acid-phosphatase and Insect-Bites-and-Stings

Acid phosphatase (Api m 3) is a major allergen in honeybee (Apis mellifera) venom, and its availability as a recombinant protein may facilitate the development of improved diagnostic tests and immunotherapies.. One objective is the determination of the complete primary structure of Api m 3 and to obtain recombinant Api m 3 on the basis of expression in insect cells. Another objective is the quantitative analysis of patient serum IgE antibody reactive to recombinant Api m 3.. The cloning of Api m 3 from venom gland cDNA and its expression as a full-length protein in eukaryotic insect cells is described. The immunoreactivity of serum IgE antibodies of honeybee venom-sensitized patients to recombinant Api m 3 was determined in an enzyme immunoassay.. PCR amplification generated a 1122-bp DNA fragment whose identity as the coding sequence of Api m 3 was verified by several means. Recombinant Api m 3, expressed in Trichoplusia ni cells, showed an expected molecular weight and enzymatic activity at pH 4.5. Analysis of tryptic fragments of purified recombinant Api m 3 by mass spectrometry confirmed its identity. In immunoassays, recombinant Api m 3 is specifically recognized by IgE antibodies of pooled serum in Western blots and by 37% of the individual sera of honeybee venom-sensitized patients in ELISA analysis.. The availability of recombinant Api m 3 provides a tool for both the development of improved diagnostic tests and the design of safer and more effective immunotherapeutic approaches for honeybee venom allergy.. The recombinant venom allergen Api m 3 is a key element in the search for an optimized component-resolved approach to honeybee venom allergy with regard to both the development of superior diagnostic tests and the improvement of allergen immunotherapy.

Topics: Acid Phosphatase; Allergens; Amino Acid Sequence; Anaphylaxis; Animals; Base Sequence; Bee Venoms; Bees; Cell Line; Cloning, Molecular; Desensitization, Immunologic; DNA, Complementary; Gene Expression; Genes, Insect; Humans; Immunoglobulin E; In Vitro Techniques; Insect Bites and Stings; Molecular Sequence Data; Moths; Sequence Homology, Amino Acid

IgE antibodies to purified proteins and peptides from honeybee venom have been measured by the RAST. Trace amounts (less than 0.1%) of the major venom protein phospholipase A2 (PLA2) grossly distorted the measurement of IgE antibody to the other venom proteins, acid phosphatase (Acid P) and hyaluronidase (HYAL), and overemphasized their importance. Reduction of antigen coupled to the cellulose paper discs, which were used in the assay, diluted out the contaminating PLA2 without apparent loss in sensitivity. The reduction of disc-bound antigen increased the competition between IgE and IgG antibodies but did not affect measurement of IgE antibodies in sera taken from 35 untreated patients who had a history of general allergic reactions to bee stings. In 54% of sera from bee venom--allergic patients, the greatest IgE antibody response was to PLA2. In all, IgE antibodies to PLA2 were present in 91% of these sera. IgE antibodies to Acid P, HYAL, or melittin were present in 60%, 51%, and 31% of sera, respectively, and accounted for the highest level of binding in 17%, 17%, and 6% of these. Only 6% of sera were positive for whole venom but negative for the isolated antigens. A low level of IgE antibody was found to peptide 401 in 6% of sera. No IgE antibodies were found to apamin. While confirming the central role played by PLA2 in bee sting allergy, these results show that other venom components are also important in some patients.

Topics: Acid Phosphatase; Antibodies; Antibodies, Anti-Idiotypic; Antibody Specificity; Apamin; Bee Venoms; Bees; Humans; Hyaluronoglucosaminidase; Hypersensitivity; Immunoglobulin E; Immunoglobulin G; Insect Bites and Stings; Melitten; Phospholipases A; Phospholipases A2; Radioallergosorbent Test

