Clin Immunol. 2001 May;107(5):914-20.

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Api m 6: a new bee venom allergen.

Kettner A, Hughes GJ, Frutiger S, Astori M, Roggero M, Spertini F, Corradin G.

Institute of Biochemistry, University of Lausanne, Lausanne, Switzerland.

BACKGROUND: Characterization of the primary structure of allergens is a prerequisite for the design of new diagnostic and therapeutic tools for allergic diseases. OBJECTIVE: The purpose of this study was the identification and characterization of a low-molecular-weight, IgE-binding, bee venom (BV) allergen. METHODS: BV proteins were separated by using size exclusion chromatography and HPLC. IgE antibody binding to purified proteins was analyzed by means of immunoblotting, and T-cell response was analyzed by means of proliferation assay. Amino acid sequence was determined with 2 approaches, namely Edman degradation and carboxy terminal analysis with mass spectrometry. RESULTS: Api m 6, which migrated as an 8-kd band in SDS-PAGE, was frequently (42%) recognized by IgE from BV-hypersensitive patients. In addition, PBMCs from BV-hypersensitive patients, as well as from a normal control subject, proliferated in response to this allergen. Api m 6 exists as 4 isoforms of 7190, 7400, 7598, and 7808 d, respectively. Amino acid sequences obtained from HPLC-purified preparations revealed that the isoforms were constituted of a common central core of 67 residues, only differing in the amino- and carboxy-terminal ends. Api m 6 showed no significant sequence homology with known proteins. CONCLUSIONS: We have identified and sequenced a new BV allergen that elicits a strong IgE and T-cell response in a large number of BV-hypersensitive patients. Api m 6 should be considered in the diagnostic and therapeutic approach of BV immunotherapy on the basis of peptides or recombinant proteins.

PMID: 11344362 [PubMed - indexed for MEDLINE]

32: Int Arch Allergy Immunol. 2001 Jan-Mar;124(1-3):180-2.

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Mechanism of IL-10-induced T cell inactivation in allergic inflammation and normal response to allergens.

Akdis CA, Joss A, Akdis M, Blaser K.

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

BACKGROUND: Induction of specific unresponsiveness (tolerance/anergy) in peripheral T cells and recovery by cytokines from the tissue microenvironment represent two key steps in specific immunotherapy (SIT) with whole allergen or antigenic T cell peptides. METHODS: Antigen-specific T cell responses and molecular mechanisms of T cell inactivation were investigated during conventional SIT, T cell epitope peptide immunotherapy and natural exposure to bee venom in allergic and hyperimmune individuals. RESULTS: T cell unresponsiveness, initiated by autocrine action of IL-10, is characterized by suppressed proliferative and cytokine responses. The unresponsive T cells can be reactivated by different cytokines that may mimic the microenvironmental cytokine influence. IL-10 initiates peripheral tolerance by blocking the CD28 costimulatory signal in T cells. Coprecipitation experiments reveal that upon stimulation CD28 and IL-10 receptor are physically associated in T cells. Accordingly, IL-10 binding to its receptor inhibits CD28 tyrosine phosphorylation, the initial step of the CD28 signaling pathway. This leads to inhibition of phosphatidylinositol 3-kinase p85 binding to CD28. IL-10 only affects T cells that receive a stimulation with low numbers of triggered T cell receptors and that require costimulatory signals by CD28. CONCLUSION: These data demonstrate the pivotal role of autocrine IL-10 and the interaction of its receptor with CD28 in the induction of T cell tolerance as an immunoregulatory mechanism controlling antigen-specific T cell responses.
Copyright 2001 S. Karger AG, Basel

Publication Types:

·       Review

PMID: 11306962 [PubMed - indexed for MEDLINE]

33: 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]

34: Eur J Immunol. 2000 Dec;30(12):3533-41.

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Expression of cutaneous lymphocyte-associated antigen on human CD4(+) and CD8(+) Th2 cells.

Akdis M, Klunker S, Schliz M, Blaser K, Akdis CA.

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

The cutaneous lymphocyte-associated antigen (CLA) represents the homing receptor involved in selective migration of memory/effector T cells to the skin. Numerous reports demonstrated distinct CLA expression on Th1 cells. However, T cells isolated from skin lesions and CLA(+) T cells circulating in peripheral blood of atopic dermatitis patients expressed high IL-5 and IL-13. Accordingly, we investigated the regulation of CLA on human type 1 and type 2 T cells. CLA was induced on freshly generated Th1 and Tc1 cells only, but not on those of type 2. Anti-CD3 stimulation was sufficient to induce CLA on Th2 cells in the absence of serum in the culture medium. In serum containing medium, IL-4 inhibited CLA and related alpha-fucosyltransferase mRNA expression. IL-12 and/or staphylococcal enterotoxin B (SEB) stimulation up-regulated CLA expression on either Th2 and Tc2 cells. On stimulation with IL-12, CLA was expressed on the surface of bee venom phospholipase A(2)-specific Th1, Th2, Th0 and T regulatory 1 clones, representing non-skin-related antigen-specific T cells. In addition, CLA could be re-induced on T cells that had lost CLA expression upon resting. These results suggest that skin-selective homing is not restricted to functional and phenotypic T cell subsets.

PMID: 11093173 [PubMed - indexed for MEDLINE]

35: 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]

36: Eur J Immunol. 2000 Jun;30(6):1638-45.

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Allergen-derived long peptide immunotherapy down-regulates specific IgE response and protects from anaphylaxis.

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

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

To evaluate a long peptide-based allergy vaccine in a murine model, CBA/J mice were sensitized with low dose alum-adsorbed phospholipase A2 (PLA2), a major bee venom allergen. Presensitized mice were treated by daily i.p. injections of a mixture of three long overlapping peptides (44- to 60-mer) spanning the entire PLA2 molecule (100 microg/peptide) for 6 consecutive days. This therapeutic approach induced a sharp drop in PLA2-specific IgE, an increase in specific IgG2a, and a marked T cell hyporesponsiveness. T cell cytokine secretion was characterized by a shift from a Th2 to a Th1 profile. Prophylactic treatment of naive mice with long peptides prior to sensitization with PLA2 induced a comparable modulation of B and T cell responses. Upon i.p. challenge with native PLA2, presensitized mice treated with the long peptide mixture were fully protected from anaphylaxis. This indicated that allergen-derived long overlapping peptides were safe and able to modulate an established Th2 response or to prevent its development. Furthermore, long peptide-based immunotherapy provided clinical protection against anaphylaxis, thus appearing as a promising approach of the therapy of allergic diseases.

PMID: 10898500 [PubMed - indexed for MEDLINE]

37: Int J Pancreatol. 2000 Feb;27(1):29-38.

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Role of various phospholipases A2 and inhibitors in the pathogenesis and prevention of pancreatic acinar cell necrosis: studies with isolated rat pancreatic acini.

Mossner J, Wessig C, Ogami Y, Keim V.

Department of Internal Medicine II, University of Leipzig, Germany. moej@server3.medizin.uni-leipzig.de

BACKGROUND: Phospholipase A2 (PLA2) may play a central role in the pathogenesis of pancreatic acinar cell necrosis. Several questions, however, are unsolved: Is acinar cell necrosis caused by PLA2 derived from infiltrating leukocytes or from pancreatic PLA2 itself? Does PLA2 cause cellular lysis by the release of lysolecithin from lecithin or by generation of free radicals? The aims of this study were to determine which form of PLA2 is responsible for cellular damage and how to inhibit its action. METHODS: Isolated rat pancreatic acini were prepared by collagenase digestion. Newly synthesized proteins were labeled by 35S-methionine. Acini were incubated in buffer to which various factors, such as porcine pancreatic PLA2 or bee venom PLA2, homogenates of either leukocytes or pancreatic homogenates, all with or without lecithin and with or without potential inhibitors (aprotinin, 4-bromophenacylbromide, BM 16.2115, quinacrine, various analogs of arachidonic acid), or free radicals (hydrogen peroxide, xanthine/ xanthine oxidase) with or without allo-purinol or dismutase/catalase were added. Cellular destruction was measured by the release of radiolabeled proteins. RESULTS: PLA2 alone, free radicals, and granulocytes were not harmful to acini within 30 min of incubation. Free radicals caused significant release of radiolabeled proteins only after 3 h of incubation; this release could be inhibited by scavengers. Incubation of pancreatic acini with PLA2 in combination with lecithin caused rapid release of radiolabeled proteins. Addition of high concentrations of enterokinase activated pancreatic homogenates both alone and with lecithin caused release of cellular proteins, suggesting that pancreatic PLA2 uses lecithin from pancreatic membranes as substrate. Almost all tested potential inhibitors of PLA2 were unable to prevent the destruction caused by either pancreatic or bee venom PLA2 and lecithin. However, HK 42, a polyunsaturated fatty acid analog, was able to reduce dose dependently the release of acinar proteins caused by pancreatic PLA2 and lecithin. CONCLUSION: Pancreatic PLA2 and not PLA2 from infiltrating leukocytes may play a role in pancreatic acinar cell necrosis. Cellular lysis is caused upon the action of lysolecithin and probably not via the action of free radicals.

PMID: 10811021 [PubMed - indexed for MEDLINE]

38: Arch Immunol Ther Exp (Warsz). 2000;48(2):107-10.

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Histamine receptor expression on peripheral blood lymphocytes is influenced by specific and nonspecific activation.

Zak-Ejmark T, Kraus-Filarska M, Malolepszy J, Jankowska R, Jutel M, Nowak IA, Nadobna G.

Department of Internal Medicine and Allergology, University Medical School, Wroclaw, Poland.

Histamine is a physiological mediator which exerts both effector and regulatory functions through its receptors on various cells. The aim of the study was to investigate changes in histamine receptor expression on peripheral blood lymphocytes affected by stimulation with both specific and nonspecific stimuli. Lymphocytes were obtained from both healthy and allergic subjects. Cells were incubated with various allergens (mixed grass pollen, Lolium perenne, Dermatophagoides pteronyssinus 1, bee venom, phospholipase A2) and nonspecific (fMLP, PMA/ionomycin, LPS) stimuli. The percentage of histamine-binding cells was determined with a fluorescence microscope after incubation with histamine-fluorescein. In control subjects histamine binding after stimulation with allergens was not significantly changed. In contrast, in allergic subjects stimulation with specific allergens resulted in significantly increased histamine binding. Nonspecific stimulation caused increased histamine binding to lymphocytes in both allergic subjects and healthy controls. We conclude that specific and nonspecific activation of lymphocytes is associated with increased expression of histamine receptors.

PMID: 10807051 [PubMed - indexed for MEDLINE]

39: Immunobiology. 2000 Jan;201(3-4):391-405.

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Lipopeptides as immunoadjuvants and immunostimulants in mucosal immunization.

Baier W, Masihi N, Huber M, Hoffmann P, Bessler WG.

Institut fur Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universitat, Freiburg, Germany.

In previous studies we have shown that lipopeptides constitute potent immunoadjuvants in mice, rabbits and other species: in parenteral immunization, lipopeptide adjuvants were comparable, or in some cases superior to Freund's adjuvant, and were devoid of the side effects of this additive. Here we demonstrate that lipopeptides also constitute adjuvants for mucosal immunizations. The serum antibody responses against the wheat storage protein gliadin, the bee venom constituent melittin, or the hen egg protein ovalbumin could in most cases be enhanced more than 100-fold by the lipopeptide P3CSK4, applied via the nasal route. An enhanced specific antibody level could also be detected in supernatants of cell cultures prepared from spleens, Peyer's patches, lungs and mesenteric lymph nodes of immunized mice. Moreover, the lipopeptide P3CSK4 enhanced chemiluminescence in mouse spleen cells and peritoneal macrophages in vitro, indicating a macrophage-activating effect. Finally, nasal application of lipopeptide increased protection against a lethal infection of influenza. Our findings are of importance for the improvement of immunizations and might lead to more effective vaccines.

PMID: 10776795 [PubMed - indexed for MEDLINE]

40: Allergy. 1999 Jul;54(7):742-7.

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The frequency of phospholipase A2 binding of basophilic granulocytes does not decrease during bee-venom-specific immunotherapy.

Irsch J, Konig C, Lohndorf A, Tesch H, Krieg T, Merk H, Radbruch A, Hunzelmann N.

Department of Genetics, University of Cologne, Germany.

BACKGROUND: The major allergenic component of bee venom is phospholipase A2 (PLA2). METHODS: In this study, PLA2 was used to analyze and enrich PLA2-binding cells from peripheral blood by high gradient magnetic cell sorting. RESULTS: In normal donors, the frequency of allergen (PLA2)-binding cells among peripheral blood mononuclear cells (PBMC) as determined by flow cytometry is below 0.1%, whereas in bee-venom-allergic patients, PLA2-binding cells are readily detectable at frequencies of up to 2.3%. In severely bee-venom-allergic patients, many basophilic granulocytes are present, as defined by anti-CD9, CD25, and CD38 mAb, comprising up to 95% of the PLA2-binding cells. From blood of allergic and normal donors, about equal absolute numbers of allergen-binding CD19/21-positive B cells can be enriched. Severe anaphylactic reactions (Mueller grade IV) and failure of or adverse reactions during immunotherapy are associated with high numbers of circulating allergen-binding basophils. Interestingly, in the patients studied, the number of PLA2-binding basophilic granulocytes did not markedly change during rush immunotherapy and up to 6 months of maintenance immunotherapy. CONCLUSIONS: The specific and reproducible enrichment of PLA2-binding cells provides a new tool for the analysis and monitoring of effector cells in bee-venom-allergic patients with immediate-type hypersensitivity.

PMID: 10442531 [PubMed - indexed for MEDLINE]

41: Int Immunol. 1999 Aug;11(8):1313-26.

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On the diversity and heterogeneity of H-2(d)-restricted determinants and T cell epitopes from the major bee venom allergen.

Texier C, Herve M, Pouvelle S, Menez A, Maillere B.

Departement d'Ingenierie et d'Etudes des Proteines, CEA-Saclay, 91191 Gif sur Yvette, France.

One of the main limitations of using synthetic peptides for immunotherapy in allergic patients is the difficulty to delineate the immunodominant T cell epitopes which are necessarily dependent on HLA molecules. We have thus addressed the question of the role of MHC II molecules in immunodominant epitopes selection in the particular case of the major bee venom allergen (API m1). To exhaustively and easily explore it, we used BALB/c mice whose H-2 haplotype is associated with high IgE and IgG responses to API m1. By means of extensive sets of synthetic peptides, we investigated the specificity of polyclonal T cells and monoclonal hybridomas from mice immunized with API m1 and delineated four immunodominant regions, restricted to either the I-E(d) or the I-A(d) molecule. All the peptides were also tested for their capacity to bind to immunopurified MHC II molecules. Eight determinants of high affinity were identified. They clustered into three distinct regions and were largely overlapping. They included all the immunodominant epitopes, but half of them were not capable of stimulating T cells. Strikingly, interacting surfaces with either the TCR or MHC II molecule greatly differed from one determinant to another. In one case, we observed that flanking regions exerted a particular action on T cell stimulation which prevented the fine epitope localization. Our results underline the diversity and complexity of MHC II-restricted determinants and T cell epitopes from the major bee venom allergen, even in a single haplotype. These data also participate in the development of alternative approaches to conventional immunotherapy.

PMID: 10421789 [PubMed - indexed for MEDLINE]

42: Clin Exp Allergy. 1999 Mar;29(3):394-401.

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IgE and T-cell responses to high-molecular weight allergens from bee venom.

Kettner A, Henry H, Hughes GJ, Corradin G, Spertini F.

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

BACKGROUND: Bee venom contains multiple allergens with a wide distribution of molecular weight. In contrast with conventional bee venom desensitization, peptide or recombinant allergen immunotherapy may have to take into account patients' individual patterns of humoral or cellular response. OBJECTIVE: To study immunoglobulin (Ig)E and T-cell responses to high-molecular weight bee venom allergens >/= 50 kDa. METHODS: Bee venom proteins were separated by size exclusion chromatography and fractions were characterized by one and two-dimensional gel electrophoresis. IgE antibody binding to bee venom fractions was analysed by immunoblotting and T-cell responses by proliferation assay. RESULTS: Among 38 bee venom-hypersensitive patients, IgE recognition pattern of bee venom allergens varied greatly. IgE bound mainly to phospholipase A2 and furthermore to several proteins >/= 50 kDa (50, 54, 69, 84 and 94 kDa). N-terminal sequences of these proteins showed no homology with known proteins. In addition, peripheral mononuclear cells from patients as well as from nonatopic donors strongly proliferated in response to those proteins. CONCLUSIONS: Although present in low amounts, high-molecular weight allergens from bee venom elicit strong IgE and T-cell responses, and may need to be considered as clinically relevant. Therefore, the development of peptide or recombinant protein-based immunotherapy for bee venom allergy may require careful characterization of such allergens.

PMID: 10202349 [PubMed - indexed for MEDLINE]

43: FASEB J. 1999 Apr;13(6):603-9.

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IL-10-induced anergy in peripheral T cell and reactivation by microenvironmental cytokines: two key steps in specific immunotherapy.

Akdis CA, Blaser K.

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

Specific immunotherapy (SIT) is widely used for treatment of allergic diseases and could potentially be applied in other immunological disorders. Induction of specific unresponsiveness (anergy) in peripheral T cells and recovery by cytokines from the tissue microenvironment represent two key steps in SIT with whole allergen or antigenic T cell peptides (PIT). The anergy is directed against the T cell epitopes of the respective antigen and characterized by suppressed proliferative and cytokine responses. It is initiated by autocrine action of IL-10, which is increasingly produced by the antigen-specific T cells. Later in therapy, B cells and monocytes also produce IL-10. The anergic T cells can be reactivated by different cytokines. Whereas IL-15 and IL-2 generate Th1 cytokine profile and an IgG4 antibody response, IL-4 reactivates a Th2 cytokine pattern and IgE antibodies. Increased IL-10 suppresses IgE and enhances IgG4 synthesis, resulting in a decreased antigen-specific IgE:IgG4 ratio, as observed normally in patients after SIT or PIT. The same state of anergy against the major bee venom allergen, phospholipase A2, can be observed in subjects naturally anergized after multiple bee stings. Together, these data demonstrate the pivotal role of autocrine IL-10 in induction of specific T cell anergy and the important participation of the cytokine microenvironment in SIT. Furthermore, knowledge of the mechanisms explaining reasons for success or failure of SIT may enable possible predictive measures of the treatment.

Publication Types:

·       Review

PMID: 10094921 [PubMed - indexed for MEDLINE]

44: J Pharmacol Exp Ther. 1999 Apr;289(1):166-72.

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Effects of petrosaspongiolide M, a novel phospholipase A2 inhibitor, on acute and chronic inflammation.

Garcia-Pastor P, Randazzo A, Gomez-Paloma L, Alcaraz MJ, Paya M.

Departamento de Farmacologia, Universidad de Valencia, Facultad de Farmacia, Valencia, Spain.

The marine product petrosaspongiolide M is a novel inhibitor of phospholipase A2 (PLA2), showing selectivity for secretory PLA2 versus cytosolic PLA2, with a potency on the human synovial enzyme (group II) similar to that of manoalide. This compound was more potent than manoalide on bee venom PLA2 (group III) and had no effect on group I enzymes (Naja naja and porcine pancreatic PLA2). Inhibition of PLA2 was also observed in vivo in the zymosan-injected rat air pouch, on the secretory enzyme accumulated in the pouch exudate. Petrosaspongiolide M decreased carrageenan paw edema in mice after the oral administration of 5, 10, or 20 mg/kg. This marine metabolite (0.01-1.0 micromol/pouch) induced a dose-dependent reduction in the levels of prostaglandin (PG)E2, leukotriene B4, and tumor necrosis factor-alpha in the mouse air pouch injected with zymosan 4 h after the stimulus. It also had a weaker effect on cell migration. The inflammatory response of adjuvant arthritis was reduced by petrosaspongiolide M, which also inhibited leukotriene B4 levels in serum and PGE2 levels in paw homogenates. In contrast with indomethacin, this marine compound did not reduce PGE2 levels in stomach homogenates. Petrosaspongiolide M is a new inhibitor of secretory PLA2 in vitro and in vivo, with anti-inflammatory properties in acute and chronic inflammation.

PMID: 10087000 [PubMed - indexed for MEDLINE]

45: J Immunol. 1999 Feb 1;162(3):1836-42.

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An altered peptide ligand specifically inhibits Th2 cytokine synthesis by abrogating TCR signaling.

Faith A, Akdis CA, Akdis M, Joss A, Wymann D, Blaser K.

Swiss Institute of Allergy and Asthma Research, Davos, Switzerland. siaf@siaf.unizh.ch

Altered peptide ligands (APL) can modify T cell effector function by their diversity in binding to the TCR or MHC class II-presenting molecules. The capacity to inhibit Th2 cytokine production by allergen-specific T cells would contribute to combating allergic inflammation. The presence of APL generated by Ala-substitutions in a synthetic dodeca-peptide spanning an immunodominant epitope of bee venom phospholipase A2 (PLA) was investigated in human T cells. Four of five substituted peptides reduced proliferation, IL-4, and IFN-gamma production by cloned PLA-specific Th0 cells proportionately. However, one APL, PLA-F82A, inhibited IL-4 but had no effect on IFN-gamma production. This uncoupling of IL-4 from IFN-gamma production was also observed on immunogenic restimulation of the cloned T cells pre-exposed to the APL/APCs. It appeared to result from lower affinity of binding to MHC class II by the APL compared with the native peptide. The APL also inhibited IL-4 production by polyclonal T cells. In consequence of the change in cytokine secretion, the production of IgG4 in vitro increased by PLA-F82A stimulation, compared with the native peptide. Exposure of the cloned T cells to either the APL or the native peptide, in the absence of professional APC, induced anergy such that proliferation and production of IL-4, IL-5, and IL-13 was abrogated on immunogenic rechallenge. Defective T cell activation appeared to result from alterations in transmembrane signaling through the TCR, specifically to lack of tyrosine phosphorylation of the tyrosine kinase, ZAP-70.

PMID: 9973449 [PubMed - indexed for MEDLINE]

46: Biochim Biophys Acta. 1998 Sep 16;1425(1):74-80.

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Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula.

Wu QX, King MA, Donovan GR, Alewood D, Alewood P, Sawyer WH, Baldo BA.

Molecular Immunology, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, NSW, Australia.

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-3H]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin 1. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin 1 using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an alpha-helical secondary structure.

PMID: 9813247 [PubMed - indexed for MEDLINE]

47: Clin Exp Allergy. 1998 Jul;28(7):839-49.

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Enzymatic activity of soluble phospholipase A2 does not affect the specific IgE, IgG4 and cytokine responses in bee sting allergy.

