IgM expressed by leukemic CD5(+) B cells binds mouse immunoglobulin light chain.

Mouse immunoglobulin (Ig) molecules have previously been shown to bind to the surface of CD5(+) B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). The results indicated that surface IgM was involved in the interaction and suggested the phenomenon was an example of the polyreactive binding capacity of the surface Ig (sIg) expressed by these malignant cells. This article describes the further characterization of the interaction between human IgM and mouse Ig molecules and subunits. Mouse Ig molecules of both kappa and lambda light chain classes bound to the B-CLL cell surface. The dissociation constant for the interaction of mouse IgG1 (K121) with the B-CLL cell surface was 3.6 x 10(-7) M. To confirm the involvement of the human IgM expressed by the B-CLL cells in the interaction, the malignant cells were stimulated in vitro to induce secretion of human IgM. Enzyme immunoassay was used to show that secreted human IgM bound to intact mouse Ig, as occurred with the cell surface analysis. The mouse Ig epitope recognized by the purified secreted human IgM was shown by Western blot analysis to be located on the light chain of the mouse Ig molecule and to be conformationally dependent. K121 light chain was cloned and expressed in E. coli and the recombinant light chain bound to the surface of CLL B cells. The results confirm that human IgM is the reactive ligand in the interaction with mouse Ig and indicate that the interaction of polyreactive IgM with mouse IgG occurs via the light chain component of IgG.

Snake inhibitors of phospholipase A(2) enzymes.

Phospholipase A(2) (PLA(2)) enzymes consist of a large family of proteins which share the same enzymatic function and display considerable sequence homology. These enzymes have been identified and characterised in mammalian tissue and snake venoms. Numerous physiological functions have been attributed to mammalian PLA(2)s and they are nontoxic. In comparison, venom PLA(2)s are toxic and induce a variety of pharmacological effects that are probably mediated via membrane receptors. Snake PLA(2) inhibitors (PLIalpha), with a similar structure to the M-type receptor, have been identified as soluble complexes in the serum of viperinae and crotalinae snakes. These inhibitors showed selective binding to crotalid group II PLA(2)s and appeared to be restricted to the serum of this snake family. Analysis of PLA(2) binding to recombinant fragments of PLIalpha indicated that the CRD region was most likely responsible for enzyme inhibition. A second type of inhibitor, PLIbeta, has been identified in serum from one viperid snake and consists of a leucine-rich structure. The third type of inhibitor, PLIgamma, was found in the serum of five snake families and contains a pattern of cysteine residues that define a three-finger structure. PLIgamma inhibitors isolated from the serum of Elapidae, Hydrophidae, Boidae and Colubridae families were able to inhibit a broad range of enzymes including the nontoxic mammalian group IB and IIA PLA(2)s, and bee venom group III PLA(2). However, differences in the binding affinities indicated specificity for particular PLA(2)s. A different representation has emerged for crotalid and viperid snakes. Their PLIgammas did not inhibit bee venom group III, mammalian group IB and IIA enzymes. Furthermore, inhibition data for the gamma-type inhibitor from Crotalus durissus terrificus (CICS) showed that this inhibitor was specific for viperid beta-neurotoxins and did not inhibit beta-neurotoxins from elapids [1]. Further studies are required to determine if this phenomenon is true for all gamma-type inhibitors from Crotalidae snakes. The relative distribution of these inhibitors, their specificities and the structural features involved in binding are discussed in this review.

Antigen binding and cytotoxic properties of a recombinant immunotoxin incorporating the lytic peptide, melittin.

BACKGROUND: The majority of immunotoxins studied to date incorporate toxins that act in the cytosol and thus need to be endocytosed by the target cell. An alternative strategy for immunotoxin development is the use of membrane active toxins, such as the pore-forming proteins. Melittin, a 26 amino acid cytolytic peptide from bee venom, is such a protein. OBJECTIVES: We report here the construction, production and functional analysis of a recombinant immunotoxin obtained by fusion of genes which encode an antibody fragment (scFv) with an oligonucleotide encoding melittin. STUDY DESIGN: The antibody fragment was derived from a murine monoclonal antibody, K121, which recognises a specific epitope (KMA) expressed on the surface of human kappa myeloma and lymphoma cells, and on human free kappa Bence Jones protein (BJP). Melittin is a 26-amino acid, membrane-lytic peptide which is a major component of bee venom. The scFv of K121 was constructed by PCR to link VH and VL genes via an oligonucleotide which encodes a flexible, hydrophilic peptide. An oligonucleotide encoding melittin and the peptide marker sequence FLAG was fused to the scFv construct using a similar linker peptide. The gene construct (scFv-mel) was inserted into the secretion vector pPOW and expressed in Escherichia coli (TOPP2). RESULTS: Expression of the recombinant scFv-mel gene and purification of the protein product was monitored by Western blot analysis. Following purification by anti-FLAG affinity chromatography, the recombinant immunotoxin (scFv-mel) was assessed for antigen binding and for cytotoxic activity by flow cytometry using antigen-expressing and non-expressing cell targets. The scFv-mel was found to exhibit binding and killing properties consistent with the specificity of the original K121 antibody. Moreover, the cytolytic activity of the scFv-mel was significantly greater on a molar basis than that of native melittin alone. CONCLUSION: The data presented here constitute the first report of a melittin-based recombinant immunotoxin and demonstrate that such a membrane active immunotoxin can be synthesised in a bacterial expression. Linking of melittin to an antibody fragment overcame the non-specific toxicity of melittin as the recombinant immunotoxin exhibited specific toxicity towards antigen-bearing target cells. The observation that the immunotoxin exhibited enhanced cytotoxic activity over the free toxin indicates the potential of this approach for the development of an effective therapeutic agent.

Insulin binding to circulating monocytes and erythrocytes in patients with non-insulin-dependent diabetes in the young.

125I-insulin binding to circulating monocytes and erythrocytes was carried out in 9 patients with non-insulin-dependent diabetes in the young (NIDDY), who belonged to families in which non-insulin-dependent diabetes was transmitted through 3 generations. The diabetics had a decreased mean maximum specific binding to monocytes 2.1 (1.1-4.1%) compared to 4.0 (2.6-6.2%) in their age, sex and weight matched reference subjects. This decreased binding was primarily due to a decrease in receptor number as all diabetics had normal affinity values (2-10 ng/ml). We have attributed the decreased binding in NIDDY to the down regulatory effect of the basal hyperinsulinemia (23.6 +/- 3.2 vs 11.7 +/- 0.5 microU/ml). By contrast the maximum specific binding to erythrocyte was similar in both groups (9.7 +/- 0.5; 8.9 +/- 0.5%; p greater than 0.5).