John K. Inman

IL-12 Induction by a Th1-Inducing Adjuvant In Vivo: Dendritic Cell Subsets and Regulation by IL-10

IL-12 induction is critical for immune responses against many viruses and intracellular bacterial pathogens. Recent studies suggest that IL-12-secreting dendritic cells (DC) are potent Th1-inducing APC. However, controversy exists concerning the function of DC subsets. Murine studies have suggested that CD8+ DC preferentially induce Th1 responses, whereas CD8− DC induce Th2 development; in this model, different DC subsets prime different responses. Alternatively, the propensity of DC subsets to prime a Th1 response could depend upon the type of initial stimulus. We used a prototypic Th1-inducing adjuvant, heat-killed Brucella abortus (HKBA) to assess stimulation of DC subsets, relationship between Ag burden and IL-12 production, and down-regulation of DC subset IL-12 production by IL-10. In this study, we show that DC were sole producers of IL-12, although most HKBA uptake was by splenic macrophages and granulocytes. More CD8− than CD8+ DC produced IL-12 after HKBA challenge, whereas only CD8+ DC produced IL-12 after injection of another Th1-promoting microbial substance, soluble Toxoplasma gondii Ags. Studies in IL-10-deficient mice revealed that IL-10 down-regulates frequency and duration of IL-12 production by both DC subsets. In the absence of IL-10, IL-12 expression is enabled in CD11clow cells, but not in macrophages or granulocytes. These findings support the concept of DC as the major IL-12 producers in spleens, but challenge the notion that CD8+ and CD8− DC are destined to selectively induce Th1 or Th2 responses, respectively. Thus, the nature of the stimulating substance is important in determining which DC subsets are activated to produce IL-12.

Benzamide-based thiolcarbamates: a new class of HIV-1 NCp7 inhibitors.

The HIV-1 nucleocapsid protein NCp7, which contains two highly conserved zinc fingers, is being used as a novel target for AIDS therapy due to its pivotal role in viral replication and its mutationally intolerant nature. Herein we report a new class of NCp7 inhibitors that possess good antiviral activity with low cellular toxicity.

Coupling of large haptens to proteins and cell surfaces: Preparation of stable, optimally sensitized erhythrocytes for hapten-specific, hemolytic plaque assays

Abstract Procedures are described for converting tertiary-butyloxycarbonyl (Boc) hydrazides of large haptenic structures to reactive acyl azides and for using these intermediates in the preparation of a sereis of highly specific immunochemical reagents. The synthesis of the initial C-terminal Boc hydrazides, which are tripeptide derivatives, is described in a companion report (Inman et al. (1973) Immunochemistry10, 153). The converted azides react readily and specifically with amino groups in mildly alkaline, aqueous media and thereby provide an excellent means for covalently coupling haptens to immunogen carriers, proteins, or cells. The azide groups have relatively uniform reactivity due to an adjacent, common, glycylglycyl structure. Thus, it has been possible to prepare groups of immunochemical reagents, representing numerous specificities, by use of a single convenient procedure. Erythrocytes bound with large haptens by this method have functioned as highly stable and sensitive indicator cells in hapten-specific hemolytic plaque and hemagglutination assays. Conditions for preparing optimally sensitized erythrocytes for quantitative studies of hapten-specific plaque forming cells have been studies. Maximum numbers of plaque forming cells were detected when about 300,000 hapten molecules were bound per indicator erythrocyte. Plaque counts decreased progressively when indicator red cells had been conjugated with appreciably more or less than this amount of hapten. It has not been previously recognized that excessively hapten derivatization of erythrocytes regularly and profoundly impedes their susceptibility to specific immune lysis. Estimates have been made of the molecular circumstances which may explain this phenomenon. Its generaly implications for complement mediated cytolysis are discussed.

Enhanced immunogenicity of protein-dextran conjugates: I. Rapid stimulation of enhanced antibody responses to poorly immunogenic molecules.

In view of our observation that anti-immunoglobulin antibody conjugated to high-molecular-weight dextran stimulates high levels of B-cell activation (Brunswick et al. J. Immunol. 1989, 143, 1239), we coupled T cell-dependent antigens to dextran. When mice were immunized, in the absence of adjuvant, with a BSA-dextran conjugate (BSA-dex), a persistent, high-titre anti-BSA IgG1 response was induced. Titres were dose-dependent and seen with as little as 10 micrograms of conjugated protein. Anti-BSA titres were detected as early as day 7, usually peaked at about day 14 and persisted for at least 4 weeks. Anti-hapten antibodies were also elicited in mice that were immunized with haptenated BSA covalently bound to dextran, and secondary responses could be induced even after inoculation of the unconjugated protein. Covalent attachment of the protein to the polymer was necessary, and the response was specific, as coinjection of BSA-dex and an unrelated antigen, goat IgG, did not elicit detectable anti-goat antibodies. The immunogenic potential of these conjugates did not depend on the ability of the dextran carrier to induce antibody, inasmuch as they stimulated high levels of anti-protein antibody in mice unresponsive to dextran. A minimum size dextran polymer was required for enhanced immunogenicity as conjugates of BSA with dextran of molecular mass 500 or 2000 kDa but not of 70 kDa gave detectable anti-BSA titres.