Wymann D, Akdis CA, Blesken T, Akdis M, Crameri R, Blaser K.

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

BACKGROUND: The soluble bee venom phospholipase A2 (PLA) represents the major allergen/antigen for allergic and hyperimmune individuals following bee sting. A number of studies implicate enzymes, and PLA in particular, as potent allergens. We have studied specific activation of T cells by enzymatically active and inactive mutants of PLA, and secretion of cytokines regulating IgE and IgG4 antibody formation. METHODS: Recombinant (r) wild type PLA (rPLA-WT) and an enzymatically inactive rPLA (rPLA-H34Q) were produced in Escherichia coli. Eleven bee venom allergic patients and three hyperimmune, healthy individuals were included in the study. After specific stimulation of PBMC with the rPLA variants, proliferative response, IFNgamma, IL-2, IL-4, IL-5, and IL-13 production, as well as total and PLA-specific IgE and IgG4 production, were analysed. RESULTS: Similar levels of specific B cell recognition, proliferative and cytokine responses were observed after stimulation with either enzymatically active or inactive rPLA. In addition, equal amounts of antigen-specific and total IgE and IgG4 antibodies were produced by stimulation with both forms of rPLA. CONCLUSIONS: The enzymatic activity of PLA does not influence the specific activation and cytokine production by T cells from bee venom-sensitized or hyperimmune individuals, or the IgE/IgG4 antibodies synthesis by B cells in vitro.

PMID: 9720818 [PubMed - indexed for MEDLINE]

48: Cytometry. 1998 Aug 1;32(4):268-73.

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Flow cytometric analysis of cell killing by the jumper ant venom peptide pilosulin 1.

King MA, Wu QX, Donovan GR, Baldo BA.

Department of Clinical Immunology, Royal North Shore Hospital, St. Leonards, New South Wales, Australia. making@doh.health.nsw.gov.au

Pilosulin 1 is a synthetic 56-amino acid residue polypeptide that corresponds to the largest allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula. Initial experiments showed that pilosulin 1 lysed erythrocytes and killed proliferating B cells. Herein, we describe how flow cytometry was used to investigate the cytotoxicity of the peptide for human white blood cells. Cells were labeled with fluorochrome-conjugated antibodies, incubated with the peptide and 7-aminoactinomycin D (7-AAD), and then analyzed. The effects of varying the peptide concentration, serum concentration, incubation time, and incubation temperature were measured, and the cytotoxicity of pilosulin 1 was compared with that of the bee venom peptide melittin. The antibodies and the 7-AAD enabled the identification of cell subpopulations and dead cells, respectively. It was possible, using the appropriate mix of antibodies and four-color analysis, to monitor the killing of three or more cell subpopulations simultaneously. We found that 1) pilosulin 1 killed cells within minutes, with kinetics similar to those of melittin; 2) pilosulin 1 was a slightly more potent cytotoxic agent than melittin; 3) both pilosulin 1 and melittin were more potent against mononuclear leukocytes than against granulocytes; and 4) serum inhibited killing by either peptide.

PMID: 9701394 [PubMed - indexed for MEDLINE]

49: Clin Exp Allergy. 1997 Sep;27(9):1016-26.

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Comment in:

·       Clin Exp Allergy. 1997 Sep;27(9):986-90.

Delineation of PLA2 epitopes using short or long overlapping synthetic peptides: interest for specific immunotherapy.

Kammerer R, Kettner A, Chvatchko Y, Dufour N, Tiercy JM, Corradin G, Spertini F.

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

BACKGROUND: Venom immunotherapy is definitely indicated in severe systemic anaphylactic reactions to bee stings, but is not devoided of risks of anaphylaxis. Safer methods of immunotherapy need to be developed. OBJECTIVE: To delineate phospholipase A2 T-cell epitopes using short 15mer vs long 40-60mer overlapping peptides, and to approach the potential interest of a venom immunotherapy based on the use of long peptides (1-60, 51-99, 90-134) mapping the whole phospholipase A2 molecule vs a restricted number of immunodominant epitopes. METHODS: Proliferation of a CD8+ T cell depleted peripheral blood mononuclear cell fraction and short-term T-cell lines from unselected bee venom hypersensitive patients in response to phospholipase A2 synthetic peptides. RESULTS: Whereas T-cell proliferation to 15mer overlapping peptides was weak, T-cell response to long overlapping peptides was in contrast vigorous in all patients, mostly directed to C-terminal peptide 90-134. Our results did not support the concept of rare dominant T-cell epitopes, and disclosed T-cell responses to multiple epitopes in several patients. No significant IgE-binding to long overlapping peptides was detected except in one patient against peptide 90-134. CONCLUSION: 15mer peptides might not be sensitive enough to fully delineate all potential T-cell epitopes scattered along the allergen. Since they do not bind IgE in vitro or only weakly, and taking into account a T-cell response frequently directed to multiple epitopes, long overlapping peptides may represent ideal tools for immunotherapy.

PMID: 9678833 [PubMed - indexed for MEDLINE]

50: Clin Exp Allergy. 1997 Sep;27(9):986-90.

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Comment on:

·       Clin Exp Allergy. 1997 Sep;27(9):1016-26.

To bee or not to be? T-cell responses to bee venom PLA2 in relation to anaphylaxis and immunotherapy.

McHugh SM.

Publication Types:

·       Comment

·       Editorial

PMID: 9678827 [PubMed - indexed for MEDLINE]

51: 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]

52: 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]

53: J Investig Allergol Clin Immunol. 1998 Mar-Apr;8(2):109-14.

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Lymphocyte subpopulations, cytokine release and specific immunoglobulin G in reactive and nonreactive beekeepers.

Annila I, Hurme M, Miettinen A, Kuusisto P, Nieminen MM.

Department of Respiratory Medicine, Medical School, University of Tampere, Finland.

Although bee venom sensitization and systemic sting reactions are common among beekeepers, the prediction of the severity of reactions has not yet been possible with laboratory tests. The present study was designed to evaluate parameters that might be clinically useful in estimation of systemic reactivity, and parameters that could differentiate allergic beekeepers from sensitized subjects. Thirty-two beekeepers were selected and placed into the following three groups: anergic (n = 10), asymptomatic sensitized (n = 11), and allergic (n = 11). Peripheral blood lymphocyte subpopulations, venom-specific immunoglobulin (Ig) E and IgG and cytokine release by peripheral blood mononuclear cells were measured. The ratio of stimulated interleukin-4 to stimulated interferon-gamma was significantly higher in sensitized beekeepers than in allergic or anergic subjects. Venom-specific IgG correlated significantly with the number of annual stings (r = 0.575) and the years spent in beekeeping (r = 0.471). No significant differences in the subpopulations of peripheral blood lymphocytes were found between the study groups. We conclude that differences in the subpopulations of peripheral blood lymphocytes are not associated with sensitization or systemic reactivity. In asymptomatic sensitized beekeepers, T helper 2 T-cell dominance is more pronounced than in allergic subjects. Bee venom specific IgG correlates directly with the degree of exposure to bee venom.

PMID: 9615305 [PubMed - indexed for MEDLINE]

54: Izv Akad Nauk Ser Biol. 1998 Mar-Apr;(2):225-9.

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[Proliferation and death of thymocytes caused by phospholipase A2 activator--melittin]

[Article in Russian]

Shaposhnikova VV, Egorova MV, Levitman MKh, Kudriavtsev AA, Sukharev VI, Korystov IuK.

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Russia.

The effects of mellitin, a component of bee venom activating phospholipase A2, on proliferation and death of the rat thymocytes were studied in a wide concentration range. Cell proliferation was estimated by the accumulation of colchicine metaphases, Necrosis was estimated by cell lysis and Trypan blue staining. Apoptosis was estimated by the type of DNA fragmentation, amount of fragmented DNA, and percentage of cells with hypodiploid DNA set. Low concentrations of mellitin (below 5 micrograms/ml) stimulated proliferation. At higher mellitin concentrations, the thymocytes die by the primary necrosis type. Mellitin did not induce apoptosis in the thymocytes within the concentration range used: on the contrary, at high concentrations, it inhibited apoptosis of the thymocytes in the control and after irradiation. Actinomycin D, inhibitor of RNA synthesis, exerted no effect on the thymocyte death in the presence of mellitin. It has been concluded that activation of phospholipase A2 may induce necrosis, rather than apoptosis, and consequently, activation of phospholipase A2 is not a necessary step in the signalling cascade that initiated apoptosis in the thymocytes.

PMID: 9609959 [PubMed - indexed for MEDLINE]

55: Allergy. 1998 Mar;53(3):233-40.

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Systemic T-cell unresponsiveness during rush bee-venom immunotherapy.

Segura JA, Assenmacher M, Irsch J, Hunzelmann N, Radbruch A.

Institute for Genetics, University of Cologne, Germany.

By rush bee-venom immunotherapy, subjects reacting allergically to the venom can be effectively anergized, although the mechanism of action is not known. Here we analyzed the systemic effects of rush desensitization on the T cells of allergic patients. In most patients, we found reduced frequencies of T cells recalled to express CD69 and the cytokines interleukin (IL)-4 and interferon-gamma (IFN-gamma) after stimulation of peripheral blood mononuclear cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin, as compared with normal donors. These frequencies are progressively reduced during immunotherapy. The frequency of cells expressing IL-2 does not change. A few patients show a different response to immunotherapy: frequencies of cells expressing CD69, IL-4, or IFN-gamma do not change, and remain similar to those of normal donors. However, the frequency of cells able to express IL-2 is increased. The analysis of cytokine expression in CD45RO+ vs CD45RO- T-cell populations revealed differences between normal and allergic donors. In allergic patients, higher frequencies of IL-4- and IFN-gamma-expressing cells among the CD45RO- subpopulation were found than in normal donors. This situation is not modified by immunotherapy. The results reveal a certain degree of heterogeneity in the response of allergic patients to bee-venom rush immunotherapy; however, all are clearly differentiated from normal controls as judged by cytokine expression of CD45RO- T cells. In most allergic patients, a considerable percentage of Th cells become unresponsive to mitogenic stimulation, and may be responsible for the desensitization itself.

PMID: 9542602 [PubMed - indexed for MEDLINE]

56: Eur J Immunol. 1998 Mar;28(3):914-25.

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Differential regulation of human T cell cytokine patterns and IgE and IgG4 responses by conformational antigen variants.

Akdis CA, Blesken T, Wymann D, Akdis M, Blaser K.

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

Bee venom phospholipase A2 (PLA) represents the major allergen and antigen in allergic and non-allergic individuals sensitized to bee sting. We have studied specific activation of peripheral T cells by different structural and conformational variants of PLA and secretion of cytokines regulating IgE and IgG4 antibody (Ab) formation. PLA molecules expressing the correctly folded tertiary structure, which show high affinity to membrane phospholipids and were recognized by Ab from bee sting allergic patients, induced high IL-4, IL-5 and IL-13 production in peripheral blood mononuclear cell cultures. In contrast, non-refolded recombinant PLA (rPLA) and reduced and alkylated native PLA (nPLA) induced more IFN-gamma and IL-2 and higher proliferative responses. Differences in proliferation and cytokine patterns among correctly folded and non-refolded PLA resulted from conformation-dependent involvement of different antigen-presenting cell (APC) types. Antigen (Ag)-presenting B cells recognized PLA only in its natural conformation, stimulated Th2 type cytokines and induced IgE Ab. Non-refolded PLA was recognized, processed and presented exclusively by monocytes and induced a Th1 dominant cytokine profile leading to IgG4 production by B cells. The possibility that production of particular cytokine patterns and Ig isotype was influenced by the enzymatic activity of PLA was excluded by using enzymatically inactive H34Q point-mutated, refolded rPLA. These findings demonstrate the decisive role of specific Ag recognition by different APC, depending on structural features, membrane phospholipid binding property and the existence of conformational B cell epitopes, in the differential regulation of memory IgE and IgG4 Ab. Furthermore, they show that a change from IgE-mediated allergy to normal immunity against a major allergen can be induced by rPLA variants that are not recognized by specific Ab and B cells but still carry the T cell epitopes. These features may enable new applications for safer immunotherapy.

PMID: 9541587 [PubMed - indexed for MEDLINE]

57: Adv Exp Med Biol. 1997;400A:365-73.

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PX-52, A novel inhibitor of 14 kDa secretory and 85 kDa cytosolic phospholipases A2.

Franson RC, Rosenthal MD.

Department of Biochemistry & Molecular Biophysics, Virginia Commonwealth University, Richmond, USA.

Previously we reported that PGBx, a prostaglandin oligomer with anti-inflammatory activity, inhibited 14 kDa phospholipase A2 (PLA2) activity and blocked arachidonic acid mobilization in prelabeled human neutrophils (Biochim. Biophys. Acta 1006:272-277, 278-286, 1989) This study describes a new inhibitor of phospholipase A2, PX-52, that also blocks agonist induced arachidonic acid mobilization in prelabeled cells. PX-52, a fatty acid polymer, inhibited hydrolysis of 14C-oleate labeled E.coli by a variety of 14 kDa PLA2s including human PMN, sperm, synovial fluid and disc, as well as porcine pancreas, N. naja, and bee venom in a dose-dependent manner with IC50s ranging from 1.0-3.7 uM. Inhibition of activity was comparable at different Ca2+ concentrations, but was relieved by increasing substrate concentration or by methylation of PX-52. Hydrolysis of [14C]-arachidonyl phosphatidylcholine by 85 kDa, cytosolic PLA2 from U937 cells was similarly inhibited by PX-52, the IC50 = 5 uM. Arachidonic acid mobilization induced by A23187 in prelabeled human PMNs was blocked by PX-52; IC50 = 10-15 uM while concentrations of up to 80 uM oleate had no effect. These results demonstrate that PX-52 inhibits the in vitro activity of secretory and cytosolic PLA2s and agonist-induced arachidonic acid release from human cells. Given its ability to block the arachidonic acid cascade, PX-52 may be useful in the control of inflammation.

PMID: 9547578 [PubMed - indexed for MEDLINE]

58: 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]

59: Eur J Immunol. 1997 Sep;27(9):2351-7.

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Glucocorticoids inhibit human antigen-specific and enhance total IgE and IgG4 production due to differential effects on T and B cells in vitro.

Akdis CA, Blesken T, Akdis M, Alkan SS, Heusser CH, Blaser K.

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

Although anti-inflammatory properties of glucocorticoids (GC) are well documented, their activity in allergic diseases is still controversial. Recently, it has been reported that GC can increase, both in vivo and in vitro, the polyclonal production of total IgE. In this study we investigated the effects of GC on the antigen (Ag)-specific IgE response in a human in vitro system with peripheral blood mononuclear cells or B cells of bee venom-sensitized individuals that allows the production of bee venom phospholipase A2 (PLA)-specific IgE and IgG4 antibodies (Ab). PLA-specific Ab were induced by simultaneously activating T cells and B cells specifically with allergen and polyclonally with anti-CD2 and soluble CD40 ligand (sCD40L) in the presence of interleukin (IL)-4. Indeed, dexamethasone and prednisolone enhanced the formation of total IgE and IgG4 in PBMC, while the production of PLA-specific IgE and IgG4 Ab was selectively inhibited in a dose-dependent manner. The suppressive effect of GC was mediated during Ag-specific stimulation and T cell-B cell interaction. This was due to GC suppressing specific T cell proliferation and cytokine production, whereas neither allergen-specific nor total IgE and IgG4 production by sCD40L/IL-4-stimulated pure B cells was affected. In contrast to GC, cyclosporine A inhibited both total and PLA-specific IgE and IgG4 secretion in peripheral blood mononuclear cells and B cell cultures. Further experiments showed that increase in nonspecific total isotype response resulted from inhibition of IL-4 uptake by cells other than B cells and sufficient availability of IL-4 to B cells for isotype switch and synthesis. Furthermore, demonstration of opposite regulatory effects of GC on specific and total isotype formation in vitro, including the inhibition of allergy-relevant Ag-specific IgE response, may contribute to a better understanding of apparently controversial observations, and explain why most allergic patients benefit from GC therapy.

PMID: 9341780 [PubMed - indexed for MEDLINE]

60: J Investig Allergol Clin Immunol. 1997 Jul-Aug;7(4):217-24.

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Mononuclear blood cell sulfidoleukotriene generation in the presence of interleukin-3 and whole blood histamine release in honey bee and yellow jacket venom allergy.

Maly FE, Marti-Wyss S, Blumer S, Cuhat-Stark I, Wuthrich B.

Institute of Clinical Chemistry, University Hospital Zurich, Switzerland.

Cross-linking IgE on basophils is known to cause both sulfidoleukotriene (sLT) generation and histamine release. We recently developed an ELISA to determine sulfidoleukotriene generation by blood mononuclear cells which employs pretreatment with IL-3 to enhance leukotriene generation (cellular antigen stimulation test, CAST). Here, we compared the CAST and whole blood histamine release in response to honey bee/yellow jacket venom (BV/YJV) in 23 patients clinically suspected of type-I allergy to these venoms. Of these, 17 were diagnosed as "definitive venom allergics," defined by a positive skin test at 100 ng/ml of venom or less. The six in whom such skin reactivity was absent were labelled "suspected venom allergics." Both venoms stimulated sulfidoleukotriene generation and histamine release also from control individuals (n = 10). In patients, insect venoms generally stimulated histamine release and sulfidoleukotriene generation in excess of the mean + 3 SD of values obtained with control individuals. However, about half of the patients reacted predominantly with either histamine release or sulfidoleukotriene generation. No overall correlation was found between threshold doses necessary to stimulate sulfidoleukotriene generation (ThsLT) and histamine release (ThHist). (Linear correlation coefficients between ThsLT and ThHist were -0.02 for honey bee venom and 0.13 for yellow jacket, n = 23). Both findings are in contrast to the concept that these responses occur in parallel. From results with "definitive venom allergics," CAST sensitivity was calculated as 100% for honey bee venom and 83% for yellow jacket, and that of the histamine release assay as 62.5% for honey bee venom and 50% for yellow jacket. Specificity of the CAST was calculated as 77% for honey bee venom and 100% for yellow jacket, and that of the histamine release assay as 44% for honey bee venom and 60% for yellow jacket. Thus, CAST results are closer to skin test results than to those of the whole blood histamine release assay.

PMID: 9330184 [PubMed - indexed for MEDLINE]

61: Naunyn Schmiedebergs Arch Pharmacol. 1997 Aug;356(2):233-9.

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Pharmacological properties of Ca2+-activated K+ currents of ramified murine brain macrophages.

Eder C, Klee R, Heinemann U.

Abteilung Neurophysiologie, Institut fur Physiologie der Charite, Humboldt Universitat, Berlin, Germany.

Using the whole-cell configuration of the patch clamp technique, calcium-activated potassium currents (I(K,Ca)) were investigated in ramified murine brain macrophages. In order to induce I(K,Ca) the intracellular concentration of nominal free Ca2+ was adjusted to 1 microM. The Ca2+-activated K+ current of brain macrophages did not show any voltage dependence at test potentials between -120 and +30 mV. A tenfold change in extracellular K+ concentration shifted the reversal potential of I(K,Ca) by 51 mV. The bee venom toxin apamin applied at concentrations of up to 1 microM did not affect I(K,Ca). Ca2+-activated K+ currents of ramified brain macrophages were highly sensitive to extracellularly applied charybdotoxin (CTX). The half-maximal effective concentration of CTX was calculated to be 4.3 nM. In contrast to CTX, the scorpion toxin kaliotoxin did not inhibit I(K,Ca) at concentrations between 1 and 50 nM. Tetraethylammonium (TEA) blocked 8.0% of I(K,Ca) at a concentration of 1 mM, whereas 31.4% of current was blocked by 10 mM TEA. Several inorganic polyvalent cations were tested at a concentration of 2 mM for their ability to block I(K,Ca). La3+ reduced I(K,Ca) by 72.8%, whereas Cd2+ decreased I(K,Ca) by 17.4%; in contrast, Ni2+ did not have any effect on I(K,Ca). Ba2+ applied at a concentration of 1 mM reduced I(K,Ca) voltage-dependently at hyperpolarizing potentials.

PMID: 9272730 [PubMed - indexed for MEDLINE]

62: J Allergy Clin Immunol. 1997 Jul;100(1):96-103.

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Modulation of T-cell response to phospholipase A2 and phospholipase A2-derived peptides by conventional bee venom immunotherapy.

Kammerer R, Chvatchko Y, Kettner A, Dufour N, Corradin G, Spertini F.

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

BACKGROUND: Immunologic mechanisms of desensitization are still incompletely understood. Safer methods of immunotherapy with reduced risks of anaphylaxis need to be developed. OBJECTIVE: To study the effects of conventional venom immunotherapy (VIT) on phospholipase A2(PLA2)-specific T cells and on T-cell reactivity to short and long synthetic peptides that map the PLA2 molecule. METHOD: Proliferation of a CD4+ cell-enriched peripheral blood mononuclear cell fraction and cytokine secretion by T cell lines from patients hypersensitive to bee venom and undergoing VIT in response to PLA2 and PLA2 synthetic peptides were measured. RESULTS: T-cell proliferation in response to three synthetic peptides, 40 to 60 amino acids long and mapping the entire PLA2 molecule with an overlap of 10 residues (1 to 59, 51 to 99, and 90 to 134) steadily increased during the first 14 weeks of VIT corresponding to the treatment period with incremental doses of antigen. These results are in contrast to the low proliferation indices obtained with short (15 amino acid-long) peptides, and the inability to characterize the immunodominant region of the molecule with short peptides. At the end of VIT (after 3 to 5 years), there was correspondingly, a marked decrease in T cell responsiveness to PLA2 and to its long synthetic peptides. This response was paralleled by a shift in the pattern of cytokine secretion by T cell lines from a T(H0)-type to a T(H1)-type pattern. CONCLUSION: After a transient increase in T-cell proliferation, late VIT was characterized by T-cell hyporesponsiveness to allergen and by modulation of cytokine secretion from a T(H0)-type to a T(H1)-type pattern. Because of their capacity to recruit multiple T-cell epitopes, long peptides mapping the entire PLA2 molecule appear to be efficient T cell stimulators and may represent potential candidates for peptide immunotherapy.

PMID: 9257793 [PubMed - indexed for MEDLINE]

63: J Pharmacol Exp Ther. 1997 Jul;282(1):123-31.

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Variabilin: a dual inhibitor of human secretory and cytosolic phospholipase A2 with anti-inflammatory activity.

Escrig V, Ubeda A, Ferrandiz ML, Darias J, Sanchez JM, Alcaraz MJ, Paya M.

Department of Pharmacology, University of Valencia and Institute of Natural Products and Agrobiology, Tenerife, Spain.

