Identification of sites on the human Fc epsilon RI alpha subunit which are involved in binding human and rat IgE.

The high affinity IgE Fc receptor (Fc epsilon RI), found on mast cells and basophils, is a tetrameric receptor complex. The extracellular portion of the Fc epsilon RI alpha subunit consists of two immunoglobulin-like domains and binds IgE in the absence of the other subunits. To localize the high affinity IgE binding site within the Fc epsilon RI alpha subunit, we generated a series of chimeric receptor constructs where one of the two immunoglobulin-like domains was either deleted or substituted with those from the human Fc gamma RIIIA alpha or the rat Fc epsilon RI alpha subunit.

Humanized Mik beta 1, a humanized antibody to the IL-2 receptor beta-chain that acts synergistically with humanized anti-TAC.

Mik beta 1 is a mouse mAb directed at the beta-subunit of the human IL-2R (Tac) that inhibits IL-2 binding and inhibits IL-2 induction of large granular lymphocytes (LGL). Mik beta 1 alone does not inhibit IL-2-induced T-cell proliferation, but acts synergistically with anti-Tac to inhibit IL-2-induced proliferation of activated T cells. To evaluate these effects for possible therapy in humans, we constructed two humanized Mik beta 1 antibodies by combining the complementarity-determining regions of the murine antibody with human framework and constant regions.

Purification and characterization of human recombinant IgE-Fc fragments that bind to the human high affinity IgE receptor.

The Fc-region of immunoglobulin E (IgE) comprising C epsilon 2, C epsilon 3, and C epsilon 4 domains is sufficient for binding to the alpha chain of the high affinity IgE-Fc receptor (Fc epsilon RI alpha). In order to identify the smallest Fc fragment capable of binding to the Fc epsilon RI alpha with high affinity, various regions of the IgE-Fc molecule were expressed in COS cells and investigated for their ability to bind Fc epsilon RI alpha. The smallest fragment that showed Fc epsilon RI alpha binding activity spans amino acids 329-547 and lacks the entire C epsilon 2 domain.

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex.

Human interleukin-2 anti-idiotypes.

We describe the generation of a monoclonal anti-idiotypic (anti-id) antibody directed against affinity purified rabbit antibodies (id) to recombinant human IL-2. This monoclonal antibody, 6A12, has the ability to inhibit the neutralization of IL-2 activity by the id, suggesting that it may bind at or near the IL-2 neutralizing site on the id. 6A12 exhibits IL-2 agonist activity on PHA-activated human T cells.

Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor.

Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70). Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit.

Structure-function analysis of human interleukin-2. Identification of amino acid residues required for biological activity.

To locate functional domains of the interleukin-2 (IL-2) protein, a cDNA clone encoding biologically active human IL-2 was mutagenized using synthetic oligonucleotides to incorporate defined amino acid substitutions and deletions in the mature protein. The IL-2 analogs were then produced in Escherichia coli and assayed for the ability to induce proliferation of IL-2-dependent cells and the ability to compete for binding to the IL-2 receptor. Our analysis of over 50 different mutations demonstrated that the integrity of at least three regions of the IL-2 molecule is required for full biological activity: the NH2 terminus (residues 1-20), the COOH terminus (residues 121-133), and 2 of the 3 cysteine residues (58 and 105).

The lack of correlation between inhibition of aggregation and cAMP levels with canine platelets.

Arachidonic acid (AA) induced aggregation of canine platelets can be inhibited by various phosphodiesterase inhibitors (PDIs) with the order of potency IBMX greater than or equal to papaverine greater than Ro 20-1724 greater than theophylline. With aggregation induced by AA plus epinephrine (EPI), only IBMX and papaverine inhibited at 100 microM. None of these PDIs affected the basal cAMP levels but all potentiated the PGE1-stimulated cAMP production, with the order of potency being Ro 20-1724 greater than papaverine greater than IBMX greater than theophylline.

The elevation of mouse thymus cell cyclic adenosine monophosphate (cAMP) by lipopolysaccharide.

The ability of certain lipopolysaccharide (LPS) preparations to elevate cyclic adenosine monophosphate (cAMP) in mouse thymus cells in the presence of Ro 20-1724, 4-(3-butoxyl 4-methoxybenzyl)-2-imidazolidinone, was not related to the source of supply, bacterial strain, or method of extraction. Under the same conditions adenosine is a potent stimulator of thymus cell cAMP and is, of course, blocked by the further addition of theophylline. When theophylline was added to the LPS preparations with Ro 20-1724, the cAMP production was also blocked.

The regulation of nucleotide metabolism of immune cells: papaverine induced nucleotide breakdown.

During a period of prelabeling of mouse thymus cells with any nucleoside at 4 degrees C, nucleoside phosphates accumulated, but no nucleic acid synthesis occurred. Elevating the temperature to 37 degrees C then led to incorporation into the respective nucleic acid reaching a maximum in 5--15 min. Papaverine inhibited this incorporation (IC50:50 muM) and caused an efflux of label into the medium as a nonphosphorylated product.

Effects of ascorbic acid on 3H-thymidine incorporation by isolated mouse thymocytes.

The 15-min incorporation of 3H-thymidine (Tdr) and 3H-uridine (Ur) into nucleic acids of freshly isolated mouse thymocytes decreased steadily with time of preincubation at 37 degrees C. Sodium ascorbate at 5 mM prevented the decline of 3H-Tdr incorporation by preventing the decrease in its uptake. No such effect was noted on the incorporation of 3H-Ur, suggesting that ascorbate might be more specific for cells in or near the S phase of the cell cycle.

Alterations in the hydrolytic activity, inhibitor sensitivity and molecular size of the rat erythrocyte cyclic AMP phosphodiesterase by calcium and hypertonic sodium chloride.

Hypertonic NaCl irreversibly reduced the sensitivity of the rat erythrocyte cyclic AMP phosphodiesterase to inhibition by Ro 20-1724 but not by papavarine. This effect could be prevented by 1 mM EDTA and mimicked by by CaCl2. The reduction in inhibitor sensitivity was associated with an increase in hydrolytic activity and a four-fold reduction in molecular weight as estimated by Sephadex G-200 chromatography.