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J K Inman

Publications and source records attributed to J K Inman.

At least 37 records · Page 2Linked to original sources
PubMed1993

Direct detection of major histocompatibility complex class I binding to antigenic peptides using surface plasmon resonance. Peptide immobilization and characterization of binding specificity.

We have developed model systems in which the binding of purified, genetically engineered, soluble analogues of major histocompatibility complex (MHC) class I molecules to immobilized antigenic peptides can be monitored in real time using surface plasmon resonance (SPR). Synthetic analogues of several peptides known to bind different mouse and human MHC class I molecules were prepared with cysteine residues substituted at appropriate positions. The analogue peptides were immobilized via the bifunctional reagent N-gamma-maleimidobutyryloxy-succinimide to amino groups generated on the dextran-modified gold surface of a biosensor flow cell. Using this approach, each position in the sequence of an H-2Ld-specific viral peptide, pMCMV (YPHFMPTNL), was used for coupling, and the resulting surfaces were tested for binding of the soluble analogue of H-2Ld, H-2Lds. In accord with our previously described H-2Ld/pMCMV three-dimensional structural model, only those residues of the peptide that remain exposed following binding (positions 4-8) can be replaced by cysteine and used for coupling. Stable binding of soluble MHC class I molecules, H-2Lds, H-2Dds, H-2Kbs, and HLA-A2s to their respective immobilized cognate peptides was detected by SPR. Specificity of the peptide/MHC interaction was characterized both by direct binding using immobilized peptides and by competition with peptides in solution, and in general was consistent with known immunological reactivity. Some peptides bound not only their cognate MHC molecule, but others at lower apparent affinity. Measurement of real time binding of MHC class I molecules to peptides immobilized through specific side chains suggests the application of a similar approach to the study of the interaction of peptides with a wide variety of peptide-binding macromolecules.

Alleles↗
PubMed1993

Exogenous antigens internalized through transferrin receptors activate CD4+ T cells.

The role of endosomes in exogenous Ag processing was investigated by targeting Ag into the endosomal transport pathway via transferrin receptors. The Ag, pigeon cytochrome c and chicken OVA, were coupled to human ferric transferrin by a heteroligation technique. The conjugates were significantly more efficient than native Ag in stimulating Ag-specific CD4+ T cells, when the APC expressed transferrin receptors. The addition of ferric transferrin eliminated the enhanced response. Paraformaldehyde-fixed APC did not present the conjugates, indicating that the conjugates still required processing to activate T cells. An augmented level of T cell activation was not observed when the APC lacked transferrin receptors or when the conjugate contained the apoenzyme form of transferrin, which does not bind the receptor. The conjugate followed an intracellular pathway similar to that for transferrin, remaining in low density vesicles. Degraded conjugate appeared rapidly in culture supernatants, within 5 min, and peaked by 20 min; under these conditions a T cell response to the conjugate was elicited that was consistent with an early processing compartment. Our results suggest that antigenic peptide fragments can be generated in the early endosomes, without delivery of these Ag to the lysosomes. Thus, various Ag may have differential processing requirements, dictated by their molecular nature, that determine the site of Ag processing.

Animals↗
PubMed1993

N-isopropyliodoacetamide in the reduction and alkylation of proteins: use in microsequence analysis.

A new reagent, N-isopropyliodoacetamide (NIPIA), for alkylation of sulfhydryl groups on proteins for microdigestion and microsequencing is described. The utility of this reagent in both of these procedures has been demonstrated. NIPIA was shown to be especially useful in microsequence analysis, where it yields high sensitivity in detection of Cys residues. This is because the phenylthiohydantoin (PTH) derivative of NIPIA-alkylated cysteine [PTH-Cys(NIPCAM)] appears as a sharp peak in a standard reverse-phase HPLC analysis of PTH amino acids, and elutes between PTH-Tyr and PTH-Pro where no other peaks are present. Thus the use of NIPIA circumvents various problems associated with HPLC analysis of PTH-Cys when other commonly used agents are employed for sulfhydryl alkylation, such as coeluting peaks or low signal levels. Procedures for the synthesis of NIPIA and other analogs, as well as PTH-Cys(NIPCAM), are also presented, and HPLC retention times for their corresponding PTH-Cys derivatives are compared.

Alkylation↗
PubMed1992

Membrane IgM-mediated signaling of human B cells. Effect of increased ligand binding site valency on the affinity and concentration requirements for inducing diverse stages of activation.