The marine product variabilin was identified as a novel inhibitor of phospholipase A2 (PLA2), which exhibited IC50 values of 6.9 microM and 7.9 microM for human synovial secretory PLA2 and U937 cells cytosolic PLA2 activities, respectively. This compound was less potent on bee venom or zymosan-injected rat air pouch enzymes and failed to affect Naja naja venom PLA2. The production of leukotriene B4 by human neutrophils stimulated with calcium ionophore A23187 was also inhibited by variabilin, which was without effect on 5-lipoxygenase, cyclo-oxygenase 1 and cyclo-oxygenase 2 activities in cell-free assays. Other functions of human neutrophils, such as degranulation and superoxide generation, were also significantly reduced in vitro. Variabilin administered topically suppressed the mouse ear edema induced by 12-O-tetradecanoylphorbol 13-acetate, whereas the ear edema induced by arachidonic acid was unaffected; this suggests an action previous to arachidonic acid metabolism. This compound administered p.o. at 30 mg/kg and 45 mg/kg significantly inhibited mouse paw edema induced by carrageenan and, at 0.01 to 1.0 micromol/pouch in the mouse air pouch injected with zymosan, exerted a marked inhibition on PGE2 and leukotriene B4 levels in exudates (ID50 values of approximately 0.028-0.029 micromol/pouch), without affecting cell migration. Our results indicate that variabilin is an inhibitor of human secretory and cytosolic PLA2 activities that controls eicosanoid production in vitro and in vivo, inhibits neutrophil degranulation and superoxide generation in vitro and shows anti-inflammatory activity after topical or p.o. administration to mice.

PMID: 9223548 [PubMed - indexed for MEDLINE]

64: Int Arch Allergy Immunol. 1997 Mar;112(3):226-30.

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The murine (H-2k) T-cell epitopes of bee venom phospholipase A2 Lie outside the active site of the enzyme. Implications with respect to a paracrine activation of Th2 cells for an IgE antibody response.

Specht C, Kolsch E.

Institute for Immunology, University of Munster, Germany.

Recombinant, enzymatic active phospholipase A2 from bee venom (PLA2) is a potent inducer of IgE antibody formation in CBA/J (H-2k) mice. In contrast, a recombinant mutant protein lacking enzymatic activity due to an amino acid exchange in the active site of the enzyme fails to induce IgE antibodies under identical immunization conditions. Peptide mapping and T-cell stimulation experiments with 18-mer overlapping peptides locate the T-helper cell-activating epitopes in the C-terminal region of the PLA2 protein. No T-cell epitopes are found in the area around position 34, the center of the enzymatically active site. The data support a model in which initially an enzymatic activation of mast cells or basophiles leads to IL-4 production which in a paracrine way drives T-helper cells, concomitantly activated by antigen, into Th2 differentiation. This ultimately favors B-cell activation for an IgE response.

PMID: 9066507 [PubMed - indexed for MEDLINE]

65: J Allergy Clin Immunol. 1997 Mar;99(3):345-53.

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Induction and differential regulation of bee venom phospholipase A2-specific human IgE and IgG4 antibodies in vitro requires allergen-specific and nonspecific activation of T and B cells.

Akdis CA, Blesken T, Akdis M, Alkan SS, Wuthrich B, Heusser CH, Blaser K.

Swiss Institute of Allergy and Asthma Research, Davos.

Investigations on the mechanisms of IgE regulation in vitro have been conducted thus far in systems that allow the synthesis of total rather than specific IgE. To study the regulatory prerequisites of antigen-specific IgE antibody production, we have established a culture system that allows the generation of bee venom phospholipase A2-specific IgE and IgG4 antibodies. Allergen-specific IgE was induced by simultaneously activating T cells and B cells specifically with allergen and polyclonally with anti-CD2 and soluble CD40 ligand in the presence of IL-4. Additional stimulation of T cells through the CD2 activation pathway by two different anti-CD2 monoclonal antibodies enhanced both the allergen-specific and the total IgE and IgG4 responses. An optimal amount of allergen (0.1 ng/ml) resulted in the induction of both allergen-specific IgE and IgG4 antibodies. Higher antigen doses reduced allergen-specific antibodies and enhanced total isotype production. This differential regulation of allergen-specific and total isotypes reflects different allergen dose-dependent mechanisms in specific and polyclonal activation of T and B cells. Although both isotypes require IL-4 for initial induction, opposite regulatory effects by T cells were observed for IgE and IgG4 antibody expression. In peripheral blood mononuclear cell cultures stimulated with soluble CD40 ligand, IL-4, and phospholipase A2, stimulation of T cells with higher amounts of anti-CD2 enhanced IgG4 in parallel to increased IL-2 and interferon-gamma secretion but inhibited IgE synthesis. These results provide evidence for differential regulation of allergen-specific and total IgE and IgG4 by antigen concentration and demonstrate the pivotal role of T cells controlling the synthesis of the IgE and IgG4 antibody isotypes.

PMID: 9058690 [PubMed - indexed for MEDLINE]

66: Eur J Immunol. 1997 Feb;27(2):515-21.

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The intensity of T cell receptor engagement determines the cytokine pattern of human allergen-specific T helper cells.

Carballido JM, Faith A, Carballido-Perrig N, Blaser K.

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

Enhanced production of T helper (Th)2 cytokines by allergen-specific Th cells plays a major role in the induction and maintenance of IgE-mediated allergic disorders. The mechanism that triggers this type of response in atopic individuals is not fully understood. Allergen-specific human Th cell clones produce interleukin (IL)-4 and low or undetectable levels of interferon (IFN)-gamma after stimulation with low concentrations of antigen. However, these Th cell clones are capable of generating significant amounts of IFN-gamma after optimal activation through their T cell receptor (TcR). Allergen-specific Th cell clones isolated from allergic individuals required higher doses of antigen to reach the plateau of proliferation and to generate Th0 cytokine responses than their counterparts isolated from nonallergic subjects. On the other hand, if allergen was replaced by anti-CD3 monoclonal antibody (mAb), both allergic and nonallergic Th cell clones attained the highest level of proliferation and significant IFN-gamma production in response to equivalent concentrations of anti-CD3 mAb. These results indicate that the strength of T cell ligation, which can be modulated by the availability of the TcR ligand, controls the balance of Thl/Th2 cytokines produced by memory Th cells in vitro. In the particular case of bee venom phospholipase A2, it is shown that the expression of allergen-specific surface Ig on antigen-presenting B cells has little influence on antigen uptake and therefore in determining the levels of T cell activation and cytokine production. Alternatively, the affinity of particular major histocompatibility complex class II molecules on antigen-presenting cells for allergen-derived peptides might determine the amount of specific ligand presented to the Th cells and play a decisive role skewing the Th cell cytokine production towards Th1 or Th2 phenotypes. These findings, which are consistent with the changes in cytokine patterns observed following clinical hyposensitization, suggest that polarized human Th2 cell subsets still retain the capacity to modulate their cytokine pattern.

PMID: 9045925 [PubMed - indexed for MEDLINE]

67: J Immunol Methods. 1996 Dec 15;199(2):119-26.

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Quantitation of the cytosolic phospholipase A2 (type IV) in isolated human peripheral blood eosinophils by sandwich-ELISA.

Zhu X, Munoz NM, Rubio N, Herrnreiter A, Mayer D, Douglas I, Leff AR.

Department of Medicine, University of Chicago, IL 60637, USA.

Sandwich enzyme-linked immunosorbent assay (sELISA) was developed for precise quantitation of cytosolic phospholipase A2 (cPLA2 type IV) concentration in isolated human peripheral blood eosinophils as an alternative to semiquantitative chemiluminescent assay employing immunoprecipitation/Western blot analysis. In this assay, monoclonal mouse anti-human cPLA2 antiserum was used as the capture antibody, polyclonal rabbit anti-human cPLA2 antiserum as the secondary antibody, and alkaline phosphatase-conjugated goat anti-rabbit IgG as the tertiary, reporter antibody. Purified human cPLA2 (0-1000 ng/ml) dissolved in Tris-HCl buffered saline was used as the standard protein. The detection limit for cPLA2 in 10(6) eosinophils was 0.109 ng/ml, and coefficients of inter- and intra-assay variation were 4.23% and 7.07%, respectively. There was no cross-reactivity with other (secretory) isoforms of PLA2 (sPLA2 types I-III) either from porcine pancreas, human synovial fluid, or bee venom. In separate studies, the recovery of cPLA2 was > 83% when eosinophil lysate was supplemented exogenously with two different concentrations of cPLA2. From a total protein content of 22.3 +/- 1.7 micrograms/10(6) cells, the baseline concentration of cPLA2 was 0.38 +/- 0.18 ng/10(6) cells in eosinophils obtained from mildly atopic donors. Immunoblotting studies confirmed the complete specificity for the type IV isoform as detected by sELISA. This sELISA method permits the precise quantitative assessment of cPLA2 in nanogram quantities per million cells, which has not previously been possible by immunoblotting analysis.

PMID: 8982353 [PubMed - indexed for MEDLINE]

68: 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]

69: Naunyn Schmiedebergs Arch Pharmacol. 1996 Nov;354(5):677-83.

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Inhibition of phospholipase A2 activities and some inflammatory responses by the marine product ircinin.

Cholbi R, Ferrandiz ML, Terencio MC, De Rosa S, Alcaraz MJ, Paya M.

Department of Pharmacology, University of Valencia, Faculty of Pharmacy, Burjassot, Spain.

The marine product ircinin has been tested for its effects on secretory and cytosolic phospholipase A2 (PLA2) activities in vitro as well as for inhibition of cellular functions in human neutrophils and inflammatory responses in mice. Ircinin inhibited Naja naja venom, human synovial recombinant, bee venom and zymosan-injected rat air pouch PLA2 with IC50 values in the microM range, similar to those of the known inhibitor scalaradial. On the other hand, ircinin was less active on cytosolic PLA2 from human monocytes and decreased potently the release of LTB4 in human neutrophils. This marine product affected weakly human neutrophil functions like superoxide generation and degranulation. In the zymosan-injected rat air pouch ircinin inhibited in vivo the activity of PLA2 present in exudates and reduced dose-dependently myeloperoxidase levels, whereas cell migration was inhibited only at the highest dose tested. This compound exerted a potent anti-oedematous effect after topical application in the mouse ear oedema test. Ircinin is a new inhibitor of PLA2 activity and our results suggest a potential role for this marine product as an inhibitor of inflammatory processes.

PMID: 8938669 [PubMed - indexed for MEDLINE]

70: Clin Exp Allergy. 1996 Oct;26(10):1112-8.

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Comment in:

·       Clin Exp Allergy. 1996 Oct;26(10):1101-4.

Influence of bee venom immunotherapy on degranulation and leukotriene generation in human blood basophils.

Jutel M, Muller UR, Fricker M, Rihs S, Pichler WJ, Dahinden C.

Medical Division, Zieglerspital, Bern, Switzerland.

BACKGROUND: Rapid clinical tolerance can be induced over several hours by very fast bee venom immunotherapy (VIT) protocols. OBJECTIVE: To investigate the mechanisms underlying VIT we examined the changes of blood basophil responsiveness during VIT. METHODS: Seven bee venom allergic patients with a history of severe systemic reactions after a bee sting were investigated. A cumulative dose of 111.1 micrograms bee venom (BV) was administered sc over 3.5 h under intensive care conditions according to an ultra-rush protocol. The release of histamine and the formation of leukotrienes in response to BV, major BV allergen Phospholipase A2 (PLA), IgE receptor cross-linking with the use of monoclonal antibodies against IgE and IgE receptor, as well as IgE independent activation in response to C5a were determined in vitro before and after ultra-rush VIT. RESULTS: We demonstrated a decrease of total histamine in peripheral blood leucocytes just after VIT. Histamine release in response to all the stimuli used is not affected by ultra-rush VIT, if expressed as per cent release of total histamine. However, the absolute amount product released in response to stimulation was decreased, particularly with allergen (BV, PLA). We also found a significant reduction of LTC4 formation after VIT in samples stimulated with specific allergen (BV, PLA). CONCLUSION: Blood basophils are a target for VIT, which induces impaired release of both preformed and newly generated mediators. However, we believe the basic mechanisms of rapid clinical tolerance induced by ultra-rush VIT remain to be investigated.

PMID: 8911695 [PubMed - indexed for MEDLINE]

71: J Clin Invest. 1996 Oct 1;98(7):1676-83.

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Epitope-specific T cell tolerance to phospholipase A2 in bee venom immunotherapy and recovery by IL-2 and IL-15 in vitro.

Akdis CA, Akdis M, Blesken T, Wymann D, Alkan SS, Muller U, Blaser K.

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

Bee venom phospholipase A2 (PLA) is the major allergen in bee sting allergy. It displays three peptide and a glycopeptide T cell epitopes, which are recognized by both allergic and non-allergic bee venom sensitized subjects. In this study PLA- and PLA epitope-specific T cell and cytokine responses in PBMC of bee sting allergic patients were investigated before and after 2 mo of rush immunotherapy with whole bee venom. After successful immunotherapy, PLA and T cell epitope peptide-specific T cell proliferation was suppressed. In addition the PLA- and peptide-induced secretion of type 2 (IL-4, IL-5, and IL-13), as well as type 1 (IL-2 and IFN-gamma) cytokines were abolished, whereas tetanus toxoid-induced cytokine production and proliferation remained unchanged. By culturing PBMC with Ag in the presence of IL-2 or IL-15 the specifically tolerized T cell response could be restored with respect to specific proliferation and secretion of the type 1 T cell cytokines, IL-2 and IFN-gamma. In contrast, IL-4, IL-5, and IL-13 remained suppressed. Treatment of tolerized T cells with IL-4 only partially restored proliferation and induced formation of distinct type 2 cytokine pattern. In spite of the allergen-specific tolerance in T cells, in vitro produced anti-PLA IgE and IgG4 Ab and their corresponding serum levels slightly increased during immunotherapy, while the PLA-specific IgE/IgG4 ratio changed in favor of IgG4. These findings indicate that bee venom immunotherapy induces a state of peripheral tolerance in allergen-specific T cells, but not in specific B cells. The state of T cell tolerance and cytokine pattern can be in vitro modulated by the cytokines IL-2, IL-4, and IL-15, suggesting the importance of microenvironmental cytokines leading to success or failure in immunotherapy.

PMID: 8833918 [PubMed - indexed for MEDLINE]

72: 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]

73: Eur J Immunol. 1996 Jun;26(6):1260-5.

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Kinetics and functional implications of Th1 and Th2 cytokine production following activation of peripheral blood mononuclear cells in primary culture.

McHugh S, Deighton J, Rifkin I, Ewan P.

Molecular Immunopathology Unit, Medical Research Council Centre, Cambridge, GB.

The importance of cytokine production in some disease processes is now widely recognized. To investigate temporal relationships between cytokines, we stimulated peripheral blood mononuclear cells (PBMC) in vitro using the T cell mitogen phytohemagglutinin (PHA) and various antigens chosen to induce predominantly Th1 (streptokinase: streptodornase or purified protein derivative) or Th2 (Dermatophagoides pteronyssinus, bee or wasp venom: allergens in sensitive subjects) responses. Cytokine production was measured by sensitive bioassays or enzyme-linked immunosorbent assays. Of the 30 subjects studied, 10 were normal and 20 individuals were allergic to either D. pteronyssinus (n = 10) or bee venom (n = 10) (examined before specific allergen immunotherapy). We examined the temporal profiles of a panel of cytokines produced in primary culture. In PHA-driven cultures, cytokines were found to be sequentially produced in the order interleukin (IL)-2, IL-4, IL-5, IL-3, interferon (IFN)-gamma, IL-10, IL-6, IL-12 and tumor necrosis factor (TNF)-alpha. The response to allergen in allergic patients was predominantly Th2 in nature, with the production of IL-4, IL-5, IL-6 and IL-10, but little or no IFN-gamma. IL-2, IL-3, TNF-alpha and IL-12 were also produced in low amounts. The response of both atopic and normal subjects to recall bacterial antigens was predominantly Th1, with high levels of IFN-gamma, IL-2 and TNF-alpha. The relevance of the order, amount and speed of production, characteristic kinetics (production, consumption, homeostatic regulation) and the cell source of the cytokines are discussed.

PMID: 8647202 [PubMed - indexed for MEDLINE]

74: J Immunol. 1996 Mar 1;156(5):1728-34.

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Biochemical characterization of antigen-specific glycosylation-inhibiting factor from antigen-specific suppressor T cells. I. Identification of a 55-kilodalton glycosylation-inhibiting factor peptide with TCR alpha-chain determinant.

Nakano T, Ishii Y, Ishizaka K.

Division of Immunobiology, La Jolla Institute for Allergy and Immunology, CA 92037, USA.

Stimulation of OVA-specific suppressor T cell (Ts) hybridoma and bee venom phospholipase A2 (PLA2)-specific Ts hybridoma with Ag-pulsed APC or by cross-linking of CD3 resulted in the formation of Ag-specific glycosylation-inhibiting factor (GIF). Affinity-purified Ag-specific GIF preparations, obtained by using Ag-coupled Sepharose or anti-TCR alpha-chain-coupled Affi-Gel, contained a 55-kDa peptide, which bound both polyclonal anti-GIF Abs and anti-TCR-alpha mAb in immunoblotting. The same hybridomas constitutively secrete 13-kDa bioactive GIF peptide that has no affinity for homologous Ag, but neither the Ag-specific GIF activity nor 55-kDa GIF peptide was detectable in culture supernatants of unstimulated cells. Northern blot analysis of mRNA from the anti-CD3-stimulated hybridoma with 32P-labeled GIF cDNA revealed only 0.6 kb mRNA, which encodes the 13-kDa nonspecific GIF. No mRNA of the 55-kDa GIF was detectable. A representative OVA-specific Th hybridoma, DO 11.10 cells contain the 0.6 kb GIF mRNA and constitutively secrete inactive GIF peptide. However, the Th hybridoma failed to secrete the 55-kDa peptide or any peptide with the TCR-alpha determinant upon stimulation with anti-CD3. It appears that the formation of the 55-kDa peptide with the TCR-alpha determinant is unique for a subset of T cells including Ts cells that form bioactive GIF.

PMID: 8596020 [PubMed - indexed for MEDLINE]

75: 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]

76: 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]

77: Arch Biochem Biophys. 1995 Dec 1;324(1):78-84.

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Bee venom phospholipase A2 is recognized by the macrophage mannose receptor.

Mukhopadhyay A, Stahl P.

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

A high affinity and a specific binding site for bee venom PLA2 was found on the surface of J774E macrophages. The binding sites for bee venom PLA2 are entirely different from the binding sites for pancreatic and snake venom PLA2 as revealed by competition experiments. Binding and uptake of bee venom PLA2 by J774E macrophages was shown to be competed by mannose-BSA, glucose-BSA, N-acetylglucosamine-BSA, but not by galactose-BSA, indicating that the binding of bee venom PLA2 is probably mediated by macrophage mannose receptor. An affinity labeling experiment revealed that the bee venom PLA2 specifically binds to a single polypeptide with a mass of approximately 180 kDa. Moreover, the affinity labeled protein component, i.e., the binding site, was not detected in the presence of excess mannose-BSA, suggesting that mannose-BSA and the bee venom PLA2 bind to the same site on macrophages. These observations were further supported by the binding of bee venom PLA2 to cells which are known to express the mannose receptor and by specific binding of bee venom PLA2 to the purified mannose receptor. These data confirm that bee venom PLA2 binding to macrophages is mediated through the mannose receptor.

PMID: 7503563 [PubMed - indexed for MEDLINE]

78: Clin Exp Allergy. 1995 Nov;25(11):1108-17.

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Selective restimulation of antigen or allergen preactivated T cells using OKT3 F(ab)2 results in the secretion of TH-1 or TH-2-like cytokine patterns.

Jutel M, Wyss-Coray T, Carballido JM, Blaser K, Muller UR, Pichler WJ.

Zieglerspital, Bern, Switzerland.

BACKGROUND: The synthesis of IgE is regulated by cytokines secreted from T-helper cells. The studies on cytokine secretion by peripheral blood mononuclear cells (PBMC) upon stimulation with antigen or allergen are difficult due to low levels of cytokines, especially of interleukin-4 (IL-4). OBJECTIVE: In this study we tried to establish a culture system, which could enable the measurement of the cytokine profiles in specifically activated cultures. METHODS: Three methods to potentiate cytokine secretion were evaluated: PBMC from bee venom or house dust mite (Dermatophagoides pteronyssinus) allergic patients as well as normal subjects were stimulated either with the major bee venom allergen phospholipase A2 (PLA) or with the major D. pteronyssinus allergen (Der p 1) or with the control antigens tetanus toxoid (TT) and purified protein derivate (PPD). After 7 days of culture the cells were restimulated either with plastic bound OKT3 F(ab)2 monoclonal antibodies (MoAbs), with the appropriate antigen + antigen presenting cells or with IL-2. The secretion of cytokines (IL-4, IFN gamma) was measured after restimulation of the cultures (day 8). RESULTS: While OKT3 F(ab)2 was unable to activate resting T cells, it could restimulate preactivated cells. Restimulation with OKT3 F(ab)2 induced higher IL-4 and IFN gamma secretion than restimulation with IL-2 or antigen. TT and PLA stimulated a similar cytokine secretion profile in normal and PLA allergic subjects with substantial levels of both IL-4 and IFN gamma. In contrast, PPD induced virtually only IFN gamma secretion. Der p 1 stimulated mainly IL-4 secretion but also IFN gamma production in some mite allergic patients. CONCLUSION: We have established a cell culture system, which combines antigen specificity with a strong cytokine inducing signal provided by anti-CD3 MoAbs. TH-1 and TH-2 characteristic cytokine patterns can be observed in short-term PBMC cultures already after 8 days of culture.

PMID: 8581844 [PubMed - indexed for MEDLINE]

79: Eur J Pharmacol. 1995 Oct 24;285(3):281-8.

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Effects of marine 2-polyprenyl-1,4-hydroquinones on phospholipase A2 activity and some inflammatory responses.

Gil B, Sanz MJ, Terencio MC, De Giulio A, De Rosa S, Alcaraz MJ, Paya M.

Department of Pharmacology, University of Valencia, Faculty of Pharmacy, Spain.

