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A A Horner

Publications and source records attributed to A A Horner.

At least 19 recordsLinked to original sources
PubMed1998

Immunostimulatory DNA is a potent mucosal adjuvant.

Most proteins delivered to mucosal surfaces fail to induce mucosal or systemic immune responses. We demonstrate that a single intranasal (i.n.) coadministration of a model antigen (beta-galactosidase, beta-gal) with immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) induces a mucosal IgA response equivalent to that induced by i.n. codelivery of beta-gal with cholera toxin (CT). Furthermore, i.n. and intradermal (i.d.) delivery of the beta-gal/ISS-ODN mix stimulates equivalent Th1-biased systemic immune responses with high-level cytotoxic T lymphocyte (CTL) activity. In contrast, i.n. immunization with beta-gal and CT results in a Th2-biased systemic immune response with poor CTL activity. Our data show that i.n. delivery of ISS-ODN provides effective adjuvant activity for the induction of both mucosal and systemic Th1-biased immune responses. This immunization approach deserves consideration in the development of vaccines against mucosal pathogens.

Adjuvants, Immunologic↗
PubMed1995

Effects of aging on the synthesis of antithrombin-binding sites on heparin chains and heparan sulphate chains in the rat.

[35S]Heparin proteoglycans were isolated from the skins and peritoneal mast cells of male rats aged 2 to 22 months. Their [35S]heparin chains were separated on antithrombin-agarose into fractions with high and low affinities for antithrombin. In skin, the proportion of 35S-labelled high-affinity heparin chains declined from 23% at 2 months to 8% at 12 months and did not change significantly between 12 and 22 months. In peritoneal mast cells, the proportion of 35S-labelled high-affinity heparin chains increased from 14% at 2 months to 21% at 4 months and then did not vary significantly until 15 months of age. By 21 months a consistent and significant decline to 8% occurred. The structures of high-affinity heparin proteoglycans did not change with age. Their decreased proportions, without change in their structure, may indicate that they are produced by a unique subset of mast cells, the proportion of which declines with age. [35S]heparan sulphate chains were isolated from skins and brains of rats in the same age range and fractionated on antithrombin-agarose. There were no significant variations in the proportions of 35S-labelled high-affinity heparan sulphate chains in skin (10%) or brain (24%) between 4 and 22 months of age.

Aging↗
PubMed1995

gamma/delta T lymphocytes express CD40 ligand and induce isotype switching in B lymphocytes.

T cells expressing gamma/delta T cell receptors home to epithelial tissue and may play a role in immunity to infectious agents and foreign antigens. In an effort to understand the role of gamma/delta T cells in directing B cell responses, we investigated the capacity of human gamma/delta T cells to express CD40 ligand (CD40L) and to drive immunoglobulin (Ig) isotype switching in B cells. A multiple step purification procedure resulted in the recovery of highly pure populations of peripheral blood CD4-CD8- gamma/delta T cells. Neither CD40L surface expression nor CD40L mRNA were detected in unstimulated gamma/delta T cells. Stimulation with phorbol ester and ionomycin induced CD40L mRNA and surface CD40L expression by gamma/delta T cells. Both the percentage of CD40L+ cells and the cell surface density of CD40L were significantly lower in gamma/delta T cells compared to unselected T cells. We further demonstrated that in the presence of neutralizing monoclonal antibody to interferon gamma (IFN-gamma), gamma/delta T cells could induce IgE synthesis in B cells, albeit to a lesser extent than unselected T cells. Furthermore, IgE synthesis driven by gamma/delta T cells was inhibited by monoclonal antibody to CD40L. These observations demonstrate that activated gamma/delta T cells express CD40L and can induce isotype switching in B cells.

Adult↗
PubMed1991

Heterogeneous distribution of antithrombin-binding sites in rat brain heparan sulphate proteoglycans.