The potential for ligand-initiated signal transduction through B cell membrane IgM is assessed in terms of ligand concentration, binding site valency, and binding site affinity for membrane Ig. Estimates of the physicochemical requirements for achieving G0* enhancement of class II MHC expression, G1 entry, and S phase entry in human B cells were made by comparing the stimulatory effects of three affinity-diverse anti-Cmu2 mAb when in bivalent (unconjugated) form, or as mAb-dextran conjugates with low binding site valency (oligovalent ligands) or high binding site valency (multivalent ligands). An increase in binding site number (and concomitant molecular mass) caused a profound reduction in both the minimal concentration and affinity requisites for B cell activation. The enhancing effect of increased binding site valency was most evident for the signaling of those most distal stages in B cell activation, i.e., G1 and S phase, which were difficult to induce with bivalent ligands. The results suggest that highly multimeric TI-2 Ag may be good immunogens because they are able to elicit a full activation response not only from infrequent high affinity B cells, but also from a substantial proportion of the many lower affinity Ag-specific B cells in virgin B cell populations. Interestingly, the activation of B cells by ligands with binding sites of high intrinsic affinity (Ka = 5 x 10(8) M-1) was less influenced by increases in binding site valency than was B cell activation by ligands with intermediate binding site affinity (Ka = 2 x 10(7) M-1). This suggests that the minimal epitope valency requirement for T cell-independent B cell activation by mIg cross-linking Ag may be dependent on the intrinsic affinity with which membrane Ig molecules on a given B cell interact with the redundantly expressed epitopes.

B-Lymphocytes↗
PubMed1990

Rapid stimulation of large specific antibody responses with conjugates of antigen and anti-IgD antibody.

Injection of mice with goat anti-mouse IgD antibody stimulates a large IgG1 anti-goat IgG antibody response, as well as polyclonal IgG1 production. To determine if this phenomenon could be used to induce large antibody responses to other Ag, covalent conjugates were produced between BSA or other Ag and H delta a/1, a mAb specific for IgD of the a allotype, and between BSA and AF3.33, a mAb specific for IgD of the b allotype. Injection of H delta a/1-BSA into BALB/c mice, which express Ig of the a allotype, or into (BALB/c x CB20)F1 mice (a x b allotype heterozygotes) induced IgG1 anti-BSA antibody responses that peaked 8 to 9 days after injection, and were more than 1000 times larger than those induced by injection of BSA alone, and 100 times larger than those induced by injecting unconjugated BSA plus H delta a/1. H delta a/1-BSA was no more immunogenic than unconjugated BSA when injected into CB20 mice, which express Ig of the b allotype, while AF3.33-BSA greatly enhanced anti-BSA antibody production in CB20, but not in BALB/c mice. Mice serially immunized with three different Ag conjugated to H delta a/1 made large antibody responses to all three Ag, provided that the mouse strain used did not recognize allotypic determinants on H delta a/1 as foreign and produce a neutralizing antibody response. Intravenous and s.c. routes of inoculation produced responses of similar magnitude and relatively low variability; responses to footpad or intramuscular inoculation were more variable, and i.p. inoculation induced smaller responses. Injection of BALB/c mice i.v. with 100 micrograms of H delta a/1-BSA induced an IgG1 anti-BSA response of 5.6 mg/ml, which was approximately 70% of the total IgG1 response. Anti-BSA responses to 30 micrograms of conjugate or less were much smaller, but could be considerably enhanced by adding unconjugated H delta a/1 to the inoculum. This system will be useful for the rapid stimulation of large antibody responses to biologically important Ag, and for investigating mechanisms of Ag processing and B and T cell activation.

Animals↗
PubMed1990

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.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1989

Surface immunoglobulin-mediated B-cell activation in the absence of detectable elevations in intracellular ionized calcium: a model for T-cell-independent B-cell activation.

We recently showed that anti-immunoglobulin conjugated to high molecular weight dextran is 1000-fold more mitogenic for B cells than unconjugated anti-immunoglobulin. This system serves as a model for T-cell-independent type 2 antigens such as haptenated Ficoll, dextran, and bacterial polysaccharides, which can also stimulate B-cell proliferation and antibody production at low concentrations. We show here that conjugated anti-immunoglobulin, at concentrations that stimulate significant increases in expression of major histocompatibility complex class II molecules and incorporation of thymidine into DNA, does not induce detectable modulation of surface immunoglobulin. These results indicate that the facilitated T-cell-independent B-cell activation by polysaccharide antigens may result from inability to modulate surface immunoglobulin, possibly resulting in persistent and/or repetitive signaling. Early large increases in Ca2+ and breakdown of inositol phospholipids presently thought to be involved in transduction of the mitogenic signal are not detectable at low concentrations of conjugated anti-immunoglobulin, raising the possibility that these biochemical events may not in fact be central to this signaling pathway.