Three 2-polyprenyl-1,4-hydroquinone derivatives (2-heptaprenyl-1,4-hydroquinone: IS1, 2-octaprenyl-1,4-hydroquinone: IS2 and 2-[24-hydroxy]-octaprenyl-1,4-hydroquinone: IS3) isolated from the Mediterranean sponge Ircinia spinosula, were evaluated for effects on phospholipase A2 activity of different origin (Naja naja venom, human recombinant synovial fluid and bee venom), as well as on human neutrophil function and mouse ear oedema induced by 12-O-tetradecanoylphorbol 13-acetate (TPA). IS1 interacted minimally with these responses. In contrast, IS2 and IS3 inhibited human recombinant synovial phospholipase A2 in a concentration-dependent manner, with minor effects on the rest of the enzymes. Both compounds slightly affected superoxide generation and degranulation in human neutrophils, whereas they decreased thromboxane B2 and leukotriene B4 synthesis and release in a mixed suspension of human platelets and neutrophils stimulated by ionophore A23187, with IC50 values in the microM range. IS3 was the most effective inhibitor of the synthesis of thromboxane B2 by human platelet microsomes and of leukotriene B4 by high speed supernatants from human neutrophils. IS2 and IS3 showed topical anti-inflammatory activity against the TPA-induced ear inflammation in mice, with similar effects on oedema and a higher inhibition of IS3 on leukocyte migration, estimated as myeloperoxidase activity in supernatants of ear homogenates. Some structure-activity relationships were established since differences in the prenylated chain attached to the hydroquinone moiety result in important modifications of these inflammatory responses.

PMID: 8575515 [PubMed - indexed for MEDLINE]

80: J Immunol Methods. 1995 Oct 12;186(1):27-36.

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Preparation of soluble recombinant T cell receptor alpha chain by using a calmodulin fusion expression system.

Ishii Y, Nakano T, Honma N, Yuyama N, Yamada Y, Watarai H, Tomura T, Sato M, Tsumura H, Ozawa T, et al.

Division of Immunobiology, La Jolla Institute for Allergy and Immunology, CA 92037, USA.

We have isolated a full length T cell receptor alpha chain (TCR alpha) cDNA derived from a bee venom phospholipase A2-specific mouse suppressor T cell hybridoma. A bacterial fusion expression system was constructed using rat calmodulin as a fusion partner for production of soluble TCR alpha. In this system, calmodulin-TCR alpha fusion protein was expressed at a high level in the soluble fraction of bacterial cell lysate, and could be purified by binding of calmodulin portion of the protein to phenyl-Sepharose. Using this system, fusion proteins containing a TCR alpha peptide corresponding to the complete extracellular region, V alpha-J alpha region or C alpha extracellular region were isolated. TCR alpha peptides were then released from the fusion proteins by digestion with thrombin which recognizes a linker sequence between calmodulin portion and TCR alpha segment. Polyclonal antibodies against constant region of TCR alpha chain (C alpha) were obtained by immunization of rabbits with the recombinant C alpha peptide. ELISA for TCR protein was established by using the polyclonal antibodies and the monoclonal antibody specific for C alpha region.

PMID: 7561145 [PubMed - indexed for MEDLINE]

81: 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]

82: Clin Exp Allergy. 1995 Sep;25(9):828-38.

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Bee venom immunotherapy induces a shift in cytokine responses from a TH-2 to a TH-1 dominant pattern: comparison of rush and conventional immunotherapy.

McHugh SM, Deighton J, Stewart AG, Lachmann PJ, Ewan PW.

Molecular Immunopathology Unit, Medical Research Centre, Cambridge, UK.

BACKGROUND: The mechanism of immunotherapy is unclear. Allergic disease is known to involve enhanced TH-2 cytokine responses to allergen. OBJECTIVE: In order to investigate the mechanisms of immunotherapy, we have examined changes in cytokine secretion before (13 patients) and during (nine patients) both rush and conventional venom immunotherapy (VIT) in bee venom allergic patients. METHODS: Peripheral blood mononuclear cells were stimulated in vitro with bee venom, non-specific antigen or mitogen and secretion of IL-4 (TH-2) and IFN gamma (TH-1) over the culture period measured. RESULTS: Untreated patients had TH-2 responses to venom and TH-1 responses to antigen and strong proliferative responses to venom. Controls showed no response (proliferation or cytokines) to venom and the normal TH-1 response to antigen. VIT resulted in marked changes in cytokine secretion to venom, with reduction of the abnormal TH-2 response and induction of a TH-1 response. The pattern differed in rush and conventional VIT. One day after rush VIT there was a significant fall in IL-4 secretion (P < 0.01), which rose by 3 weeks then declined. In conventional VIT there was a gradual reduction of IL-4 production significant after 2 months and undetectable by 6 months. IFN gamma secretion was induced by VIT. Proliferative responses mirrored the IL-4 changes. One day after rush VIT there was a loss of T cells, monocytes and NK cells from peripheral blood. CONCLUSION: This study shows that immunotherapy shifted cytokine responses to allergen from a TH-2 to a TH-1 dominant pattern, suggesting direct effects on T cells. How these cytokine changes relate to clinical desensitization is not clear. In the longer term they would result in an isotype switch from IgE to IgG. Early changes in cytokine or chemokine production might downregulate mast cell or basophil reactivity and explain the rapid desensitization in rush VIT.

Publication Types:

·       Clinical Trial

PMID: 8564721 [PubMed - indexed for MEDLINE]

83: 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]

84: Clin Exp Allergy. 1995 Aug;25(8):704-12.

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Tryptase and histamine release due to a sting challenge in bee venom allergic patients treated successfully or unsuccessfully with hyposensitization.

Eberlein-Konig B, Ullmann S, Thomas P, Przybilla B.

Dermatologische Klinik und Poliklinik, Ludwig-Maximilians-Universitat, Munich, Germany.

BACKGROUND: Hyposensitization with been venom leads to full protection in most, but not all patients with IgE-mediated systemic reactions to bee stings. OBJECTIVE: To determine the relationship of clinical reactivity to the release of mediators and to changes of antibody concentrations in the peripheral circulation at a bee sting challenge test. METHODS: Blood was sampled before (1 min) and at 15, 60 and 180 min after a sting challenge from 19 patients on hyposensitization. Of these six still reacted and 13 were protected. Histamine, mast cell tryptase, bee venom-specific IgE and IgG in the serum, and histamine release from peripheral blood leucocytes (PBL) upon exposure to bee venom were determined. RESULTS: Tryptase above the detection level was found only at 15 (60) min in 4/6 (1/6) patients who reacted. After the sting challenge there was a significant increase of the histamine levels in patients who reacted at 15 min (P < 0.05) and in patients who did react at 60 and 180 min (P < 0.01). The total histamine content of PBL was significantly decreased after 15 and 60 min in patients who reacted (P < 0.01) and in those that did not (P < 0.05). Bee venom-induced histamine release was significantly reduced in patients reacting and those that did not at 15 min (P < 0.05), and was significantly decreased in reactors also at 60 and 180 min (P < 0.05/0.01). Specific IgG antibodies showed a minor decrease (P < 0.05) after the sting challenge in both groups, whereas specific IgE did not change significantly. CONCLUSION: These results indicate that bee venom anaphylaxis is associated with the release of mediators from both mast cells as well as basophils. Successful hyposensitization does not induce a state of immunological non-reactivity, but rather alters the magnitude and the pattern of mediator release.

PMID: 7584681 [PubMed - indexed for MEDLINE]

85: J Immunol. 1995 Apr 15;154(8):4187-94.

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Bee venom immunotherapy results in decrease of IL-4 and IL-5 and increase of IFN-gamma secretion in specific allergen-stimulated T cell cultures.

Jutel M, Pichler WJ, Skrbic D, Urwyler A, Dahinden C, Muller UR.

Ziegler Hospital, Bern, Switzerland.

The mechanisms of bee venom immunotherapy (VIT) are largely unknown. The aim of this study was to follow the changes of T cell cytokine secretion during the course of VIT. Ten bee venom-allergic patients with a history of severe systemic reactions, positive skin tests, and bee venom (BV)-specific serum IgE Abs were treated as follows: on the first day, a cumulative dose of 111 micrograms, starting with 0.1 microgram, was administered s.c. under intensive care conditions. Further injections of 100 micrograms BV were given on day 7, day 21, and thereafter at intervals of 4 wk. Blood samples were obtained just before the initiation of VIT, after the last injection on the same day, and before the subsequent BV injections on days 7, 21, and 50 of VIT. Peripheral blood mononuclear cells (PBMC) were stimulated with phospholipase A (PLA), the major BV allergen, or with a control Ag tetanus toxoid (TT). Cytokine secretion was measured 24 h after restimulation of the cultures with solid-phase bound OKT3 F(ab')2 mAbs after 7 days of culture. In PLA-stimulated cultures, VIT resulted in decreased IL-4 and IL-5 and increased IFN-gamma secretion. In TT-stimulated cultures, we observed similar levels of cytokines before and during VIT. We conclude that ultra-rush VIT changes allergen-specific T cell reactivity.

PMID: 7706753 [PubMed - indexed for MEDLINE]

86: 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]

87: Eur J Immunol. 1995 Feb;25(2):538-42.

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Carbohydrate-dependent, HLA class II-restricted, human T cell response to the bee venom allergen phospholipase A2 in allergic patients.

Dudler T, Altmann F, Carballido JM, Blaser K.

Swiss Institute of Allergy and Asthma Research, Davos.

The T cell-independent antibody response to polysaccharide antigen (Ag) is believed to result from their inability to bind major histocompatibility complex (MHC) restriction elements. However, recent studies using glycosylated analogues of known immunogenic peptides revealed that glycopeptides can interact with MHC molecules and are able to elicit specific T cell responses in experimental animals. This raises questions about the possible role which carbohydrates can play in T cell responses following natural exposure to glycoprotein antigens. Analyzing the fine specificity of the human T cell response against the major bee venom allergen phospholipase A2 (PLA), a 16-20-kDa protein glycosylated at a single site (Asn13), we have identified several T cell clones which proliferate in response to the glycoprotein but not to its non-glycosylated variants. Neither the carbohydrate moiety alone nor the combination of carbohydrate and non-glycosylated protein could substitute for the intact glycoprotein. Antibody directed against the carbohydrate moiety inhibited Ag-induced proliferation of these clones whereas control clones with known peptide specificity were not affected, providing additional evidence for the involvement of carbohydrates in T cell recognition. Moreover, peripheral blood mononuclear cells of two individuals from whom glycosylation-dependent T cell clones have been isolated showed significantly higher proliferation in response to glycosylated compared to non-glycosylated Ag, suggesting that glycosylation can contribute in some cases extensively to the immunogenicity of a glycoprotein Ag. Thus, this report shows that glycosylation-dependent Ag recognition by T cells can also occur following natural exposure to a glycoprotein.

PMID: 7875217 [PubMed - indexed for MEDLINE]

88: J Allergy Clin Immunol. 1995 Feb;95(2):587-96.

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IgE+ cells in the peripheral blood of atopic, nonatopic, and bee venom-hypersensitive individuals exhibit the phenotype of highly differentiated B cells.

Donohoe PJ, Heddle RJ, Sykes PJ, Fusco M, Flego LR, Zola H.

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

We have analyzed IgE+ cells in peripheral blood of atopic donors, donors hypersensitive to bee venom, and nonatopic control donors with two- and three-color flow cytometry. Although the percentage of IgE+ cells varied among these groups, the overall phenotypic patterns were similar. Most IgE+ cells do not display typical B-cell markers, such as CD19, CD20, and CD21. A significant proportion of these cells stain for CD38, indicating that they are more differentiated. IgE+ cells express Fc gamma RII and CD45RO, an isoform associated with an advanced level of differentiation. The majority of IgE+ cells do not coexpress other surface immunoglobulin isotypes. In the case of bee venom-hypersensitive donors, we have been able to identify a small population of IgE+ cells with a specificity for phospholipase A2, a major immunogenic component of bee venom. The phospholipase A2+ cells display a phenotype similar to that of the IgE+ cells.

PMID: 7531730 [PubMed - indexed for MEDLINE]

89: Int Arch Allergy Immunol. 1994 Jul;104(3):262-9.

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Lysine residues in bee venom phospholipase A2 are important for binding to human monoclonal or polyclonal antibodies of the IgG4 isotope.

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

Swiss Institute of Allergy and Asthma Research, Davos.

Discontinuous antigenic sites on bee venom phospholipase A2 (PLA) have been mapped using human monoclonal antibodies or human polyclonal serum antibodies (hpAbs) of the IgG4 isotype from beekeepers or of the IgE isotype from individuals allergic to PLA. Lysine residues of PLA have been specifically modified by acetylation or acylation by treatment with citraconic anhydride of their epsilon-amino groups to analyze their role in antigen-antibody binding. After the modifications, the binding of PLA to the human monoclonal antibodies is lost, whereas the binding to IgG4 hpAbs is significantly decreased. In contrast, the effect on the binding of PLA to IgE hpAbs appears to be more heterogeneous. The data indicate the importance of lysine residues as being part of B cell epitopes in PLA-specific antibodies of the IgG4 isotype, but less so for those of the IgE isotype.

PMID: 7518267 [PubMed - indexed for MEDLINE]

90: J Allergy Clin Immunol. 1994 Apr;93(4):758-67.

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Regulation of IgE and IgG4 responses by allergen specific T-cell clones to bee venom phospholipase A2 in vitro.

Carballido JM, Carballido-Perrig N, Oberli-Schrammli A, Heusser CH, Blaser K.

Swiss Institute of Allergy and Asthma Research, Davos.

An in vitro antibody response to bee venom phospholipase A2 (PLA) from peripheral blood mononuclear cells of bee sting-sensitized individuals was achieved after stimulation with PLA and pokeweed mitogen. This stimulation resulted in a secretion of TH1-associated cytokines and induced PLA-specific and nonspecific IgG4 antibody production but not IgE production. The addition of interleukin-4 (IL-4) to this system decreased the secretion of IgG antibodies, whereas secretion of polyspecific IgE was induced. The mitogen was not required if peripheral blood mononuclear cells were enriched with autologous, PLA-specific, resting T-cell clones in the presence of the antigen. In these experiments the cytokine profile of the particular clone determined the antibody class generated. Low ratios of IL-4 to interferon-gamma, induced by the antigen alone or obtained by neutralizing anti-IL-4 antibodies, enhanced IgG4 antibody formation, whereas IgE levels increased at high ratios of IL-4 to interferon-gamma. These results suggest a complementary regulation of the main isotypes, IgE and IgG4, implicated in allergic and protective hyperimmune responses.

PMID: 8163785 [PubMed - indexed for MEDLINE]

91: Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2824-8.

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Antigen-binding glycosylation inhibiting factor from a human T-cell hybridoma specific for bee venom phospholipase A2.

Gomi H, Tagaya Y, Nakano T, Mikayama T, Ishizaka K.

Division of Immunobiology, La Jolla Institute for Allergy and Immunology, CA 92037.

We obtained human T-cell hybridomas that are specific for bee venom phospholipase A2 (PLA2) and constitutively secrete glycosylation inhibiting factor (GIF). Upon crosslinking of CD3, the hybridoma produced GIF having affinity for PLA2. When affinity-purified PLA2-binding GIF was used as an immunogen, monoclonal antibodies specific for the antigen-binding GIF were obtained. Monoclonal antibody 110BH3 bound the antigen-binding GIF but failed to bind the 13-kDa nonspecific GIF, as determined by both bioassay and ELISA. In contrast, 388F1, a monoclonal antibody against nonspecific GIF, gave ELISA signals with both the nonspecific GIF and the antigen-binding GIF. Gel filtration of affinity-purified antigen-binding GIF revealed the presence of a 72- to 80-kDa protein which gave ELISA signals with both 110BH3 and 388F1 and contained GIF bioactivity. Upon reduction and alkylation, the antigen-binding GIF dissociated into a 62- to 64-kDa protein which gave positive ELISA with antibody 110BH3 but no signal with antibody 388F1, and a 15-kDa protein, which gave ELISA signal with the 388F1 but not with 110BH3. Immunoblotting of a PLA2-binding GIF preparation revealed that under reducing conditions, the antigen-binding GIF dissociated a 13-kDa peptide which reacted with polyclonal antibodies against recombinant GIF. The results indicate that the 13-kDa nonspecific GIF is a subunit of antigen-binding GIF. The PLA2-binding GIF has affinity for an epitope, representing amino acid residues 19-28 in PLA2 which appears to be an external structure in the antigen.

PMID: 7511819 [PubMed - indexed for MEDLINE]

92: Int Immunol. 1993 Aug;5(8):833-42.

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Epitope specificity of bee venom phospholipase A2-specific suppressor T cells which produce antigen-binding glycosylation inhibiting factor.

Mori A, Thomas P, Tagaya Y, Iijima H, Grey H, Ishizaka K.

Division of Immunobiology, La Jolla Institute for Allergy and Immunology, CA 92037.

From the spleen cells of BALB/c mice primed with bee venom phospholipase A2 (PLA2), we established seven T cell hybridomas which constitutively secreted glycosylation inhibiting factor (GIF), expressed both CD3 and TCR alpha beta, and responded to antigen-pulsed antigen presenting cells (APC) for the formation of IgE-binding factor. Upon stimulation with antigen-pulsed APC, four of the seven hybridomas produced GIF having affinity for native PLA2. The antigen-binding GIF could suppress the anti-hapten antibody response of BALB/c mice to dinitrophenyl (DNP)-PLA2 conjugates in a carrier-specific manner and bound to immunosorbents coupled with either the mAb 14-12 or anti-TCR alpha chain, H28-710. Analysis of the epitope specificity of the TCR on the GIF-producing T hybridomas indicated that all of the hybridomas which could produce antigen-binding GIF upon antigenic stimulation recognized the synthetic peptide representing amino acid residues 19-34 in PLA2 molecules in the context of the product of the I-Ad subregion and the antigen-binding GIF formed by the cells had affinity for the peptide. The 3-D structure of bee venom PLA2 indicates that the sequence of amino acid 14-24 forms a loop in the PLA2 molecule and represents an external structure of the antigen, while peptide 25-37 forms an alpha helix. Evidence was obtained which suggests that the sequence of 25-34 contains amino acid residues interacting with Ia molecules, while peptide 19-24 contains residues involved in the interaction of p19-34-Ia complexes with TCR on the hybridomas. It was also found that not only the synthetic peptide 19-34, but also the peptides 13-28 and 19-30 inhibited the binding of antigen-binding GIF to PLA2-coupled Sepharose, while peptide 25-40 failed to do so. The results collectively indicate that the antigen-binding GIF and TCR on the cell source of the factor interact with a common epitope which is exposed on the surface of a nominal antigen.

PMID: 7691164 [PubMed - indexed for MEDLINE]

93: J Immunol. 1993 Apr 15;150(8 Pt 1):3582-91.

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T cell epitope specificity in human allergic and nonallergic subjects to bee venom phospholipase A2.

Carballido JM, Carballido-Perrig N, Kagi MK, Meloen RH, Wuthrich B, Heusser CH, Blaser K.

Swiss Institute of Allergy and Asthma Research, Davos.

Phospholipase A2 (PLA) is a biochemically fully defined glycoprotein, representing the main allergen of bee venom. We have established CD4+ T cell clones specific to PLA from subjects allergic and nonallergic to bee sting. By screening the epitope specificity of these clones with 62 synthetic overlapping dodecapeptides representing the PLA molecule, two immunogenic epitopes, PLA81-92 and PLA113-124, were identified. Additional screening, using longer peptides of up to 18 residues, revealed a third epitope at position PLA 45-62. These epitopes are recognized by specific T cell clones in association with HLA-DP and -DQ molecules, although HLA-DR-associated responses to PLA exist. Primary in vitro proliferation of unfractionated PBMC from bee sting allergic, hyposensitized, or hyperimmune subjects to PLA-derived peptides revealed the same immunogenic sites as found in the T cell clones. Among the different groups of individuals, proliferative responses to the PLA molecule and fragments thereof were generally higher in allergic patients than in nonallergic subjects. Thus, at least three linear epitopes are involved in T cell recognition of PLA, independently whether or not subjects are allergic.

PMID: 7682244 [PubMed - indexed for MEDLINE]

94: Biochemistry. 1993 Apr 6;32(13):3506-10.

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Structure-immunogenicity relationship of melittin and its N-terminal truncated analogs.

King TP, Coscia MR, Kochoumian L.

Rockefeller University, New York, New York 10021.

Melittin is an amphipathic 26-residue peptide from bee venom. We showed previously that, in the murine system, melittin has one major B-cell epitope in the hydrophilic region of residues 21-26 and one T-cell epitope in the hydrophobic midregion of 11-19. In this paper we compared the immunogenicity and the biophysical properties of a series of melittin analogs which differ by stepwise two-residue truncation in the N-terminus of residues 2-10. All analogs retain the B- and T-cell epitopes of melittin. However, the analogs which have more than two residues deleted at the N-terminus are nonimmunogenic for antibody responses although they are immunogenic for T-cell responses. The analogs were found to differ in their hemolytic activity, helical content, and oligomer formation in different solvents. These results support the hypothesis that the immunogenicity of melittin for antibody response is associated with its binding to cell membranes followed with oligomer formation but its immunogenicity for T-cell response is not.

PMID: 7681691 [PubMed - indexed for MEDLINE]

95: Immunology. 1993 Apr;78(4):513-9.

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Elimination of IgE regulatory rat CD8+ T cells in vivo differentially modulates interleukin-4 and interferon-gamma but not interleukin-2 production by splenic T cells.

Diaz-Sanchez D, Noble A, Staynov DZ, Lee TH, Kemeny DM.

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

Intraperitoneal immunization of Hooded Lister rats with a soluble antigen such as bee venom phospholipase A2 (PLA2), or ovalbumin (OVA) together with the toxic lectin, ricin, eliminates a population of early-activated CD8+ T cells which regulate IgE production. These early-activated CD8+ T cells are eliminated because they bear increased ricin-binding glycoproteins on their surface. This immunization regimen produces a vigorous and long-lived IgE response. The effect of this treatment on the capacity of splenic T cells to secrete interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and to generate IL-4 RNA message was assessed. IFN-gamma production by phytohaemagglutinin (PHA)- or ionomycin and phorbol myristate acetate (PMA)-stimulated splenocytes or purified splenic T cells from animals immunized with antigen and ricin was substantially reduced as compared with animals which were given saline or antigen alone (P < 0.001 Student's t-test). At the height of the primary IgE response IFN-gamma production by PHA-stimulated splenocytes was positively correlated with the number of CD8+ T cells (r = 0.90, P < 0.001) and inversely related to the level of serum IgE (r = -0.77, P < 0.020); serum IgE was inversely related to the number of CD8+ T cells (r = -0.92, P < 0.001). The reduced capacity of spleen cells from ricin and antigen immunized rats to produce IFN-gamma was first seen 7 days after immunization. The fall in the ability of splenocytes to secrete IFN-gamma closely paralleled the rise in serum IgE. IL-2 was assayed using an IL-2-dependent cell line which responded to rat IL-2 but not IL-4. Production of IL-2 by splenocytes taken from rats immunized with ricin+antigen was not significantly different to that produced by comparable cells obtained from animals immunized with antigen alone or saline. However, the levels of IL-4 mRNA, detected in ionomycin and PMA-stimulated splenocytes using a quantitative polymerase chain reaction (PCR) procedure, were three- to fourfold higher in ricin and antigen immunized animals as compared with control animals. Following boosting with antigen and ricin the levels of IL-4 message detected increased a further three- to fourfold. These data show that the potentiated IgE response produced by immunization with antigen+ricin is associated with a decreased ability of splenocytes to produce IFN-gamma and an increased capacity to make IL-4.