APRIL and BAFF Promote Increased Viability of Replicating Human B2 Cells via Mechanism Involving Cyclooxygenase 2

Of relevance to both protective and pathogenic responses to Ag is the recent finding that soluble molecules of the innate immune system, i.e., IL-4, B cell-activation factor of the TNF family (BAFF), and C3, exhibit significant synergy in promoting the clonal expansion of human B2 cells following low-level BCR ligation. Although IL-4, BAFF, and C3dg each contribute to early cell cycle entry and progression to S phase, only BAFF promotes later sustained viability of progeny needed for continued cycling. The present study sought to further clarify the mechanisms for BAFF’s multiple functions. By comparing BAFF and a proliferation-inducing ligand (APRIL) efficacy at different stages in the response (only BAFF binds BR3; both bind transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation Ag, the early role was attributed to BR3, while the later role was attributed to TACI/B cell maturation Ag. Importantly, BAFF- and APRIL-promoted viability of cycling lymphoblasts was associated with sustained expression of cyclooxygenase 2 (COX-2), the rate-limiting enzyme for PGE2 synthesis, within replicating cells. Supernatants of cultures with BAFF and APRIL contained elevated PGE2. Although COX-2 inhibitors diminished daughter cell viability, exogenous PGE2 (1–1000 nM) increased the viability and recovery of lymphoblasts. Increased yield of viable progeny was associated with elevated Mcl-1, suggesting that a BAFF/APRIL → TACI → COX-2 → PGE2 → Mcl-1 pathway reduces activation-related, mitochondrial apoptosis in replicating human B2 cell clones.

Small-molecule inactivation of HIV-1 NCp7 by repetitive intracellular acyl transfer

The zinc fingers of the HIV-1 nucleocapsid protein, NCp7, are prime targets for antiretroviral therapeutics. Here we show that S-acyl-2-mercaptobenzamide thioester (SAMT) chemotypes inhibit HIV by modifying the NCp7 region of Gag in infected cells, thereby blocking Gag processing and reducing infectivity. The thiol produced by SAMT reaction with NCp7 is acetylated by cellular enzymes to regenerate active SAMTs via a recycling mechanism unique among small-molecule inhibitors of HIV.

A deeply recessed active site in angiotensin-converting enzyme is indicated from the binding characteristics of biotin-spacer-inhibitor reagents

Two biotinylated derivatives of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, were synthesized. Compounds BL11 (epsilon-biotinamidocaproyl-lisinopril) and BL19 (epsilon-biotinamidocaproyl-beta-alanyl-beta-alanyl-lisinopril) have, respectively, 11 and 19 atoms of spacing structure between the biotin and the inhibitor moieties. Both compounds were found to be potent inhibitors of mouse kidney ACE, but they lost this ability in the presence of streptavidin in free solution. However, BL19 (but not BL11), when complexed to ACE, retained enough residual binding strength for streptavidin to allow the complex to be specifically removed from solution by streptavidin-agarose beads. It was thus possible to employ BL19 for the affinity isolation of ACE from crude mixtures. These results indicate that the bound determinant of lisinopril must lie at least 11 A below the outer surface of the ACE molecule.

In vivo antiviral activity of novel human immunodeficiency virus type 1 nucleocapsid p7 zinc finger inhibitors in a transgenic murine model.

Control of human immunodeficiency virus through the use of inexpensive chemotherapeutics, with minimal side effects and decreased potential for engendering resistant virus, is a long-term therapeutic goal. In principle, this goal can be accomplished if viral replication in reservoirs of chronically and latently infected cells is addressed. As a first step, we have developed novel antiviral compounds based on a 2-mercaptobenzamide thioester chemotype, including the pyridinioalkanoyl thioesters, which specifically target the zinc fingers of the human immunodeficiency virus nucleocapsid protein (NCp7). Using these compounds in a murine transgenic model, in which infectious human immunodeficiency virus is induced from an integrated provirus, we show inhibition of transgenic spleen cell p24 expression with potencies comparable to acute infection assays using human peripheral blood lymphocytes. More importantly, transgenic mice treated in vivo with two 2-mercaptobenzamide thioesters expressed significantly lower plasma p24, and splenocytes from these animals produced fewer infectious virions. Thus, these thioesters may provide an effective means for inhibiting the expression of human immunodeficiency virus from integrated viral reservoirs.

Synthesis of large haptenic compounds having a common functional group that permits covalent linkage to proteins, cell surfaces, and adsorbents.

Abstract Details are given for the synthesis and characterization of two series of tripeptide derivatives that were prepared by joining various, aromatic-ring-based structures to an N-terminal, N-acetyl- l -tyrosyl or β-alanyl residue through an azo or amide linkage, respectively. The remainder of the tripeptide derivative was, in all cases, glycyglycine tertiary-butyloxycarbonyl hydrazide. The latter, C-terminal feature allows these compounds to be stored in a stable form and to be converted readily to reactive acyl azides by methods commonly employed in peptide synthesis. The azides provide a suitable means for covalently linking the derivatized tripeptides to amino groups on carriers and cell surfaces under mild conditions in aqueous media (see Inman et al. , 1973). The linked groups serve very effectively as enlarged or spaced-out haptens in studies of immunological specificity. The design and advantageous features of these compounds and their corresponding haptenic groups are discussed.

Demonstration of a simple method for reducing losses of tryptic peptides during automated sequencing.

Abstract Braunitzer, Schrank and Ruhfus (Hoppe-Seylers Z. Physiol. Chem., 351 , 1589 (1970)) reported improvement in yields of amino acid phenylthio-hydantoins from the automated sequencing of a lysine-containing peptide initially treated with 4-sulfophenylisothiocyanate (SPITC). Using this approach we obtained essentially complete sequences for six peptides, containing between 5 and 26 amino acid residues, which C-terminate in lysine or aminoethylcysteine. We obtained only limited sequence information on the same peptides when they had not been pretreated with SPITC. Equally satisfactory results were obtained with the acid form or the sodium salt of SPITC which were prepared by the method reported here. A volatile buffer system was used successfully in place of Quadrol.