Animals↗
PubMed1988

Picogram quantities of anti-Ig antibodies coupled to dextran induce B cell proliferation.

To investigate the properties which enable type 2 Ag, as exemplified by dextran and Ficoll, to stimulate high levels of antibody responses in the relative absence of T cells, we conjugated anti-IgD and anti-IgM mAb to both dextran and Ficoll and examined their B cell-activating properties. Such conjugated anti-Ig antibodies stimulated both early and later stages of B cell activation at picogram concentrations, which are at least 1000-fold lower than that required for B cell stimulation by unconjugated anti-Ig antibodies, and the level of proliferation they stimulated was on average 10-fold greater. Furthermore, concentrations of anti-Ig dextran (100 pg/ml) which modulated little sIgD from the B cell surface were strong inducers of enhanced B cell expression of MHC class II molecules. Conjugation of Fab fragments of anti-IgD or nonmitogenic anti-IgM mAb to dextran rendered them as mitogenic as dextran conjugated to strongly stimulatory anti-IgD or anti-IgM antibodies. The ability of dextran and Ficoll to serve as effective carrier molecules for anti-IgD was not related solely to their large m.w., because anti-IgD coupled to polymerized BSA (m.w. 1.5 X 10(6), was only 10- to 50-fold more potent than unconjugated anti-IgD antibodies at stimulating B cell DNA synthesis. These results suggest, therefore, that the unique ability of picogram concentrations of haptenated type 2 Ag to stimulate Ig secretion in the absence of T cells may be a function of their ability to promote effective cross-linking without resulting in the modulation of sIg. This would enable such Ag to mediate repetitive B cell signaling, a situation that cannot be achieved by unconjugated anti-Ig antibodies which result in modulation of sIg at their mitogenic concentrations. These compounds therefore may be employed to study B cell activation stimulated by sIg cross-linking at concentrations that may more closely reflect those which are achieved under physiologic conditions by type 2 Ag.

Animals↗
PubMed1986

Controlled primary functionalization of agarose affinity supports by carboxymethylation and subsequent addition of spacer units.

Experimental details are given for the O-carboxymethylation of cross-linked agarose beads with chloroacetate in aqueous sodium hydroxide, formation of a tandem 2-amino-ethylamide derivative, and coupling of beta-alanine residues to the aminoethyl groups using a new reagent, N-phthalimidyl N'-ethylsulfonylethoxycarbonyl-beta-alaninate. An ideal support for attaching affinity ligands results. Extent of derivatization was controlled by the time and temperature of the first reaction. Functional group densities were estimated by using hydrogen ion binding measurements for carboxyls and a new reagent, N-succinimidyl 3-[2-(4-nitrophenylamino)ethyldithio]propionate, for assaying reactive amino groups on solid supports. Synthesis and use of the new reagents are described.

Chemical Phenomena↗
PubMed1984

Hapten-specific B cell blockade of the immune response to a thymus-independent-1 antigen produced by concomitant administration of a thymus-independent-2 antigen.

CBA/N mice harbour an X-linked B cell defect which is transmitted by CBA/N female mice to their hybrid male progeny. These mice mount normal responses to thymus-dependent (TD) and some thymus-independent (TI-1) antigens, while the response to TI-2 antigens is absent. Hapten-specific plaque-forming cell (PFC) responses to TD antigens can be blockaded by concomitant exposure of these mice to TI-2 antigens bearing the same hapten. This paper investigates in defective mice the blockade of their response to TNP3-LPS (trinitrophenylated lipopolysaccharide, a TI-1 antigen), imposed by DNP59-Ficoll (dinitrophenylated Ficoll, a TI-2 antigen). The effectiveness of the blocking agent, DNP59-Ficoll, differed in various inbred mouse strains: CBA/N X C3H/HeN F1 male greater than CBA/N female greater than CBA/N X C3H/HeN F1 female. The role of T cells in the observed hapten-specific blockade phenomenon was investigated using athymic CBA/N nude mice and a B cell tolerogen. Our findings indicate that T cell participation is not essential for the blockade of CBA/N PFC responses and they suggest that direct blockade of TI- and TD-responsive B cell populations is likely to occur.

Animals↗
PubMed1983

Amidination.