PMID: 8495968 [PubMed - indexed for MEDLINE]

96: Immunology. 1993 Feb;78(2):226-36.

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Ricin enhances IgE responses by inhibiting a subpopulation of early-activated IgE regulatory CD8+ T cells.

Diaz-Sanchez D, Lee TH, Kemeny DM.

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

Ricin, a toxic lectin from castor beans greatly enhances IgE responses to bee venom phospholipase A2 (PLA2) in high and low IgE responder strains of rat. The increase in IgE is accompanied by a 60% reduction in the number of CD8+ but not CD4+ T cells in the spleen. Optimal enhancement of IgE by ricin occurs when it is given at the same time as the antigen or 24 hr later, suggesting that it acts on cells which were activated as a consequence of immunization. Radio ligand-binding studies with 125I ricin were used to compare the number of ricin binding sites on CD4+ and CD8+ T cells. No difference was seen in either the affinity or the number of receptors for ricin on the CD4+ and CD8+ T cells of unimmunized rats. In contrast, CD8+ T cells taken from rats which had been immunized with 10 micrograms of PLA2 24 hr earlier demonstrated considerably more ricin receptors (3.9 x 10(7) +/- 2.2 x 10(6) binding sites/cell) than CD4+ T cells (3.19 x 10(6) +/- 1.08 x 10(6) binding sites/cell). However the affinity of the receptors for ricin was unchanged. Cytofluorographic analysis with fluorescein isothiocyanate (FITC)-labelled ricin confirmed these observations and indicated that increased ricin binding occurred on a subpopulation of CD8+ T cells. The effect of CD8+ T cells on IgE regulation was investigated by adoptive transfer. 1 x 10(8) highly purified (> 98%) splenic CD8+ T cells collected from Brown Norway rats 3 days after immunization with 10 micrograms of PLA2 were adoptively transferred to naive, syngeneic recipients. The IgE antibody response to PLA2 + A1(OH)3 seen in these animals was reduced by 91%. Adoptive transfer of CD4+ T cells from the same donor animals did not induce suppression and nor did adoptive transfer of CD8+ T cells from animals given both ricin and PLA2. However, when recipients of CD8+ T cells taken from rats immunized with PLA2 were immunized with a different antigen [ovalbumin (OVA)] and A1(OH)3 the IgE antibody response was also suppressed, although to a lesser extent (66%). These results show that co-administration of ricin and PLA2 depletes a subpopulation of ricin-sensitive, early activated CD8+ T cells and that these CD8+ T cells are potent suppressors of the primary IgE response.

PMID: 8097181 [PubMed - indexed for MEDLINE]

97: Pneumonol Alergol Pol. 1993;61(7-8):346-51.

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[Evaluation of diagnostic value of skin test, venom specific antibodies against EgE and basophil histamine release test in hymenoptera allergy]

[Article in Polish]

Nittner-Marszalska M, Malolepszy J, Medrala W.

Katedry i Kliniki Chorob Wewnetrzynch i Alergologii AM, Wroclawiu.

The study compares results of skin test and assessments of venom specific IgE levels (FAST) in patients with Hymenoptera sting allergic reactions. Results of the two diagnostic methods show considerable correlation. Positive correlation occurred in 90% of the patients (92.4% bee venom sensitive allergic patients and 80% wasp venom sensitive patients). Negative correlation occurred in 2% of the patients. In the remaining 8% of the group the results did not correlate. Basophil histamine release test was performed in 13 patients with allergic reactions after stinging. 77% of the basophil histamine release test results showed positive correlation with the other two tests. No correlation was found between the size skin test, the class of the FAST test, and the result of basophil histamine release test. No relationship was found either, between results of the described test and the severity of sting reaction classified to Mueller scale.

PMID: 7691338 [PubMed - indexed for MEDLINE]

98: 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]

99: J Pharmacol Exp Ther. 1992 Aug;262(2):866-73.

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Fuscoside: an anti-inflammatory marine natural product which selectively inhibits 5-lipoxygenase. Part I: Physiological and biochemical studies in murine inflammatory models.

Jacobson PB, Jacobs RS.

Department of Biological Sciences, University of California, Santa Barbara.

The biological and biochemical pharmacology of fuscoside, a novel anti-inflammatory marine natural product isolated from the Caribbean gorgonian Eunicea fusca, has recently been characterized using murine (part I) and human (part II) models of inflammation. Topically applied fuscoside (FSD) effectively inhibits phorbol myristate acetate (PMA)-induced edema in mouse ears at levels comparable with indomethacin over a 3.3-hr exposure period, and is significantly more efficacious than indomethacin over 24 hr in the PMA model. Histological preparations and quantification of the neutrophil-specific marker, myeloperoxidase, demonstrate that FSD inhibits neutrophil infiltration into PMA-induced regions of edema and inflammation. In systemic studies, where FSD is injected i.p. before the topical application of PMA, negligible effects on ear inflammation are observed. FSD does not inhibit bee venom or human synovial fluid phospholipase A2 up to concentrations of 500 microM. In calcium ionophore-activated cultures of mouse peritoneal macrophages, FSD selectively and irreversibly inhibits leukotriene C4 biosynthesis (IC50 = 8 microM), yet has negligible effects on prostaglandin E2 production. FSD is also without effect on the conversion of arachidonic acid to prostaglandin E2 by ram seminal vesicle cyclooxygenase. Chromatographic and spectroscopic studies suggest that FSD is not metabolized, and that drug uptake/binding by macrophages is time dependent, saturable and independent of active transport mechanisms. These studies represent the first report of an anti-inflammatory marine natural product that selectively inhibits leukotriene biosynthesis.

PMID: 1501127 [PubMed - indexed for MEDLINE]

100: 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]

101: J Allergy Clin Immunol. 1992 Jul;90(1):42-51.

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Mapping human T cell epitopes on phospholipase A2: the major bee-venom allergen.

Dhillon M, Roberts C, Nunn T, Kuo M.

Department of Medicine, Queen's University, Kingston, Ontario, Canada.

Phospholipase A2 (PLA2), the major bee-venom allergen, was purified by gel filtration, inactivated by denaturing, and carboxymethylating its cysteine residues. Peripheral blood mononuclear cells from an individual (HLA-DR2 [15], Dw52, DQ1 and DQ3) allergic to bee stings were used to generate cell lines specific for PLA2 and a control antigen, tetanus toxoid. These lines were 90% CD3+, 64% CD4+ and 20% CD8+ by fluorocytometry analysis. T-lymphocyte epitope mapping done with 12 overlapping synthetic peptides of PLA2 revealed two immunodominant epitopes. These epitopes correspond to amino acid sequences 50 to 69 and 83 to 97 of PLA2. Cytokine interleukin-4 and Interferon-gamma secretion was studied from PLA2- and tetanus toxoid-specific cell lines. Interleukin-4 secretion was common to both cell lines but only tetanus-toxoid cell lines secreted interferon-gamma. No interferon-gamma was found to be secreted by PLA2-specific cell line in response to stimulation by PLA2 or the two immunodominant peptides.

PMID: 1378459 [PubMed - indexed for MEDLINE]

102: 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]

103: J Protein Chem. 1992 Jun;11(3):275-80.

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Mast cell degranulating (MCD) peptide analogs with reduced ring structure.

Buku A, Reibman J, Pistelli A, Blandina P, Gazis D.

Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029-6574.

Mast cell degranulating (MCD) peptide, a component of bee venom, is a 22 amino acid peptide with two disulfide bridges. In this first structure-activity study of MCD peptide, three analogs were synthesized and tested: two analogs shortened by omitting sequences 6-10 and 8-13, respectively, and one analog lacking the disulfide bridge between cysteine residues 5 and 19. These analogs were synthesized by solid-phase methods and were compared to MCD peptide in two assays for inflammation: histamine release from mast cells and superoxide anion release from neutrophils. All three analogs produced histamine release, although with only about one fifth of the activity of MCD peptide. Superoxide anion-releasing activity, however, did not parallel histamine release. MCD peptide did not release superoxide anion, while the 6-10 and 8-13 deletion analogs were strong and weak stimulants, respectively, of this anion. CD spectra showed that the secondary structures of the three analogs were very similar to that of MCD peptide, so that a change in secondary structure cannot completely explain the changes in releasing activities. Charge differences between the two deletion analogs and MCD peptide may explain some of the differences in activity. This is the first demonstration that the various activities of MCD peptide can be separated, and provides a lead through which the purported antiinflammatory activity of MCD peptide may possibly be explored in the future.

PMID: 1382440 [PubMed - indexed for MEDLINE]

104: Neurosci Res. 1992 Apr;13(3):207-16.

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K+ channel involvement in induction of synaptic enhancement by mast cell degranulating (MCD) peptide.

Kondo T, Ikenaka K, Fujimoto I, Aimoto S, Kato H, Ito K, Taguchi T, Morita T, Kasai M, Mikoshiba K.

Institute for Protein Research, Osaka University, Japan.

A bee venom, mast cell degranulating peptide (MCD), which induces long-term potentiation (LTP) of synaptic transmission in hippocampal slices, was found to possess multiple functions. They include (1) binding and thereby inhibiting a voltage-dependent K(+)-channel in brain membranes, (2) incorporation in a lipid bilayer to form voltage-dependent and cation-selective channels by itself, and (3) activation of a pertussis toxin (Ptx)-sensitive GTP-binding proteins. In this study, we prepared several derivatives and analogues of MCD and investigated which function is more closely related to the induction of LTP. Another bee venom, apamin, formed ion channels in a lipid bilayer which were indistinguishable from those formed by MCD. D-MCD, an optical isomer of MCD, activated a Ptx-sensitive GTP-binding protein. However, these peptides did not induce LTP in the hippocampal slices. A snake venom, dendrotoxin-I (DTX-I), bound to the same K(+)-channels as MCD and did induce LTP. These results suggest that the most potent aspect of MCD involved in LTP inducibility is its interaction with the voltage-dependent K(+)-channel.

PMID: 1376885 [PubMed - indexed for MEDLINE]

105: J Immunol. 1992 Feb 1;148(3):729-37.

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Glycosylation-inhibiting factor from human T cell hybridomas constructed from peripheral blood lymphocytes of a bee venom-sensitive allergic patient.

Thomas P, Gomi H, Takeuchi T, Carini C, Tagaya Y, Ishizaka K.

La Jolla Institute for Allergy and Immunology, CA 92037.

Human T cell hybridomas, which constitutively secrete glycosylation inhibiting factor (GIF), were constructed from PBL of an allergic individual who was sensitive to honey bee venom. PBMC of the patient were stimulated with either denatured or cyanogen bromide-treated bee venom phospholipase A2 (PLA2), and Ag-activated cells were propagated by IL-2 in the presence of human recombinant lipocortin I. T cells obtained in the cultures were fused with a HAT-sensitive mutant of the human lymphoblastoid cell line CEM. Approximately one-third of hybridoma clones constitutively secreted GIF. The GIF-producing hybridomas were CD3+ and bore TCR-alpha beta. GIF formed by unstimulated hybridomas lacked affinity for bee venom PLA2. Upon cross-linking of CD3, however, a majority of the GIF-producing hybridomas formed IgE-binding factors and GIF, the latter of which had affinity for bee venom PLA2. Both nonspecific GIF and Ag-binding GIF from the hybridomas bound to an immunosorbent coupled with the anti-lipomodulin mAb 141-B9. Using an affinity-purified GIF as an immunogen, we established mouse B cell hybridomas that secreted monoclonal anti-human GIF. In order to characterize human nonspecific GIF, one of the GIF-producing hybridomas was adapted to a serum-free medium, and culture supernatant was fractionated by DEAE-Sepharose column chromatography and by gel filtration. The majority of nonspecific GIF in the culture supernatant was recovered from DEAE-Sepharose by elution of the column with 10 mM Tris-HCl buffer, pH 8.0, containing 50 mM NaCl. Affinity-purification of GIF in the DEAE Sepharose fraction by using anti-GIF-coupled Affigel, and analysis of the purified GIF by SDS-PAGE revealed that human GIF is a single polypeptide chain of 14 to 15 kDa. Gel filtration of both crude and affinity-purified GIF preparations confirmed the molecular size of the cytokine.

PMID: 1730869 [PubMed - indexed for MEDLINE]

106: Rom J Intern Med. 1992 Jan-Mar;30(1):63-7.

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The in vitro action of a succussed substance on the proliferative response of human lymphocytes stimulated with phytohemagglutinin.

Chirila M, Hristescu S, Manda G, Neagu M, Olinescu A.

N. Gh. Lupu Institute of Internal Medicine, Bucharest, Romania.

Human peripheral blood lymphocytes from healthy controls, immunodepressed patients presenting chronic bacterial infections or neoplasias and from allergic patients were stimulated in vitro with phytohemagglutinin (PHA) in culture medium supplemented or not with 1 x 10(-7), 1 x 10(-15) or 1 x 10(-30) succussed dilutions or bee venom or phosphorus in tridistilled water. The most significant inhibition due to DNA incorporation was noted in lymphocytes from allergic patients cultivated in media supplemented with 1 x 10(-30) succussed substance dilution in the presence of PHA. The cells from immunodepressed patients did not show a significant inhibition at 1 x 10(-30) dilution. Hypothetically, we try to explain these findings as the expression of the changes induced by the succussed solution on the water molecule which in turn, influences the chemical structure of the cellular membrane and implicitly, its functions.

PMID: 1496261 [PubMed - indexed for MEDLINE]

107: Arch Immunol Ther Exp (Warsz). 1992;40(2):107-11.

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Specific allergen induced motility of peripheral blood mononuclear cells and polymorphonuclear leukocytes in insect sting allergy.

Zak-Nejmark T, Malolepszy J, Jutel M, Nittner-Marszalska M, Nadobna G.

Department of Internal Medicine and Allergology, Medical Academy, Wroclaw, Poland.

The motility of peripheral blood mononuclear cells (MNC) and polymorphonuclear (PMN) leukocytes from normal and bee venom allergic subjects was investigated by a modified Boyden micropore filter method. The study comprised MNC locomotion in bee venom and histamine gradients and PMN locomotion in bee venom and fMLP gradients. We demonstrated statistically significant increase in MNC and PMN motility towards bee venom in allergic patients group. This effect disappeared after the preincubation of MNC with anti-human IgE antibodies. We observed no such effect in PMN leukocytes. Increased MNC motility in histamine gradient was observed only in control subjects group. Similarly significant increase in PMN locomotion towards fMLP was found in both allergic and control subjects. The results here demonstrated suggest that a specific allergen might be a chemoattractant for peripheral blood MNC and PMN leukocytes from atopics and could be capable to induce non-infectious inflammatory reactions as a result of its interaction with these sensitive cells.

PMID: 1299166 [PubMed - indexed for MEDLINE]

108: Agents Actions. 1991 Sep;34(1-2):70-2.

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AGN 190383, a novel phospholipase inhibitor with topical anti-inflammatory activity.

De Vries GW, Lee G, Amdahl L, Wenzel M, Garst M, Wheeler LA.

Department of Biological Sciences, Allergan, Inc., Irvine, CA 92715.

AGN 190383 is a 5-hydroxy-2(5H)-furanone ring analog of the marine natural product manoalide. When applied topically, AGN 190383 inhibits phorbol ester induced mouse ear edema. It is a potent inhibitor of bee venom phospholipase A2 and blocks the release of arachidonic acid from calcium ionophore A23187 stimulated human neutrophils. AGN 190383 also inhibits both hormone-operated and depolarization-dependent calcium mobilization in GH3 cells, as well as fMLP stimulated increases in free cytosolic calcium in human PMNs. Furthermore, it is also able to block the release of the neutral protease elastase from stimulated neutrophils. The effects of AGN 190383 on arachidonic acid metabolism and leukocyte function may account, in part, for its anti-inflammatory activity in vivo.

PMID: 1793055 [PubMed - indexed for MEDLINE]

109: FEBS Lett. 1991 Aug 5;287(1-2):15-8.

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Mast cell degranulating (MCD) peptide and its optical isomer activate GTP binding protein in rat mast cells.

Fujimoto I, Ikenaka K, Kondo T, Aimoto S, Kuno M, Mikoshiba K.

Institute for Protein Research, Osaka University, Yamadaoka Suita, Japan.

The MCD peptide in bee venom induces degranulation in mast cells. The internal calcium concentration of mast cells increased and remained high following MCD stimulation. This calcium increase was blocked by pertussis toxin (Ptx) treatment, suggesting that MCD peptide activates Ptx-sensitive G-protein. Even in the absence of external calcium in the incubation medium, the calcium concentration increased by MCD treatment, but soon returned to the original level. D-MCD, the optical isomer of the MCD peptide, also increased the internal calcium concentration through a Ptx-sensitive pathway. We suggest that cationic clusters at one side of the surface are more important in activating the G-protein than the alpha-helix conformation.

PMID: 1908786 [PubMed - indexed for MEDLINE]

110: J Immunol. 1991 Apr 15;146(8):2664-70.

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Murine IgE and IgG responses to melittin and its analogs.

Fehlner PF, Kochoumian L, King TP.

Rockefeller University, New York, NY 10021.

Melittin, a bee-venom peptide of 26 amino acids, induces IgE and IgG responses in man and animals. The antibody response was shown previously to be specific primarily for the C-terminal 6 residues and its T cell epitope in H-2d restricted mice was shown to be in residue 11-19 of melittin. To study the relationship of peptide structure and immunogenicity in mice, we have prepared a series of melittin analogs varied in length and composition at the C-terminus. Immunogenicity of the analogs for IgG and IgE responses was found to correlate with two factors: a peptide length of more than 24 residues and the presence of a hydrophilic C-terminal region preferably with two to four cationic groups. These factors result in the ability of peptide to bind to cell membranes. Analogs that possess these features are good immunogens whereas those lacking any of these features are weak immunogens.

PMID: 1707915 [PubMed - indexed for MEDLINE]

111: 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]

112: Immunology. 1991 Feb;72(2):297-303.

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Generation of a long-lived IgE response in high and low responder strains of rat by co-administration of ricin and antigen.

Diaz-Sanchez D, Kemeny DM.

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

Certain strains of rats infested with the nematode parasite Nippostrongulus brasiliensis mount vigorous, persistent immunoglobulin E (IgE) responses. In the absence of parasites, adjuvants such as Bordatella pertussis or Al(OH)3 are needed to produce IgE responses to soluble antigens. These are short-lived, even in high IgE responder strains. In this study we have produced long-lived IgE responses in both low (Wistar) and high (Brown Norway) IgE responder strains of rats by repeated injections of ricin, a toxic lectin from castor beans, and phospholipase A2 (PLA2), a bee venom protein. Total IgE levels rose from 30 +/- 20 ng/ml to 39,000 +/- 7500 ng/ml in the Wistar rats compared with an increase from 120 +/- 100 ng/ml to 47,000 +/- 8000 ng/ml in the Brown Norway rats. An even greater (10(4)-fold) increase was seen in PLA2-specific IgE antibody levels. total and PLA2-specific IgE started to fall 6 weeks after treatment was stopped in the Wistar and after 12 weeks in the Brown Norway rats. The duration of the response was 204 and 248 days, respectively. The IgE-enhancing properties of ricin were compared in low, mid (Hooded Lister) and high IgE responder rats. Total IgE and PLA2-specific IgE but not IgG antibody (Ab) responses were enhanced in all animals given ricin and PLA2 but not in animals given ricin or PLA2 alone. The increase was greater in Wistar rats (48-fold) than in Brown Norway rats (eightfold) and by Day 24 the levels of both total and PLA2-specific IgE in three different strains were indistinguishable. PLA2-specific IgE antibody-secreting cells were detected in the spleen at a frequency of 1:5000. These results show: (i) that repeated immunization of rats with antigen and ricin produce a very large IgE response which was long-lived; (ii) that this response was indistinguishable in different IgE responder strains of rat; and (iii) that the IgE response declines earlier in low IgE responder strains of rats.

PMID: 2016124 [PubMed - indexed for MEDLINE]

113: J Immunol. 1991 Feb 1;146(3):799-806.

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Murine T cell responses to melittin and its analogs.

Fehlner PF, Berg RH, Tam JP, King TP.

Rockefeller University, New York, NY 10021.

The 26-residue peptide melittin from bee venom elicits high IgG1 and IgE responses in selected strains of mice. The antibody responses were shown previously to be specific mainly for the region of residue 20-26. The T cell epitope of melittin in H-2d-restricted mice is now found to be primarily in residue 11-19, corresponding to an alpha-helical amphiphilic segment of the molecule. Melittin-specific T cell lines have varying responses to different structural analogs of the melittin T cell epitope, and the results indicate that the antigenicity of T cell epitope peptides depend more on their primary structure than on their secondary structure. Melittin-specific T cell clones are found to be CD4+ and secrete IL-4, and are restricted to presentation on I-Ad or I-Ed. The I-Ad- or I-Ed-restricted clones differ in their responses to different analogs of melittin.

PMID: 1703178 [PubMed - indexed for MEDLINE]

114: Int Arch Allergy Appl Immunol. 1991;95(2-3):191-4.

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Appearance of macrophage migration inhibition factor in patients with systemic reactions to bee venom.

Goldberg A, Livni E, Mekori YA.

Unit of Allergy and Clinical Immunology, Meir General Hospital, Kfar Saba, Israel.

Cellular responses in bee venom (BV) allergy is a controversial issue. Previous studies could not reach an agreement whether this mechanism is activated as a result of allergic sensitization to bee venom. All previous works have used lymphocyte proliferation as their method to analyze cell-mediated immunity. In the present work, we tried to explore whether the production of macrophage migration inhibition factor (MIF), which is another in vitro correlate with cellular responses, is increased in these patients. We also examined which of the major antigenic components of BV played a significant role in the cellular response. Peripheral blood lymphocytes from 10 patients with systemic allergic reactions to bee sting and 9 healthy volunteers were examined for their ability to induce positive MIF responses. Macrophage inhibition was significantly increased in allergic patients when tested with BV, phospholipase A2 (PLA2) and with melittin. Positive MIF responses to other components were also more common in allergic patients than in the control group. Our results indicate that cellular response to BV is expressed in patients with systemic allergic reaction to BV. When major antigenic components of BV are examined, PLA2 seems to play the major role in inducing this response.