Antibodies to individual bee venom antigens were studied in detail in nine bee sting-allergic patients who received venom immunotherapy without side effects, in two patients who failed to reach maintenance, and in two whose sensitivity returned. The study was confined to patients who had IgE antibodies to at least one of four purified bee venom antigens at the start of treatment. IgE and IgG antibodies to phospholipase A2 (PLA2), hyaluronidase (HYAL), and acid phosphatase (ACID P) and IgE antibodies to melittin (MEL) were measured, and changes in the antibody levels were followed during bee venom immunotherapy. Two contrasting patterns of antibody response were seen in the nine successfully treated patients. In five patients there was a rise in serum IgG antibodies to the same antigens as the IgE antibodies. In two patients' serum IgE antibody to HYAL or ACID P fell without a marked IgG antibody response to these antigens, although high levels of IgG antibody to PLA2 were present in both. Although the first pattern is consistent with a "blocking" role for IgG antibody, clearly the second is not. Not all patients can be conveniently divided into these two categories, and two patients did not show any significant change in either IgG or IgE antibody but were nevertheless able to tolerate the maintenance dose of 100 micrograms of venom. Two patients who failed to reach the maintenance dose of 100 micrograms because of their allergic reactions to the injections of venom were distinguished by (1) very high serum IgE antibody and (2) a low ratio of IgG/IgE antibody. Passive immunization with IgG antibody from a hyperimmune beekeeper was, however, protective in these patients, although it did not raise their overall serum IgG antibody level very much. We are unable to explain either the failure of conventional therapy or the beneficial effect of passive immunization in these two patients. Two bee sting--allergic beekeepers lost their sensitivity to stings, but later, when their sera contained IgE antibody to another bee venom antigen, they reacted to stings and inhalation of beehive dander. These data suggest that either falling IgE antibody or IgG- "blocking" antibody could be responsible for providing clinical protection to bee venom--allergic subjects. Renewed clinical sensitivity was observed when the IgE response was modulated, with patients making IgE antibody first to one antigen and then to another.

Topics: Acid Phosphatase; Adolescent; Adult; Animals; Antigens; Bee Venoms; Bees; Female; Follow-Up Studies; Humans; Hyaluronoglucosaminidase; Immunoglobulin E; Immunoglobulin G; Insect Bites and Stings; Male; Middle Aged; Phospholipases A; Phospholipases A2

In 8 patients with lymphadenosis benigna cutis (LABC) cytochemical and in 2 of them immunocytological studies have been performed. 1) In patients with LABC we find ectopic organoid proliferations of "lymphfollicle"-like structures within the dermis which predominately consist of small lymphocytes and large reticulum cells. Immunocytological differentiation of the lymphocytes leads to the characterization of B- and T-lymphocytes in a ration 2:1. 2) Large reticulum cells represent a peculiarly remarkable cell class in infiltrates of LABC. Because of their typical arrangement disseminated within the lymphocytic infiltrate they have been designated as "starry sky" cells. Cytochemically they are characterized by an unusual high content of nonspecific esterases and acid phosphatase, most of them show phagocytized basophilic bodies. Because of their shape, arrangement and enzymcytochemical behaviour these cells can be referred to as typical for the LABC disease. 3) Monocytes cannot be found within the "lymphfollicles". Mast cells and connective tissue cells are rarely observed. Polymorphonuclear granulocytes can be demonstrated in great numbers in any part of the involved cutis when there is an insect bite in history. 4) As a reaction of the ectopic proliferation of lymphoreticular tissue within the dermis there is an activation of the surrounding connective tissue with an increase of the alkaline phosphatase activity within these cells, new formation of collagen fibres and strong proliferation of alkaline phosphatase positive capillaries. 5) Etiopathologically it is stressed, that in LABC for example an insect bite induces stimulation of hematopoietic potentialities of undifferentiated mesenchymal germ centres within the cutis takes place, leading to the development of ectopic of "lymphfollicle" like structures.

Topics: Acid Phosphatase; Adult; Aged; Alkaline Phosphatase; Animals; B-Lymphocytes; Capillaries; Child; Child, Preschool; Connective Tissue Cells; Esterases; Humans; Insect Bites and Stings; Lymphadenitis; Mast Cells; Middle Aged; Monocytes; Reticulum; Skin Diseases; T-Lymphocytes

Topics: Acid Phosphatase; Adult; Alanine Transaminase; Alkaline Phosphatase; Aspartate Aminotransferases; Blister; Body Fluids; Cantharidin; Cathepsins; Dermatitis, Contact; Epidermolysis Bullosa; Erythema Multiforme; Glucuronidase; Humans; Insect Bites and Stings; L-Lactate Dehydrogenase; Leukocytes; Male; Pemphigus; Phenolphthaleins; Skin