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Amidines↗
PubMed1982

Functional antigen binding by the defective B cells of CBA/N mice.

CBA/N mice have an X-linked B cell defect which prevents them from responding to nonmitogenic thymic independent (TI-2) antigens such as dinitrophenylated DNP-Ficoll (1,2). The F1 male progeny of CBA/N female mice express the same defect. Spleen cell suspensions from such defective mice (CBA/N X C3H/HeN F1 males) could not respond to DNP-Ficoll following in vitro immunization and subsequent transfer into irradiated, syngeneic, F1 male recipients as expected. In contrast, normal CBA/N X C3H/HeN F1 female spleen cells could respond and effect a "rescue"; they mounted strong plaque-forming cell responses 7 days after in vitro exposure to DNP-Ficoll and subsequent transfer into irradiated F1 male recipients. Defective F1 male spleen cells, however, could bind significant quantities of 125I-DNP-Ficoll after in vitro exposure. Extensive washing of these spleen cells could not reverse this binding. Such DNP-Ficoll-exposed and washed F1 male spleen cells could, after transfer, aid normal untreated F1 female cells in their rescue function. The defective F1 male spleen cells could convey immunogenic quantities of DNP-Ficoll to the "rescuing" F1 female cells. Mitomycin treatment of F1 male cells did not interfere with their conveyor function. Goat anti-mouse mu serum impeded the passive antigen conveyor function of defective F1 male cells as did prior exposure to high concentrations of free DNP hapten. Our data support the view that the B cell defect of CBA/N X C3H/HeN F1 male mice does not relate to antigen binding, but rather to an inability to be effectively triggered by certain cell-bound polymeric antigens.

Animals↗
PubMed1981

Rapid removal of acetimidoyl groups from proteins and peptides. Applications to primary structure determination.

Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies.

Amino Acid Sequence↗
PubMed1981

Parallel cross-reactivity patterns of 2 sets of antigenically distinct cytochrome c peptides: possible evidence for a presentational model of Ir gene function.

B10.A mice were immunized with either the carboxyl terminal peptide fragment 81-104 of pigeon cytochrome c or its acetimidyl derivative and an immune response was seen with strong preference for the immunogen. Strain distribution studies and blocking with an anti-Ia monoclonal antibody indicated that the same immune response (Ir) gene and restriction element were utilized in both responses. The specificity of the responses were evaluated by restimulating in vitro with a set of cytochrome c fragments from various species. Even though the derivatized and native fragments were poorly cross-reactive, the same phylogenetic pattern was seen when pigeon cytochrome c fragment 81-104 primed cells were tested with the set of underivatized fragments and when acetimidyl pigeon cytochrome c fragment 81-104 primed cells were tested with the same set of derivatized fragments. Primed cells from a 2nd major histocompatibility complex congenic strain of mice, B10.A(5R), displayed equivalent discrimination between derivatized and native forms but showed a markedly different phylogenetic pattern of cross-reactivity. These data indicate that the immune system recognizes 2 sites on the nominal antigen. One site, which accounts for the common hierarchy and is under Ir gene control, contains residues Gln-100, and possibly other carboxyl terminal residues. The 2nd site, which effects the distinction between native and derivatized fragments, contains at least 1 lysine other than at the carboxyl terminal. The implications of these data for theories of T cell recognition and Ir gene function are discussed.

Animals↗
PubMed1981

Immune memory to a nonmitogenic, thymic independent antigen in mice: variation among inbred strains and possible relationship to oncogenesis.

Immune memory to DNP-Ficoll, a nonmitogenic, thymic-independent (TI-2) antigen, was demonstrated in several inbred strains of mice. Direct and indirect splenic plaque-forming cell responses were measured in mice given a secondary challenge with DNP-Ficoll and in appropriate control mice. Strong IgM memory, but no IgG memory, was observed in SJL/J, AKR/J, and C58/J mice. C57L/J mice gave both a strong IgM memory response and a relatively strong IgG memory response to DNP-Ficoll. C57BL/6N, C57BR/cdJ, and A/HeJ mice were unable to mount significant IgM or IgG memory responses to this antigen under an identical immunization schedule. These results are indicative of marked genetic variation in mice in the capacity for B memory cell expression. They also identify a provocative but unexplained association between positive IgM memory responses to DNP-Ficoll in SJL/J, AKR/J, and C58/J mice and a propensity to develop lymphoreticular neoplasia. This association is observed when there is no clear evidence for IgG memory.

Animals↗