PMID: 1937921 [PubMed - indexed for MEDLINE]

115: Biochim Biophys Acta. 1990 Aug 24;1027(2):185-90.

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Interaction of wasp venom mastoparan with biomembranes.

Katsu T, Kuroko M, Morikawa T, Sanchika K, Yamanaka H, Shinoda S, Fujita Y.

Faculty of Pharmaceutical Sciences, Okayama University, Japan.

Mastoparan-induced changes in the K+ permeability of rat peritoneal mast cells, human erythrocytes, Staphylococcus aureus and Escherichia coli were examined. Mastoparan did not efficiently increase the K+ permeability of cells except for S. aureus. The release of membrane phospholipids was also observed from S. aureus cells in the concentration range of the permeability enhancement. Mastoparan stimulated histamine release from mast cells, independently of a small efflux of K+. Mastoparan became markedly effective to E. coli cells whose outer membrane structure was chemically disrupted beforehand, showing that the peptide can enhance the permeability of the cytoplasmic membranes of both Gram-positive and -negative bacteria. In experiments using liposomes, mastoparan increased the permeability of the liposomes composed of egg phosphatidylethanolamine and egg phosphatidylglycerol, which are the lipid constituents of the cytoplasmic membrane of E. coli cells, while it showed a weak activity to the liposomes composed of egg phosphatidylcholine and cholesterol. The latter result related closely to the fact that this peptide acted weakly on erythrocytes and mast cells in which acidic lipids constitute a minor portion. Mastoparan decreased the phase transition temperature of dipalmitoylphosphatidylglycerol liposomes, but it did not affect that of dipalmitoylphosphatidylcholine liposomes. These results indicate that mastoparan penetrated into membranes mainly containing acidic phospholipids and disrupted the membrane structure to increase the permeability. The action of the wasp venom mastoparan was compared with that of a bee venom melittin.

PMID: 2204429 [PubMed - indexed for MEDLINE]

116: Arzneimittelforschung. 1990 Aug;40(8):912-3.

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Antioxidant activity of and interleukin production affected by honey bee venom.

Rekka E, Kourounakis L, Kourounakis P.

Department of Pharmaceutical Chemistry, School of Pharmacy, University of Thessaloniki, Greece.

Honey bee venom is found to inhibit significantly nonenzymatic lipid peroxidation. It also possesses a considerable hydroxyl radical scavenging activity, evaluated by its competition with dimethyl sulfoxide for HO.. These results, in relation to the in vitro suppression mainly of interleukin-1 production offered by honey bee venom, may further support that antioxidant activity is involved in the anti-inflammatory activity of honey bee venom.

PMID: 2242083 [PubMed - indexed for MEDLINE]

117: Infect Immun. 1990 Aug;58(8):2678-82.

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Effect of staphylococcal delta-toxin and bee venom peptide melittin on leukotriene induction and metabolism of human polymorphonuclear granulocytes.

Raulf M, Alouf JE, Konig W.

Lehrstuhl fur Medizinische Mikrobiologie und Immunologie, Ruhr-Universitat Bochum, Federal Republic of Germany.

The abilities of delta-toxin from Staphylococcus aureus and melittin to induce and modulate the generation of leukotriene from human polymorphonuclear granulocytes (PMNs) were studied. Stimulation of PMNs with melittin (10 micrograms) induced leukotriene formation, whereas stimulation with delta-toxin did not. Preincubation of the PMNs with delta-toxin modulated the subsequent generation of leukotriene from PMNs induced by Ca ionophore A23187 or opsonized zymosan. The generation of leukotriene B4 (LTB4), induced by the Ca ionophore A23187, was increased when the PMNs were preincubated with delta-toxin for 5 min. When opsonized zymosan was used as a secondary stimulus to activate the delta-toxin-pretreated PMNs, LTB4 generation decreased. In contrast, melittin showed no significant modulatory effect on the generation of leukotriene from PMNs. In addition, preincubation of PMNs with delta-toxin inhibited the conversion of LTB4 to omega-oxidation products. Our data suggest that peptides with similar structures, e.g., delta-toxin and melittin, induce and modify leukotriene generation in different manners.

PMID: 2164512 [PubMed - indexed for MEDLINE]

118: Infect Immun. 1990 Jun;58(6):1653-9.

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Effect of Staphylococcus aureus delta-toxin on human granulocyte functions and platelet-activating-factor metabolism.

Kasimir S, Schonfeld W, Alouf JE, Konig W.

Medizinische Mikrobiologie und Immunologie, AG Infektabwehrmechanismen, Ruhr Universitat, Bochum, Federal Republic of Germany.

The production of delta-toxin is supposed to be responsible for various pathophysiological effects during infection with Staphylococcus aureus. We compared the effects of delta-toxin with the structurally related bee venom toxin melittin on granulocyte functions and inflammatory mediator release. Delta-toxin and melittin induced a rapid Ca2+ influx, as was shown by fluorescence detection. Furthermore, oxygen radical production, as determined by luminol-enhanced chemiluminescence, was triggered by delta-toxin (0.15 to 15 micrograms/ml), whereas melittin showed only marginal effects. Release of lysozyme and beta-glucuronidase was observed only at high concentrations of 15 micrograms of melittin and delta-toxin per ml. Preincubation (15 min) of neutrophils with both toxins resulted in the formation of 3H-platelet-activating factor (3H-PAF) from 3H-lyso-PAF. After 5 min of incubation, the exogenously added lyso-PAF was converted to PAF (delta-toxin, 80 +/- 2%; melittin, 27 +/- 12% of total radioactivity; n = 3, mean +/- standard error of the mean) and 1-O-alkyl-2-acyl-glycerophosphorylcholine (alkyl-acyl-GPC) (corresponding values, 20 +/- 3% and 51 +/- 14% of total radioactivity). The newly generated PAF was rapidly metabolized to lyso-PAF and alkyl-acyl-GPC during the subsequent incubation period of 60 min. In the absence of any toxin, no formation of PAF from lyso-PAF was observed. Further studies indicated that the metabolism of PAF into lyso-PAF and alkyl-acyl-GPC was inhibited in the presence of delta-toxin. Melittin had no significant effects on PAF metabolism. Neither delta-toxin nor melittin modulated the uptake of PAF and lyso-PAF significantly. Our data provide evidence that delta-toxin has an effect on the activity of neutrophil granulocytes with regard to its proinflammatory capacity.

PMID: 2341170 [PubMed - indexed for MEDLINE]

119: J Immunol Methods. 1990 Mar 9;127(2):221-33.

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A method to generate antigen-specific suppressor T cells in vitro from peripheral blood T cells of honey bee venom-sensitive, allergic patients.

Carini C, Iwata M, Warner J, Ishizaka K.

Department of Medicine, Johns Hopkins University School of Medicine, Good Samaritan Hospital, Baltimore, MD 21239.

Peripheral blood mononuclear cells of patients allergic to honey bee venom were stimulated with denatured bee venom phospholipase A2, and the antigen-activated T cells were propagated for 4 days by human IL-2 in the presence or absence of recombinant human lipocortin I. Upon antigenic stimulation with the denatured phospholipase A2 and autologous monocytes or by cross-linking of CD3 by anti-CD3 antibody, the activated T cells, which had propagated by IL-2 alone, formed N-glycosylated IgE-binding factors and glycosylation enhancing factor (GEF), while those propagated in the presence of lipocortin formed unglycosylated IgE-binding factors and glycosylation inhibiting factor (GIF). The GEF and GIF formed by the antigen- or anti-CD3-stimulated T cells had affinity for bee venom phospholipase A2 and could be purified by using anti-lipomodulin Sepharose. In the mouse lymphocyte system, the major cell source of GIF is antigen-specific suppressor T cells, and the antigen-binding GIF from the cells suppressed the in vivo antibody response in an antigen (carrier)-specific manner. In view of the findings in the mouse system, the present results may provide an immunological maneuver to generate allergen-specific suppressor T cells, and to obtain allergen-specific suppressor factor from T cell populations in the peripheral blood of allergic patients.

PMID: 2138201 [PubMed - indexed for MEDLINE]

120: Br J Pharmacol. 1990 Feb;99(2):350-4.

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Anti-inflammatory activity of bee venom peptide 401 (mast cell degranulating peptide) and compound 48/80 results from mast cell degranulation in vivo.

Banks BE, Dempsey CE, Vernon CA, Warner JA, Yamey J.

Department of Physiology, University College London.

1. The relationship between the anti-inflammatory activity of the bee venom peptide 401 in the carrageenin-induced oedema of the rat hind paw and its mast cell degranulating activity has been reinvestigated. 2. Mast cell degranulation caused by compound 48/80 (10 mg kg-1) or by allergen challenge in rats sensitized to Nippostrongylus brasiliensis also suppressed rat hind paw oedema in the same test. 3. The anti-inflammatory activities of peptide 401 and compound 48/80 were partially suppressed by pretreatment of rats with mepyramine and methysergide, at doses (2.5 mg kg-1) that completely suppressed skin reactions to these mast cell-derived amines. Pretreatment of rats with compound 48/80 also suppressed the apparent anti-inflammatory actions of peptide 401 and of compound 48/80. 4. Injection of peptide 401 together with carrageenin increased the inflammatory response in the rat hind paw. 5. The anti-inflammatory activity of peptide 401 and of compound 48/80 in the carrageenin-induced swelling of the rat hind paw arises from mast cell degranulation in vivo.

PMID: 2328399 [PubMed - indexed for MEDLINE]

121: J Allergy Clin Immunol. 1990 Feb;85(2):505-9.

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Evidence for leukotrienes in animal venoms.

Czarnetzki BM, Thiele T, Rosenbach T.

Department of Dermatology, University Clinics, Munster, West Germany.

To elucidate further the pathomechanisms of cutaneous whealing in response to insect or sea-animal stings, commercial sources of bee and wasp venoms and of sea nettle nematocyst extracts, as well as crude bee and wasp venoms, were examined for the presence of histamine, leukotrienes (LT) C4, LTB4 (radioimmunoassay and reverse-phase high-pressure liquid chromatography), and neutrophil chemotactic activity (NCA). Histamine was present in all samples with the highest levels in the crude insect venoms. The same held for LTC4 with highest quantities in a liquid commercial bee venom extract and in the crude venoms. Relatively large amounts of LTB4 were recovered from the sea nettles with a correspondingly high NCA. Although small levels of LTB4 were found in the other samples, there was no clear correlation to the NCA on a quantitative basis. The demonstration of these mediators offers an explanation for the occurrence of local immediate and persistent wheals at sites of bee, wasp, and sea nettle stings in nonsensitized individuals.

PMID: 1968071 [PubMed - indexed for MEDLINE]

122: Zentralbl Bakteriol. 1989 Oct;271(4):521-31.

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Inhibition of in vitro and in vivo mast cell degranulation by Taenia crassiceps metacestodes in vitro incubation products.

Seifert B, Geyer E.

Fachbereich Biologie, Philipps-Universitat, Marburg.

In vitro released products of T. crassiceps metacestodes (TcIP) harvested from the peritoneal cavity of NMRI mice were tested for inhibitory effects on the in vitro degranulation of peritoneal mast cells (MCs) of normal mice (NMRI) and rats (Wistar) and on the in vivo degranulation of rat (Wistar) skin MCs (PCA-assay). In vitro degranulation was elicited chemically (compound 48/80, polymyxin B or the bee venom peptide, mellitin). In vivo degranulation was triggered immunologically (anaphylactic systems ovalbumin/anti-ovalbumin or Fasciola hepatica crude fluke extract antigen/serum of fluke-infected rats (Wistar]. In vitro degranulation of murine peritoneal MCs or the in vitro histamine release of rat peritoneal MCs normally induced chemically was significantly inhibited when the MCs were preincubated with the TcIP or with serum of T. crassiceps-infected NMRI mice from day 35 post infection and thereafter. In vitro degranulation of peritoneal MCs of infected mice was strongly inhibited beginning on day 10 after infection. Also in vivo degranulation of the IgE-sensitized rat skin MCs was significantly reduced by intradermal injection of the TcIP before (6, 3 and 1 h) antigen challenge and by preinjection (1 h) of serum from infected mice (day 80 p.i.)-The inhibitory effect was also demonstrated after immunoadsorption of mouse serum proteins naturally contaminating the TcIP. Heating (100 degrees C/15 min), even in the presence of 0.25 M HCl, did not suppress the inhibitory activity.

PMID: 2479392 [PubMed - indexed for MEDLINE]

123: Allergy. 1989 Sep;44(7):453-9.

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Relevance of basophil histamine release changes during venom immunotherapy.

Stephan V, Kuhr J, Urbanek R.

University Children's Hospital, Freiburg, W. Germany.

We investigated the effect of rush and long-term venom immunotherapy on histamine release parameters in bee venom allergic patients. Ten patients received rush venom immunotherapy, and histamine release data were obtained immediately before and after treatment. 17 patients were assessed by histamine release 24 to 63 months after termination of long-term venom immunotherapy. A control group of 10 non-allergic subjects was included in this study. Histamine released from whole blood was determined in a sensitive radio-enzymatic assay using a single isotope technique. Bee venom phospholipase A-induced histamine release from whole blood proved to be a test procedure of high specificity and sensitivity. Eight of 10 untreated patients and no control subject showed significant antigen-induced histamine release. Results obtained from patients immediately after successful rush venom immunotherapy showed an important decrease (mean 45.9%) of total histamine content of basophil leukocytes in all patients. Antigen-induced maximum histamine release was found to be increased in one, decreased in two and unchanged in seven patients. In patients who received long-term immunotherapy cell sensitivity to phospholipase A was significantly lower than in a group of untreated patients (P less than or equal to 0.002). These results suggest that even years after discontinuation of immunotherapy, histamine release parameters reflect patients' protection from systemic sting reactions as assessed by sting challenges. Histamine depletion of basophils induced by rush immunotherapy may play an important role in patients' protection immediately after termination of the rush regimen.

PMID: 2479278 [PubMed - indexed for MEDLINE]

124: Biochem Biophys Res Commun. 1989 Aug 30;163(1):155-60.

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Mast cell degranulating peptide forms voltage gated and cation-selective channels in lipid bilayers.

Ide T, Taguchi T, Morita T, Sato M, Ikenaka K, Aimoto S, Kondo T, Hojo H, Kasai M, Mikoshiba K.

Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Japan.

Mast cell degranulating peptide, a toxin of bee venom, is a polypeptide composed of 22 amino acids. Exposure of the asolectin bilayer to the peptide results in the formation of channels that are more permeable to K+ than Cl-. These channels are activated when the voltage of the cis side, to which the peptide is added, is made positive.

PMID: 2476125 [PubMed - indexed for MEDLINE]

125: Agents Actions. 1989 Jun;27(3-4):425-7.

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Rheumatoid arthritis synovial fluid phospholipase A2 activating protein (PLAP) stimulates human neutrophil degranulation and superoxide ion production.

Bomalaski JS, Baker D, Resurreccion NV, Clark MA.

V.A. Medical Center, Medical College of Pennsylvania, University of Pennsylvania, Philadelphia 19104.

Rheumatoid arthritis is characterized by excessive eicosanoid production, and phospholipase enzymes are the rate limiting step in eicosanoid synthesis. We have shown previously that cells from patients with rheumatoid arthritis express enhanced phospholipase A2 enzyme activities. Recently, we have isolated a phospholipase A2 activating protein termed PLAP from rheumatoid synovial fluid. This novel human protein shares biochemical and antigenic similarities with melittin, a bee venom phospholipase activating protein. Because melittin has been shown to induce neutrophil degranulation and superoxide formation, and because exuberent release of lysosomal enzymes and superoxide have been implicated in the pathogenesis of rheumatoid arthritis, we examined the role of PLAP on inducing these neutrophil functions.

PMID: 2552770 [PubMed - indexed for MEDLINE]

126: 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]

127: 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]

128: Int Arch Allergy Appl Immunol. 1989;88(1-2):129-31.

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Characteristics of histamine secretion induced by neuropeptides: implications for the relevance of peptide-mast cell interactions in allergy and inflammation.

Pearce FL, Kassessinoff TA, Liu WL.

Department of Chemistry, University College London, UK.

A variety of basic, sensory neuropeptides induced the release of histamine from rat peritoneal mast cells. However, a wide range of other polycationic agents, such as compound 48/80, the mast-cell-degranulating peptide from bee venom and polylysine also activated this cell type. Histamine release induced by all of these agents had characteristic features in common. In each case, the process was extremely rapid, essentially independent of added calcium or phospholipids, not mediated through cell-fixed antibodies, and inhibited by antagonists of the so-called polyamine receptor. The release was also very species- and tissue-specific. All of the compounds were most active against rat serosal mast cells. Tissue mast cells of this species and peritoneal mast cells of other rodents showed graded responses while guinea pig and human mast cells were unreactive. On the basis of these results, the possible role of peptide-mast cell interactions in neurogenic models of inflammation is discussed.

PMID: 2468610 [PubMed - indexed for MEDLINE]

129: Hautarzt. 1988 Oct;39(10):662-70.

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[The basophilic histamine liberation test as a diagnostic method in Hymenoptera venom allergy]

[Article in German]

Przybilla B, Ring J, Wielgosch J.

Dermatologische Klinik und Poliklinik, Ludwig-Maximilians-Universitat Munchen.

In 181 patients with systemic reactions to hymenoptera stings as a result of immediate-type allergy to bee or wasp venom confirmed by history, skin testing and demonstration of hymenoptera venom-specific IgE antibodies on radioallergosorbent testing (RAST), in vitro histamine release from peripheral basophilic granulocytes following stimulation with bee or wasp venom was evaluated. Suspensions of washed peripheral leukocytes at a density of 2 x 10(6)/ml were incubated with four different concentrations of hymenoptera venoms. Histamine was measured by spectrofluorometry, and histamine release was calculated as a percentage of the total histamine content. Tests performed with peripheral leukocytes obtained from 18 non-allergic individuals served as controls. In both bee venom allergy and wasp venom allergy the corresponding allergen induced concentration-dependent histamine release. Upper normal limits of histamine release were defined for the venom concentrations used. When these were used as reference values the basophil histamine release test exhibited a specificity of 94% and a sensitivity of 82% in the diagnosis of bee venom allergy, and a specificity of 83% and a sensitivity of 68% in the diagnosis of wasp venom allergy. Since there was no relevant significant correlation between the results of the basophil histamine release test on one hand and the severity of sting reactions and the prick test and RAST results on the other, it seems that the histamine release test determined additional parameters of sensitization. Thus, the method is a valuable adjunct to the in vitro methods available for the diagnosis of hymenoptera venom allergy.

PMID: 2466814 [PubMed - indexed for MEDLINE]

130: J Rheumatol. 1988 Jul;15(7):1126-8.

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Bee venom, adjuvant induced disease and interleukin production.

Hadjipetrou-Kourounakis L, Yiangou M.

Laboratory of General Biology, Aristotelian University of Thessaloniki, Greece.

Interleukin production and the in vitro mitogenic responses from honey bee venom treated normal rat splenocytes were reduced considerably compared to controls. Addition of interleukin-1 (IL-1) or interleukin-2 (IL-2) supernatants to these cultures in vitro resulted in an increase of their responses to normal levels. These results suggest that in vivo honey bee venom treatment affects the production of IL-1 by macrophages directly. Honey bee venom treatment affects adjuvant induced disease development by inhibiting certain macrophage functions and thus indirectly inhibiting the activation of T and B cells, and possibly the activation of an endogenous virus which might be involved in adjuvant induced disease induction.

PMID: 3262759 [PubMed - indexed for MEDLINE]

131: Int Arch Allergy Appl Immunol. 1988;86(2):234-7.

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Histamine-binding lymphocytes as a measure of reactivity to bee venom.

Ruby J, Czarny D, Muller HK.

Department of Pathology and Immunology, Monash University Medical School, Melbourne, Australia.

Reduced lymphocyte histamine-binding after in vitro exposure of whole blood samples to bee venom appears related to allergen reactivity. The reactivity demonstrated using this method matched the clinical findings and was used to monitor declining responses to allergen during desensitization therapy for hypersensitivity to bee venom. After successful desensitization to bee venom, patients' lymphocytes did not show decreased histamine binding following in vitro exposure to venom. We conclude that lymphocyte histamine-binding sites are down-regulated by allergen-mediated histamine release in hypersensitive subjects, and that this response diminishes with immunotherapy. This report describes a novel method for the in vitro assessment of reactivity to bee venom.

PMID: 3391688 [PubMed - indexed for MEDLINE]

132: Clin Allergy. 1988 Jan;18(1):39-44.

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Low specific IgE, IgG and lymphocyte reactivity in a group of patients developing anaphylaxis following a honey-bee sting.

Lomnitzer R, Lanner A, Rabson AR.

Medical Research Council Human Cellular Immunology Research Unit, School of Pathology, South African Institute for Medical Research, Johannesburg.

Ten patients who developed severe generalized reactions following a honey-bee sting were investigated for the presence of specific IgE and IgG antibodies, and for lymphocyte reactivity following in-vitro honey-bee venom (HBV) stimulation. Five of the patients (high responders) showed high HBV-specific IgE and IgG levels, whereas the other five patients (low responders) showed low HBV-specific IgE and IgG levels. Mononuclear cells from the high responder group incorporated significant amounts of 3H-thymidine when activated with pure bee venom, whereas insignificant lymphocyte proliferation was observed in the low-responder group. It is concluded that, amongst HBV-sensitive patients, a group of low responders exists in whom the mechanism of anaphylaxis cannot be explained.

PMID: 3349591 [PubMed - indexed for MEDLINE]

133: J Lipid Res. 1987 Oct;28(10):1166-76.

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Effect of cholesterol enrichment on 12-hydroxyeicosatetraenoic acid metabolism by mouse peritoneal macrophages.

Mathur SN, Field FJ.

Department of Internal Medicine, University of Iowa, Iowa City 52242.

The metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) was investigated in mouse peritoneal macrophages enriched in cholesterol by incubation with acetylated low density lipoproteins. After incubating with labeled arachidonic acid, cholesterol-rich cells released more 12-HETE into the medium than unmodified macrophages. With time, however, 12-HETE decreased in the medium of both cell preparations suggesting re-uptake of this monohydroxyfatty acid and perhaps further metabolism. When control macrophages were incubated with radiolabeled 12-HETE for 2 hr, almost 70% of the cell-associated 12-HETE label was incorporated into phospholipids. In contrast, in cholesterol-rich cells, only 31% of the 12-HETE label was incorporated into phospholipids. Bee venom phospholipase completely hydrolyzed the label, suggesting that the monohydroxyfatty acid was esterified at the sn-2 position of the phospholipid. In cholesterol-rich cells, 69% of the 12-HETE was diverted into neutral lipids. Two major neutral lipids were identified in cholesterol-rich macrophages. One neutral lipid band which migrated with an Rf value of 0.34 contained the hydroxylated fatty acid esterified to a glyceride. The other neutral lipid band having an Rf value of 0.49 contained cholesterol and by further analysis was found to contain predominantly cholesteryl-12-HETE. The labeled fatty acids in these two neutral lipids were mostly oxidized products of 12-HETE in contrast to the native 12-HETE observed in the phospholipids. Cholesterol-rich macrophages released 25% more products of 12-HETE metabolism than control macrophages. Two major products were observed in the medium which eluted in the area of a standard di-HETE, LTB4, on high performance liquid chromatography (HPLC) analysis. We propose that the reincorporation of 12-HETE into these neutral lipids and the increased capacity for further metabolism of this biologically potent hydroxyfatty acid could be a mechanism by which the cholesterol-rich macrophage maintains its membrane function, and regulates the amount of 12-HETE in the pericellular space.

PMID: 3119755 [PubMed - indexed for MEDLINE]

134: Biochem Pharmacol. 1987 Jun 15;36(12):1941-5.

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Signal response transduction in rabbit neutrophil leucocytes. The effects of exogenous phospholipase A2 suggest two pathways exist.

Lackie JM, Lawrence AJ.

Rabbit neutrophils stimulated by chemotactic peptide (fMLP) or phorbol ester (PMA) respond with a metabolic burst which can be assayed by following luminol-enhanced chemiluminescence. Depending upon the agonist used, exogenous bee-venom phospholipase A2 (PLA2) will enhance or inhibit the response. Neutrophil activation by fMLP is enhanced by PLA2 or by the addition of arachidonic acid, but unaffected by lysophosphatide. The cellular response to PMA is markedly inhibited by PLA2 or by lysophosphatide, though not completely abrogated, but is enhanced by arachidonic acid. The lysophosphatide inhibition overrides the arachidonic acid potentiation of the PMA-induced response. Neither PLA2 nor arachidonic acid alone will activate the cells; it seems that agonist is essential. We interpret these results to mean that at least two signal-response transduction systems are involved in agonist-induced metabolic activation of rabbit neutrophil leucocytes.

PMID: 3109432 [PubMed - indexed for MEDLINE]

135: Eur J Immunol. 1987 Jun;17(6):757-61.

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Expression of Fc epsilon receptors on activated human T lymphocytes.

Prinz JC, Endres N, Rank G, Ring J, Rieber EP.

Our results clearly demonstrate that the low-affinity receptor for IgE (Fc epsilon R) is an activation antigen transiently expressed on a subpopulation of human T lymphocytes. It can be selectively induced by stimulation with certain antigens or lectins, but it is not found on resting T cells. The increased numbers of activated Fc epsilon R+ T cells observed after stimulation of peripheral blood mononuclear cells (PBMC) from bee venom allergic patients with the specific allergen phospholipase A2 (PLA2) suggest that Fc epsilon R+ T cells might very well be involved in the regulation of the human IgE response against the respective antigen. These results were obtained by the use of two monoclonal antibodies, M-L25 and M-L47, which were raised against the human low-affinity Fc epsilon R in our laboratory. After stimulation of PBMC with phytohemagglutinin a peak of 7.6 +/- 6% Fc epsilon R+ T cells was observed on day 3, with pokeweed mitogen of 0.8 +/- 0.8% on days 2 and 3, and with concanavalin A of 0.6 +/- 0.7% Fc epsilon R+ T cells on day 2. Stimulation of PBMC with tetanus toxoid (TT) induced Fc epsilon R on maximally 0.6 +/- 0.8% of the total T cells (day 4), stimulation with purified protein derivative from tuberculin (PPD) on 0.2 +/- 0.6% of the T cells (day 2). In contrast to these antigens, stimulation of PBMC from bee venom allergic patients with PLA2 induced as a peak 2.5 +/- 2.5% of the total T cells to express Fc epsilon R (day 5), although the stimulated T cell population was much smaller than with TT or PPD, as was shown by their stimulation indices. The allergen-stimulated Fc epsilon R+ T cells were exclusively T4+. The Fc epsilon R-expression index was determined, which for a specific antigen or lectin correlates the percentage of Fc epsilon R+ T cells to the stimulated T cell population, respectively.

PMID: 2954825 [PubMed - indexed for MEDLINE]

136: J Allergy Clin Immunol. 1987 Feb;79(2):407-9.

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Bee venom allergy in a patient with urticaria pigmentosa.

Price LA, Safko M.

Publication Types:

·       Case Reports

PMID: 3819222 [PubMed - indexed for MEDLINE]

137: Biochem Biophys Res Commun. 1986 Aug 29;139(1):222-7.

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Differential cytolysis of murine spleen, bone-marrow and leukemia cells by melittin reveals differences in membrane topography.

Killion JJ, Dunn JD.

L1210 leukemia cells are 2-4 fold more sensitive to the cytolytic effects of melittin, the membrane-active toxin of bee venom, than normal DBA/2 mouse spleen and bone-marrow cells. Lysis of the normal cells was abolished when either 75 mM galactosamine, glucosamine or 100 microM beta-lactoglobulin was added to the melittin-cell reaction, but lysis of the leukemia cells was unaffected. The amino-groups appeared necessary for blocking melittin-mediated lysis since glucose, galactose and the N-acetyl derivatives were not inhibitory. Bone-marrow cells were more readily protected from lysis than spleen cells. Since melittin-inhibitor complexes were not detected by gel chromatography and the inhibitor could be added to the cell suspension after melittin, the evidence suggests that bone-marrow cells are rich in membrane binding sites for carbohydrates that decrease in mature spleen cells and are virtually absent after neoplastic transformation.

PMID: 3767954 [PubMed - indexed for MEDLINE]

138: 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]

139: Inflammation. 1986 Jun;10(2):175-82.

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Bee venom melittin blocks neutrophil O2- production.

Somerfield SD, Stach JL, Mraz C, Gervais F, Skamene E.

Bee venom (BV) is used in folk medicine to treat arthritis. It has antiinflammatory effects in animal models of rheumatic disease. We have studied the effects of BV on human neutrophil production of superoxide (O2-) and hydrogen peroxide, finding potent, nontoxic, dose-dependent production inhibition. Melittin, the major fraction of BV (50-70%) shows high-affinity calmodulin binding (Kd 3 nM). Drugs which bind calmodulin, such as trifluoperazine, inhibit O2- production by human neutrophils. For these reasons we have investigated the effect of melittin and other BV peptides on O2- production by human peripheral blood leukocytes. We show that melittin inhibited O2- production both pre- and poststimulation in contrast to other BV fractions which were without effect. Oxygen radicals and their derivatives from inflammatory cells are implicated in the tissue damage occurring during inflammation. The inhibition is due to a direct effect on cells, and not indicator medium, dismutation, toxic or scavenging effects. We propose that melittin may serve as a prototype small (mol wt 1280), cationic, amphipathic, calmodulin-binding, membrane-active, superoxide-production-inhibiting peptide, providing a model for peptides which could have a role in in vivo regulation of radical production.

PMID: 3011670 [PubMed - indexed for MEDLINE]

140: J Rheumatol. 1986 Apr;13(2):477.

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Bee venom and arthritis.

Somerfield SD.

Publication Types:

·       Letter

PMID: 3014144 [PubMed - indexed for MEDLINE]

141: Immunology. 1986 Mar;57(3):455-9.

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Functional characterization of mast cells generated in vitro from the mesenteric lymph node of rats infected with Nippostrongylus brasiliensis.

Shanahan F, Lee TD, Denburg JA, Bienenstock J, Befus AD.

We have examined the biochemical and functional characteristics of mast cells grown in tissue culture from the mesenteric lymph node (MLN) of rats infected with the nematode Nippostrongylus brasiliensis and compared them with mast cells isolated from the small intestinal mucosa and peritoneal cavity of infected animals. Cultured mast cells (MC) and isolated intestinal mucosal mast cells (MMC) had a similar histamine content, and both contained type II protease (RMCP II) which was absent from peritoneal mast cells (PMC). PMC, MMC and cultured MC each responded to immunologically induced histamine secretion, but MMC and cultured MC were hyporesponsive to calcium ionophores and unresponsive to widely used PMC secretagogues including compound 48/80 and bee venom peptide 401. MMC and cultured MC also differed from PMC in their lack of responsiveness to the anti-allergic agent disodium cromoglycate. Thus, MC cultured from the MLN are distinct from PMC but have a biochemical and functional phenotype similar to that of intestinal MMC.

PMID: 2420704 [PubMed - indexed for MEDLINE]

142: Jpn J Med Sci Biol. 1986 Feb;39(1):9-20.

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Bactericidal activity of the membrane fraction isolated from phagocytes of mice and its stimulation by melittin.

Kondo E, Kanai K.

The membrane fraction prepared from mouse peritoneal exudate cells was incubated with mycobacteria, staphylococci, or E. coli in acetate buffer of pH 5.6 to follow the fate of viable bacilli. The membrane fraction exhibited bactericidal effect on mycobacteria and staphylococci, but not on E. coli. The activity to kill mycobacteria, as well as the endogenous phospholipase A2 activity, of the membrane fraction was markedly enhanced by melittin, a basic peptide from bee venom, and inhibited by indomethacin and EDTA. The role of the enzyme activity in the bactericidal activity was discussed.

PMID: 3090322 [PubMed - indexed for MEDLINE]

143: 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]

144: J Immunopharmacol. 1986;8(2):165-204.

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Stimulation of arachidonic acid release and eicosanoid biosynthesis in an interleukin 2-dependent T cell line.

Abraham RT, McKinney MM, Forray C, Shipley GD, Handwerger BS.

Previous studies have provided pharmacologic evidence that T lymphocyte function may be regulated in part by the intracellular production of various arachidonic acid (AA) metabolites in response to cellular stimulation. However, the specific AA metabolic capabilities of homogeneous T cell populations have not been clearly defined. In the present studies, we have employed an accessory cell-free T cell line, HT-2, as a model system for the examination of stimulus-induced eicosanoid biosynthesis in T lymphocytes. HT-2 cells were biosynthetically labeled with [3H]-AA and challenged briefly with various agents that stimulate the hydrolytic release of AA from cellular phospholipids. The bee venom peptide melittin stimulated a profound AA release response in the cells and the concomitant synthesis of both cyclooxygenase (PGF2 alpha, PGE2 and PGD2) and lipoxygenase (5-,12-,15-HETE and possibly 5-,12-diHETE) metabolites of AA. The formation of PGs was blocked by 5 microM indomethacin, demonstrating that this cell line contains cyclooxygenase activity functionally similar to that described in macrophages and other cell types. The high activity of melittin in this system was shown to result largely from a synergy between the peptide itself and a persistent bee venom phospholipase A2 contaminant. However, experiments with melittin freed of detectable phospholipase A2 activity by heating, and with synthetic homopolymers of (L)-lysine and (L)-arginine demonstrated that HT-2 cells contain sufficient endogenous, stimulus-responsive phospholipase A2 to provide both the cyclooxygenase and lipoxygenase pathways of AA metabolism ith substrate. In contrast, Ca++ ionophores, which are known to stimulate AA release and metabolism in certain cell types, stimulated only AA release but no detectable eicosanoid biosynthesis in HT-2 cells. Experiments with exogenous bacterial phospholipase C suggested that this cell line can also generate free AA for eicosanoid biosynthesis from membrane-derived 1,2-diacylglycerol. These results indicate that multiple intracellular pathways of AA metabolism are present HT-2 cells, and that the stimulus-induced release of AA and the production of eicosanoid second messengers may result from activation of either phospholipase A2 or phospholipase C.

Publication Types:

·       Review

PMID: 3088127 [PubMed - indexed for MEDLINE]

145: J Clin Invest. 1985 Nov;76(5):1971-7.

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Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking.

Snyder LM, Fortier NL, Trainor J, Jacobs J, Leb L, Lubin B, Chiu D, Shohet S, Mohandas N.

To further define the conditions for forming spectrin-hemoglobin cross-linking in human erythrocyte membranes and to examine its possible effects on membrane function, we incubated normal human erythrocytes for up to 3 h in concentrations of H2O2, varying from 45 to 180 microM, in an azide phosphate buffer, pH 7.4. The chemical changes observed indicated that methemoglobin formation occurred early and at a low concentration (45 microM). Morphologic changes characterized by increased echinocyte formation occurred in a dose-dependent fashion. In addition, decreased cell deformability commensurate with increased membrane rigidity was found. Finally, an increase in cell recognition as determined by monocyte phagocytosis and adherence in vitro, as well as decreased phosphatidylcholine accessibility to bee venom phospholipase A2, was found in H2O2-treated erythrocytes compared with controls. Both of these latter changes were closely correlated with the extent of spectrin-hemoglobin cross-linking. In addition to these protein-mediated interactions, lipid peroxidation also occurred after H2O2 exposure, as shown by generation of fluorescent amino propene derivatives. The addition of the antioxidant, butylated hydroxytoluene, decreased the fluorescent derivatives, but did not prevent the effects on membrane function. This suggests that lipid peroxidation, though present, was not necessary for the membrane changes found. In contrast, spectrin-hemoglobin aggregation and the alterations in membrane function were completely prevented by prior exposure of the erythrocytes to carbon monoxide.

PMID: 4056060 [PubMed - indexed for MEDLINE]

146: Biochem Pharmacol. 1985 Jul 1;34(13):2391-4.

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Inhibition of prostaglandin biosynthesis by the mast-cell-degranulating agent compound 48/80 but not by the mast-cell-degranulating peptide (peptide-401) from bee venom.

Dempsey CE, Banks BE, Roberts NA, Vernon CA.

PMID: 4015681 [PubMed - indexed for MEDLINE]

147: Acta Physiol Pharmacol Bulg. 1985;11(2):50-5.

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Further investigation on the antiinflammatory properties of adolapin--bee venom polypeptide.

Koburova KL, Michailova SG, Shkenderov SV.

Adolapin is a basic polypeptide (M. W. 11500) isolated from bee venom. It showed marked antiinflammatory and analgetic properties and inhibited cyclooxygenase. It was found that adolapin inhibited also the activity of bee venom phospholipase A2 (7 nmole/ml producing about 80% inhibition of 2.5 nmole/ml phospholipase). In addition it inhibited the lipoxygenase from human platelets (4.5 nmole/ml inhibited about 80% of the activity of 0.8 mg protein/ml). Adolapin (20 micrograms/kg) caused an elevation of c-GMP level in rat spleen and brain as well as a decrease of c-AMP in rat spleen. Adolapin was tested by the "tail flick" method which allowed the demonstration of its analgetic action. The partial inhibition of the analgetic effect of adolapin induced by naloxon, proved the participation of a central mechanism of action. Similar to other nonsteroid analgetics, adolapin displayed antipyretic effect (40 micrograms/kg caused an inhibition of the mean temperature rise about 62%.

PMID: 2996298 [PubMed - indexed for MEDLINE]

148: Peptides. 1985;6 Suppl 3:431-6.

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Amino acid sequence of bumblebee MCD peptide: a new mast cell degranulating peptide from the venom of the bumblebee Megabombus pennsylvanicus.

Argiolas A, Herring P, Pisano JJ.

A new 28 amino acid peptide, we recently isolated from the venom of the bumblebee Megabombus pennsylvanicus. has been characterized. The peptide, Met-Cys-Ile-Cys-Lys-Asn-Gly-Lys-Pro-Leu-Pro-Gly-Phe-Ile-Gly-Lys-Ile-Cys- Arg-Lys-Ile-Cys-Met-Met-Gln-Gln-Thr-His(NH2), has been named bumblebee mast cell degranulating (MCD) peptide due to its ability to degranulate rat peritoneal mast cells, and its resemblance to the bee venom MCD peptide. Bumblebee MCD peptide, unlike bombolitins, the other mast cell degranulating heptadecapeptides of bumblebee venom, is not lytic and releases histamine at a dose as low as 0.05 micrograms/ml (1.6 X 10(-8) M).

PMID: 2421265 [PubMed - indexed for MEDLINE]

149: Inflammation. 1984 Dec;8(4):385-91.

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Bee venom inhibits superoxide production by human neutrophils.

Somerfield SD, Stach JL, Mraz C, Gervais F, Skamene E.

Investigation of the antiinflammatory properties of bee venom demonstrates that it inhibits production of superoxide anion by human neutrophils in a potent, selective, nontoxic, dose-dependent fashion, both pre- and poststimulation by particulate and soluble activators of the neutrophil oxidative metabolism burst. The effect is not due to receptor competition, superoxide dismutase, and/or catalase activity, scavenging, or indicator media effects. These findings may explain the antiinflammatory effects of whole bee venom in experimental systems, its widespread use in folk medicine, and lead to the development of potent, new antiinflammatory substances for therapeutic use in man.

PMID: 6097547 [PubMed - indexed for MEDLINE]

150: 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]

151: 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]

152: Br J Pharmacol. 1984 Apr;81(4):693-701.

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A comparison of histamine secretion from peritoneal mast cells of the rat and hamster.

Leung KB, Pearce FL.

Functional mast cells have been obtained by peritoneal lavage of the rat and hamster. Both cell types released histamine on stimulation with appropriate dilutions of anti-rat IgE and anti-hamster serum. The maximum response evoked by each reagent was significantly greater for the hamster cells. The release was non-cytotoxic and was in each case blocked by the corresponding soluble antigen. The rat and hamster cells responded to concanavalin A and the lectin from lentil. Phosphatidylserine (PS) potentiated the release only from the rat cells. In the absence of the lipid, the hamster cells were more reactive. The lectin from wheat germ, in the presence of PS, evoked histamine secretion only from the rat cells. Both populations were refractory to the lectin from soybean and to protein A. Rat peritoneal cells were more responsive to the basic secretagogues compound 48/80 and peptide 401 (the MCD-peptide from bee venom). These differences were less marked in the case of polylysine and polyarginine. The two cell populations responded to the calcium ionophores A23187, ionomycin and chlortetracycline. The hamster cells were significantly more sensitive to the former two liberators but markedly less reactive to chlortetracycline. Adenosine 5'-triphosphate (ATP) and dextran were potent histamine liberators from the rat cells but were totally ineffective against the hamster. Acetylcholine and carbamylcholine had no effect on either cell type. These results are discussed in terms of the functional heterogeneity of mast cells from different sources.

PMID: 6202354 [PubMed - indexed for MEDLINE]

153: Eksp Med Morfol. 1984;23(3):143-8.

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[Antipyretic effect of a polypeptide from bee venom--adolapin]

[Article in Bulgarian]

Koburova K, Mikhailova S, Shkenderov S.

PMID: 6440766 [PubMed - indexed for MEDLINE]

154: J Biol Chem. 1983 Nov 25;258(22):13697-702.

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Facilitation of phospholipase A2 activity by mastoparans, a new class of mast cell degranulating peptides from wasp venom.

Argiolas A, Pisano JJ.

The effect of mastoparan, Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-LeuNH2, and related peptides on the release of arachidonic acid from egg yolk lecithin liposomes, rat peritoneal mast cells, and cultured human fibroblasts was studied. In unsonicated liposomes, labeled with 1-stearoyl-2[1-14C]arachidonyl-sn-glycero-3-phosphocholine, 5 X 10(-5) M mastoparan caused a 12-, 15-, and 50-fold increase in the production of arachidonic acid catalyzed by phospholipase A2 from bee venom, eastern diamondback rattlesnake and porcine pancreas, respectively. The stimulant effect of mastoparan and related peptides was dose-dependent and further enhanced by sonication of liposomes. In contrast, melittin, while stimulating the production of arachidonic acid by phospholipase from bee venom, was inactive with the rattlesnake and pancreatic enzymes. Melittin was also only weakly active with liposomes containing stearic acid in place of arachidonic acid. Like melittin, mastoparans stimulated phospholipase activity in tissue homogenates and caused a dose-dependent release of arachidonic acid from rat peritoneal mast cells and cultured human fibroblasts prelabeled with [14C]arachidonic acid. The heptapeptide fragments mastoparan 1-7 and mastoparan 8-14, and succinylated mastoparan were ineffective. The results suggest that mastoparan and related peptides in insect venoms act, at least in part, by stimulating phospholipase activity.

PMID: 6643447 [PubMed - indexed for MEDLINE]

155: J Allergy Clin Immunol. 1983 Oct;72(4):399-406.

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Diagnosis of Polistes wasp hypersensitivity.

Grant JA, Rahr R, Thueson DO, Lett-Brown MA, Hokanson JA, Yunginger JW.

Patients referred from the Houston, Texas, metropolitan area were evaluated for allergic reactions to insect stings. Forty-eight persons reported at least one systemic reaction caused by a Polistes paper-nest wasp sting. Honey bees, imported fire ants, and other types of Hymenoptera were identified in that order by 19 other subjects with systemic allergic reactions. Life-threatening airway obstruction and/or hypotension were noted by most of our patients. Wasp venom skin testing was positive in 65% of subjects reporting sensitivity to this insect. Skin testing was correlated quantitatively with basophil histamine release, and qualitatively with RAST assays using Polistes wasp venom. Venoms from common species of Polistes were highly cross-reactive as shown by RAST and basophil histamine release. Patients having a positive history and laboratory response (by skin testing, histamine release, or RAST) to Polistes wasp venom also were positive to bee venom about 20% of the time and to another vespid (hornet or yellow jacket) over 50% of the time.

PMID: 6194197 [PubMed - indexed for MEDLINE]

156: FEBS Lett. 1983 Feb 21;152(2):227-30.

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Conformational change of mastoparan from wasp venom on binding with phospholipid membrane.

Higashijima T, Wakamatsu K, Takemitsu M, Fujino M, Nakajima T, Miyazawa T.

The conformational change upon binding with phospholipid membrane has been studied of mastoparan from wasp venom, a tetradecapeptide causing the degranulation of mast cells. The 270-MHz 1H-NMR spectra and CD spectra indicate that the mastoparan molecule takes the alpha-helical conformation in methanol solution, but a much less ordered form in aqueous solution. On binding with phospholipid membrane, the alpha-helical conformation is formed even in aqueous medium. Such a conformational change is primarily due to the interaction between the aliphatic side chains of mastoparan and the hydrophobic interior of phospholipid membrane, in contrast to the case of melittin from bee venom.

PMID: 6825849 [PubMed - indexed for MEDLINE]

157: Int Arch Allergy Appl Immunol. 1983;72(3):234-8.

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A comparison of histamine secretion from isolated peritoneal mast cells of the mouse and rat.

Barrett KE, Pearce FL.

The effect of different histamine liberators on isolated peritoneal mast cells of the mouse and rat has been examined. Both cell types responded to the lectin, concanavalin A, and the release was in each case potentiated by phosphatidylserine. The rat cells released histamine on treatment with dextran but the mouse cells were essentially refractory to the polysaccharide. The mouse cells were significantly more responsive to the actions of the ionophore A23187 and adenosine 5'-triphosphate but much less reactive towards the polycations, compound 48/80 and peptide 401 (the MCD-peptide from bee venom). The reactivity of the mouse cells towards the latter two agents was enhanced in the absence of extracellular calcium. These results further emphasize the functional heterogeneity of mast cells from different sources.

PMID: 6194120 [PubMed - indexed for MEDLINE]

158: J Immunol. 1982 Jun;128(6):2481-6.

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Mucosal mast cells. II. Effects of anti-allergic compounds on histamine secretion by isolated intestinal mast cells.

Pearce FL, Befus AD, Gauldie J, Bienenstock J.

Functional mast cells have been isolated from the lamina propria of the small intestine of rats infected with the nematode Nippostrongylus brasiliensis. The cells released histamine on challenge with specific antigen, anti-rat IgE, concanavalin A, and calcium ionophores but were less responsive than peritoneal mast cells (MMC) from the same animals. Intestinal mucosa mast cells (PMC) were refractory to the action of the basic secretagogues peptide 401 from bee venom and compound 48/80. The anti-allergic compounds disodium cromoglycate (less than or equal to 10(-3) M), AH 9679 (less than or equal to 10(-4) M), and theophylline (less than or equal to 10(-2)) did not inhibit antigen-induced histamine secretion by MMC, although these compounds were effective against PMC. In contrast, doxantrazole (10(-5) to 10(-3) M) inhibited the secretion of histamine from both MMC and PMC in a comparable dose-dependent fashion. Thus, we have established that mast cells from different sites are functionally heterogeneous not only in their response to various stimuli for histamine secretion, but also in their responses to different pharmacologic modulators of secretion. It cannot be assumed that anti-allergic compounds effective against mast cells in one tissue site or organ will be equally efficacious against mast cells in other sites. The extent of this functional heterogeneity must be established, and its investigation may provide new insights into the biochemical events involved in mast cell secretion.

PMID: 6176639 [PubMed - indexed for MEDLINE]

159: J Immunol. 1982 Jun;128(6):2475-80.

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Mucosal mast cells. I. Isolation and functional characteristics of rat intestinal mast cells.

Befus AD, Pearce FL, Gauldie J, Horsewood P, Bienenstock J.

We have developed a procedure for the dispersion of mast cells from the intestinal lamina propria (LP) and epithelium of rats infected with the intestinal nematode, Nippostrongylus brasiliensis. The dispersed cells are morphologically and histochemically similar to intestinal mucosal mast cells (MMC) in situ and are distinguishable from peritoneal mast cells (PMC). MMC derived from the LP or epithelium of parasitized animals secrete histamine in response to the specific parasite antigens as well as anti-IgE. Unlike PMC, these cells are unresponsive to the basic secretagogues 48/80 and bee venom peptide 401. Similarly, bee venom peptide 401 conjugated with dansyl chloride binds to PMC and mast cells in the thymus and intestinal serosa, but not to mast cells in or derived from the intestinal LP and epithelium. Studies on PMC treated by the intestinal cell isolation procedure show that the functional characteristics of the MMC cannot be solely attributed to the isolation procedure. Thus, MMC have been isolated and shown to be morphologically, histochemically, and functionally different from PMC, as suggested by previous in vivo studies of the normal intestine.

PMID: 6176638 [PubMed - indexed for MEDLINE]

160: 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]

161: Nauchnye Doki Vyss Shkoly Biol Nauki. 1982;(4):41-5.

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[Regulatory action of bee venom on the humoral immunity system]

[Article in Russian]

Orlov BN, Romanova EB.

PMID: 7200806 [PubMed - indexed for MEDLINE]

162: Int Arch Allergy Appl Immunol. 1982;68(4):320-5.

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Studies of chemically modified honeybee venom. II. Immunogenicity and suppression of reaginic antibody formation.

Mueller U, Reisman R, Wypych J, Arbesman C.

Honeybee venom allergens modified by formaldehyde treatment (F), acetoacetylation (A), and coupling to polyethylene glycol (P) were studied with regard to their immunogenicity and their IgE-suppressive potential in rabbits and mice. F indiced a strong IgG response in rabbits and especially mice, but only a moderate IgE response in the mouse. Its IgE-suppressive potential in mice was comparable to that of unmodified bee venom. A induced only a weak IgG and IgE response. Its IgE-suppressive potential was greater than that of unmodified bee venom. P was nonimmunogenic and had only a marginal IgE-suppressive effect. F and A seem promising for the treatment of bee sting-allergic individuals.

PMID: 6212550 [PubMed - indexed for MEDLINE]

163: Adv Exp Med Biol. 1982;149:521-7.

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Unique functional characteristics of mucosal mast cells.

Befus AD, Pearce FL, Goodacre R, Bienenstock J.

Mast cells have been isolated from the intestine (IMC) of rats previously infected with the nematode, Nippostrongylus brasiliensis. Functional studies on IMC have shown that they are responsive to antigen and possess surface IgE but, in contrast to peritoneal mast cells (PMC), IMC are unresponsive to the basic secretagogues, 48/80 and bee venom peptide 401, and hyporesponsive to ionophores. Furthermore, sodium cromoglycate, AH9679 and theophylline inhibited secretion by PMC but not IMC, whereas doxantrazole inhibited secretion by both cells. Histochemical investigations established that there is mast cell heterogeneity in the human intestine as well as in the rat. Since GALT and BALT are important in intestinal mastocytosis it is important to determine whether the distinct functional properties of IMC reflect a distinct precursor population, inducer cell, or other factors in GALT or BALT.

PMID: 6183933 [PubMed - indexed for MEDLINE]

164: Int Arch Allergy Appl Immunol. 1981;64(2):201-9.

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Flow-cytometric analysis of human basophil degranulation. III. Degranulation induced by allergens and antibodies in hay fever and bee venom allergic patients.

Nakagawa T, Moyseyenko O, de Weck AL.

Human basophil degranulation responses to grass pollen and whole bee venom as allergens and to anti-IgE and anti-IgG4 as antibodies in hay fever and bee venom allergic patients were assessed using a flow-cytometric system. The concordance of basophil degranulation with skin tests and RAST assays reached more than 90%. These allergens and antibodies could degranulate basophils in a dose-dependent manner; however, there was a wide variation in the response of basophils obtained from different individuals. Moreover, a significant correlation was observed between the degree of degranulation by allergens and anti-IgE, or by allergens and anti-IgG4, but this was not the case between serum IgE concentrations and basophil response to anti-IgE. These data suggest that 'releasability' of basophils upon challenge by allergens and antibodies is also an important parameter in determining the degree of hypersensitivity in each subject.

PMID: 7007256 [PubMed - indexed for MEDLINE]

165: Eur J Pharmacol. 1980 Sep 5;66(4):339-45.

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Differential reactivity of isolated mast cells from the rat and guinea pig.

Ennis M, Pearce FL.

The effects of various chemical histamine liberators on isolated rat peritoneal, rat mesenteric and guinea-pig mesenteric mast cells were examined. All three cell types responded, but to different degrees, to calcium ionophores and surface active agents. The rat mesenteric cells also reponded, but less effectively than the peritoneal cells, to compound 48/80, peptide 401 from bee venom and ATP. Rat mesenteric cells were essentially refractory to the action of dextran and guinea-pig cells were almost totally unresponsive to the named secretagogues. These results show that there are marked functional differences between the mast cells examined and suggest that isolated tissue cells may usefuly complement rat peritoneal cells in the study of anaphylactic and anaphylactoid reactions.

PMID: 6158454 [PubMed - indexed for MEDLINE]

166: Hoppe Seylers Z Physiol Chem. 1980 Apr;361(4):525-35.

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[Basic peptides in bee venom, VI. Structure-activity studies on the anti-inflammatory effects of derivatives and fragments of the MCD-peptide (author's transl)]

[Article in German]

Martin W, Hartter P.

Mastcell-degranulating peptide from bee venom exhibits anti-inflammatory activity in the Carrageenine-inflammation model on mice and rats. Its dose/response relationship corresponds to that of dexamethasone. In the 125I-rat serum albumin-test the substance shows an inhibitory effect of 87% on the developing Carrageenine edema at a dose of 1 mg/kg; in contrast the bee venom peptides apamin and melittin exhibit small edema supressing effects. The anti-inflammatory characteristics of MCD-peptide have to be considered with regard to its high basicity and specific molecular structure, determined by two disulfide-bridges for the derivaties with acetylated amino groups respectively with alkylated cysteine residues show no significant activity. A pentapeptide in the C-terminal region of MCD-peptide (Lys-Ile-Cys-Gly-Lys)2 inhibits the carrageenin edema to 55% at a dose of 10 mg/kg.

PMID: 7380393 [PubMed - indexed for MEDLINE]

167: Hoppe Seylers Z Physiol Chem. 1980 Apr;361(4):503-13.

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[Basic peptides from bee venom, IV. Synthesis of the mast cell-degranulating peptide by liquid-phase fragment condensation (author's transl)]

[Article in German]

Hartter P.

The synthesis of the mast cell-degranulating peptide by liquid-phase fragment condensation is described. After the carboxyterminal of the peptide is condensated with polyethylene-glycol (Mr 10000) the following fragments are coupled stepwise on the polymer, a soluble carrier in dichloromethane by the dicyclohexylcarbodiimide/hydroxybenzotriazole-method. Pos. 17-21 Boc-Lys(Z)-Ile-Cys(SiPr)-Gly-Lys(Z) (I) Pos. 12-16 Boc-Pro-His(Trt)-Ile-Cys(Trt)-Arg(Tos) (II) Pos. 8-11 Boc-His(Trt)-Val-Ile-Lys(Z) (III) Pos. 5-7 Boc-Cys(SiPr)-Lys(Z)-Arg(Tos) (IV) Pos. 1-4 Boc-Ile-Lys(Z)-Cys(Trt)-Asn(Mbh) (V) It is practical to crystallize the polyethyleneglycol peptide-coupling products from ethanol after each step. Most of the protecting groups can be removed by treatment of the complete polyethylene-glycol-peptide in trifluoroacetic acid/HBr. In methanol, saturated with ammonia, the peptide is removed in the amid-form from the carrier. The guanidyl-blocking group disappears by solving the peptide in liquid HF. The crude peptide is converted into the tetra-S-sulfonate derivate by oxidative sulfitolysis and purified by ion-exchange and gel chromatography. After reduction by mercaptoethanol a cautious air-reoxidation of the SH- to the SS-peptide followed. Rechromatography on ion-exchange and dextran gels yields a peptide with good biological activity in rat cell histamin-liberation and inflammation inhibition compared with the natural recombinated product.

PMID: 7380391 [PubMed - indexed for MEDLINE]

168: Enzyme. 1980;25(5):322-8.

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Effect of tumor promoters on induction of aryl hydrocarbon hydroxylase in human lymphocytes.

Kyner D, Christman JK, Acs G.

The induction of aryl hydrocarbon hydroxylases in lymphocytes is dependent on their activation. The tumor-promoting phorbol esters which induce blast formation and DNA synthesis in lymphocytes enable polycyclic aromatic hydrocarbons to induce aryl hydrocarbon hydroxylases. Melittin, the major constituent of bee venom, acts synergistically with these phorbol esters in enhancing both lymphocyte activation and hydroxylase synthesis. Since aryl hydrocarbon hydroxylases convert procarcinogens to carcinogens these results suggest that tumor promotion by phorbol esters may be associated with their ability to affect the induction of these enzymes. This hypothesis is supported by the finding that phorbol and phorbol esters which lack tumor-promoting activity fail to enhance induction of aryl hydrocarbon hydroxylases in lymphocytes. However mezerein, although rather ineffective in promoting tumors, activates lymphocytes and permits polycyclic aromatic hydrocarbons to induce their hydroxylases effectively.

PMID: 7449754 [PubMed - indexed for MEDLINE]

169: Biochem J. 1979 Sep 1;181(3):623-32.

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Further studies on the structural requirements for polypeptide-mediated histamine release from rat mast cells.

Jasani B, Kreil G, Mackler BF, Stanworth DR.

Structure-activity studies have been performed on a series of naturally occurring and 'tailor-made' polypeptides, by measurement of ability to induce selective histamine release from normal rat peritoneal mast cells in vitro. Compounds investigated include corticotropin and melittin derivatives, mast-cell-degranulating peptide from bee venom, polymyxin B, bradykinin and various synthetic poly(amino acids) and short-chain peptides. It was confirmed that a cluster of four basic residues (lysine or arginine) was optimal for histamine release by corticotropin and melittin polypeptides, provided that the C-terminal carboxyl group was substituted (by, for instance, amidation). In contrast, the presence of a free C-terminal carboxyl group or nearby dicarboxylic acid residues led to a considerable diminution in histamine-releasing activity. Likewise, polypeptides comprised essentially of acidic amino acids were inactive. On the basis of these observations it has been possible to predict that synthetic peptides comprising a particular sequence within the Fc region of human immunoglobulin E, the immunoglobulin class particularly involved in mediation of allergic reactions of the immediate type, would possess potent histamine-releasing activity when similarly made to react with normal rat mast cells. The further study of such a structure should throw new light on the molecular basis of allergen-antibody triggering of mast cells.

PMID: 92986 [PubMed - indexed for MEDLINE]

170: Z Hautkr. 1979 Feb 1;54(3):75-81.

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[Capabilities and limitations of in-vitro allergy diagnostics (on the model of bee-venom allergy and on Neurodermatitis in relation to IgE-T-lymphocyte interaction)]

[Article in German]

Jarisch R.

PMID: 311110 [PubMed - indexed for MEDLINE]

171: Biull Eksp Biol Med. 1977 Jul;84(7):78-80.

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[Histamine-liberating action of MCD-peptide from bee venom]

[Article in Russian]

Gushchin IS, Miroshnikov AI, Martynov VI.

Histamine release (HR) from the isolated rat mast cells induced by MCD-peptide, mellitin (from the Apis mellifica venom) and compound 48/80 was studied. The dose-response curve, the latent period and temperature dependence of HR induced by MCD-peptide were similar to those of HR induced by compound 48/80. The HR induced by MCD-peptide proved to be an energy-dependent process that was independent of cyclic adenosine-3',5'-monophosphate.

PMID: 70248 [PubMed - indexed for MEDLINE]

172: Nature. 1977 Jun 23;267(5613):713-4.

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Succinyl bee venom melittin is a leukocyte chemotactic factor.

Wilkinson PC.

PMID: 876392 [PubMed - indexed for MEDLINE]

173: Hoppe Seylers Z Physiol Chem. 1977 Mar;358(3):331-7.

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[Basic peptides in bee venom, III. Synthesis of peptide fragments from the sequence of the mast-cell-degranulating peptide (author's transl)]

[Article in German]

Hartter P.

The synthesis by conventional methods of the following three peptides is described: MCD(8-11) Boc-His(Trt)-Val-Ile-Lys(Z) (III) MCD(5-7) Boc-Cys(SiPr)-Lys(Z)-Arg(Tos) (IV) and MCD(1-4) Boc-Ile-Lys(Z)-Cys(Trt)-Asn(Mbh) (V). These peptides are fragments of the mast cell degranulating peptide from bee venom. The purity of the fragments synthesized was examined by thin-layer chromatography, amino acid and elementary analysis. Including the fragments Boc-Lys(Z)-Ile-Cys(SiPr)-Gly-Lys(Z) (I) and Boc-Pro-His(Trt)-Ile-Cys(Trt)-Arg(Tos) (II), which were described earlier, the synthesis of the mast-cell-degranulating peptide on a polyethylene-asparagine support appears possible.

PMID: 856709 [PubMed - indexed for MEDLINE]

174: Hoppe Seylers Z Physiol Chem. 1976 Dec;357(12):1683-93.

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[Basic peptides in bee venom, II. Synthesis of two pentapeptides from the sequence of the mast-cell-degrading peptide (author's transl)]

[Article in German]

Hartter P.

The synthesis of two protected pentapeptides is described. The peptides are fragments of the sequence of a mast-cell degranulating peptide from bee venom. The fragments Boc-Lys(Z)-Ile-Cys(SiPr)-Gly-Lys(Z)(I) and Boc-Pro-His(Trt)-Ile-Cys(Trt)-Arg(Tos) (II) were synthesized conventionally. The deprotection of the alpha-amino group by HCl/acetic acid of Boc-Ile-Cys(SiPr)-Gly-Lys(Z) was accompanied by a disulfide exchange at the cysteine residue. After the hydrolysis of fragment II with 6N HCl, allo-isoleucine could be detected by gas chromatography and amino-acid analysis.

PMID: 1017794 [PubMed - indexed for MEDLINE]

175: Clin Allergy. 1976 May;6(3):293-300.

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Comparison of the allergenic properties of bee venom and whole bee body extract.

Light WC, Reisman RE, Rosario NA, Arbesman CE.

The allergenic properties of bee venom and whole bee body extract were compared by in vivo and in vitro tests. The majority of patients with known bee sting sensitivity had positive intracutaneous skin test reactions with bee venom and had bee venom specific IgE in their sera. Of seventeen patients with ppsitive bee venom skin tests, nine had positive tests with whole bee body extract. Of thirty sera containing elevated levels of bee venom specific IgE obtained from untreated patients, fourteen sera contained whole body specific IgE but in much lower titres. In RAST inhibition experiments using both bee venom and whole bee body extract as coupling antigens, bee venom was a more potent inhibiting antigen than whole body extract. From these experiments we conclude that bee venom is a more potent allergen than whole bee body extract.

PMID: 59640 [PubMed - indexed for MEDLINE]

176: J Biol Chem. 1976 Apr 25;251(8):2279-89.

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Evidence for a phospholipid requirement in the specific binding of glucocorticoids to receptors of fibroblasts and thymic lymphocytes.

Schulte HF, Nielsen CJ, Sando JJ, Pratt WB.

The specific steroid binding capacity of soluble preparations from mouse fibroblasts and rat thymic lymphocytes is inactivated by incubation with phospholipases. Receptor binding is drastically reduced by very low concentrations of boiled phospholipase A preparations from bee venom and snake venoms. The enzyme effect is calcium-dependent and is blocked by both phospholipid and a substrate analog that is a competitive inhibitor of phospholipase A. The specific binding capacity is also sensitive to digestion by phospholipase C. Two possible mechanisms are considered for the phospholipase A effect: (a) the receptor protein may be associated with a phospholipid component which is required for specific hormone binding; (b) phospholipase A may be producing detergent products that are indirectly inactivating the receptor. Examination of the effects of lysophosphatide on the receptor and assay of lipid phosphate in the receptor preparation do not support a mechanism based solely on detergent effects. Because phospholipase C, which does not produce detergent products, also inactivates the binding, we propose that the phospholipases may be digesting the phospholipid which is a requisite component of the glucocorticoid receptor.

PMID: 177409 [PubMed - indexed for MEDLINE]

177: Arch Biochem Biophys. 1976 Feb;172(2):661-71.

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Allergens of honey bee venom.

King TP, Sobotka AK, Kochoumian L, Lichtenstein LM.

PMID: 56920 [PubMed - indexed for MEDLINE]

178: 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]

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

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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]

180: Clin Allergy. 1972 Dec;2(4):317-23.

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Allergenic and biological activities of melittin from honey bee venom.

Mackler BF, Russell AS, Kreil G.

PMID: 4629300 [PubMed - indexed for MEDLINE]

181: Am J Vet Res. 1972 Sep;33(9):1867-74.

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Pathologic features in mice hyposensitized to bee venom.

Rothenbacher H, Benton AW.

PMID: 5053166 [PubMed - indexed for MEDLINE]

182: Nippon Igaku Hoshasen Gakkai Zasshi. 1970 Mar;29(12):1494-500.

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[Radioprotection by bee venom]

[Article in Japanese]

Kanno I, Ito Y, Okuyama S.

PMID: 5266622 [PubMed - indexed for MEDLINE]

183: Am J Physiol. 1969 Oct;217(4):965-8.

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Chromatographic fractions of bee venom: cytotoxicity for mouse bone marrow stem cells.

Cole LJ, Shipman WH.

PMID: 5824335 [PubMed - indexed for MEDLINE]

184: Hoppe Seylers Z Physiol Chem. 1969 May;350(5):536-46.

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[Amino acid sequence of MCD-peptide, a specific mast cell-degranulating peptide from bee venom]

[Article in German]

Haux P.

PMID: 5789872 [PubMed - indexed for MEDLINE]

185: Naunyn Schmiedebergs Arch Exp Pathol Pharmakol. 1968;260(2):127-8.

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MCL-peptide, a selectively mastocytolytic factor isolated from bee venom.

Habermann E, Breithaupt H.

PMID: 4239165 [PubMed - indexed for MEDLINE]

186: Naunyn Schmiedebergs Arch Exp Pathol Pharmakol. 1968;261(3):252-70.

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[MCD-peptide from bee venom: isolation, biochemical and pharmacolgical properties]

[Article in German]

Breithaupt H, Habermann E.

PMID: 4235474 [PubMed - indexed for MEDLINE]

187: Acta Physiol Scand. 1967 Dec;71(4):357-67.

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Histamine release from rat mast cell granules induced by bee venom fractions.

Fredholm B, Haegermark O.

PMID: 4172487 [PubMed - indexed for MEDLINE]

188: Acta Physiol Scand. 1967 Dec;71(4):257-69.

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Studies on morphological changes and histamine release induced by bee venom, n-decylamine and hypotonic solutions in rat peritoneal mast cells.

Bloom GD, Haegermark O.

PMID: 4172483 [PubMed - indexed for MEDLINE]

189: Acta Physiol Scand. 1967 Apr;69(4):304-12.

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Histamine release from rat mast cells induced by a mast cell degranulating fraction in bee venom.

Fredholm B, Haegermark O.

PMID: 4166435 [PubMed - indexed for MEDLINE]

190: Biochem Pharmacol. 1966 Dec;15(12):2037-43.

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Studies on a mast cell degranulating factor in bee venom.

Fredholm B.

PMID: 5973173 [PubMed - indexed for MEDLINE]

191: Farmakol Toksikol. 1966 Jul-Aug;29(4):488-9.

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[The effect produced by bee venom on certain enzymes and biologically active groups in the tissues of mice in vivo]

[Article in Russian]

Korablev MV, Simorot RP, Kurbat NM.

PMID: 4294241 [PubMed - indexed for MEDLINE]