Heparan sulphates with high binding affinity for antithrombin (HA-HS), labelled in vivo with [35S]sulphate, were extracted from rat brains and purified by chromatography on DEAE-cellulose and on antithrombin-agarose. HA-HS proteoglycans (HA-HSPG) were then separated from HA-HS chains on Sepharose CL-6B. The total HA-HSPG product was rechromatographed on antithrombin-agarose. Six HA-HSPG subfractions with differing degrees of affinity for antithrombin were recovered and treated with NaOH to release their chains. Rechromatography of these six 35S-labelled HS chain preparations on antithrombin-agarose showed that their proportions of chains with no affinity for antithrombin (NA-HS chains) ranged from 36 to 71%. There was a reciprocal relationship between the proportion of NA-HS chains in each HA-HSPG subfraction and the degree of affinity for antithrombin of the rest of its chains (assessed relative to 3H-labelled HA-heparin chains with which they were co-chromatographed). Similar characteristics of antithrombin-binding-site distribution apply to HA-heparin proteoglycans from rat skin studied previously [Horner (1987) Biochem. J. 244, 693-698]. The data suggest that the sites at which 3-O-sulphation of some glucosamine N-sulphate residues occurs in the Golgi complex of brain cells (probably endothelial cells) which synthesize HA-HSPGs (as in mast cells, which synthesize HA-heparin PGs) are distributed sparsely but not randomly.

Animals↗
PubMed1990

Rat heparan sulphates. A study of the antithrombin-binding properties of heparan sulphate chains from rat adipose tissue, brain, carcase, heart, intestine, kidneys, liver, lungs, skin and spleen.

Adult male rats were given [35S]sulphate intraperitoneally. Heparan [35S]sulphate (HS) chains were recovered from adipose tissue, brain, carcase, heart, intestine, kidneys, liver, lungs, skin and spleen by digestion with Pronase, precipitation with cetylpyridinium chloride, digestion with chondroitin ABC lyase and DNAase and gradient elution from DEAE-Sephacel. Purity was confirmed by agarose-gel electrophoresis and degradation with HNO2. Fractionation by gradient elution from antithrombin-agarose indicated that the proportion of HS with high binding affinity for antithrombin (HA-HS) ranged from 4.7% (kidneys) to 21.5% (brain). On a mass basis the major sources of HA-HS were carcase, skin and intestine. HA-HS from intestine was arbitrarily divided into subfractions I-VI, with anticoagulant activities ranging from 1 to 60 units/mg [by amidolytic anti-(Factor IIa) assay] and from 4 to 98 units/mg [by amidolytic anti-(Factor Xa) assay], indicating that the antithrombin-binding-site densities of HA-HS chains covered a wide range, as shown previously for rat HA-heparin chains [Horner, Kusche, Lindahl & Peterson (1988) Biochem. J. 251, 141-145]. HA-HS subfractions II, IV and VI were mixed with samples of HA-[3H]heparin chains and rechromatographed on antithrombin-agarose. Affinity for matrix-bound antithrombin did not correlate with anticoagulant activity, e.g. HA-HS subfraction IV [38 anti-(Factor Xa) units/mg] was co-eluted with HA-heparin chains [127 anti-(Factor Xa) units/mg].

Adipose Tissue↗
PubMed1989

Molecular-size-dependent variations in the proportions of chains with high binding affinities for antithrombin in rat skin heparin proteoglycans.

Approximately half of all rat skin heparin proteoglycans have polysaccharide chains that have no sites with high binding affinity for antithrombin. The rest have chains with high-affinity antithrombin-binding-site densities ranging from zero to five sites per chain, with a high degree of variation. Proteoglycans vary in size because of diversity in the number of chains per molecule; the relationship between proteoglycan size and high-affinity antithrombin-binding-site density has not been studied previously. Polydisperse heparin proteoglycans from rat skin, labelled biosynthetically with 35S, were fractionated by gel filtration on Bio-Gel A-150m and arbitrarily divided into five fractions of decreasing average molecular size. Fractionation of these products on antithrombin-agarose showed that the proportion of proteoglycans with high affinity for antithrombin decreased from 39% to 25% as molecular size decreased. However, as the molecular size of high-affinity proteoglycans decreased, the proportion of their chains that had high affinity increased from 29% to 59%. Therefore molecular size is a significant factor in determining the proportion of high-affinity chains in heparin proteoglycans. A model of heparin biosynthesis is proposed in which areas of specific enzyme activity that control the synthesis of the antithrombin-binding-site sequence are sparsely and nonrandomly distributed on mast-cell Golgi membranes. It is postulated that the likelihood of a developing proteoglycan encountering one of these hypothetical areas is molecular-size-dependent.

Animals↗
PubMed1988

Determination of the range in binding-site densities of rat skin heparin chains with high binding affinities for antithrombin.

Rat skin heparin proteoglycans vary markedly in the proportions of their constituent polysaccharide chains that have high binding affinity for antithrombin. As the proportion of such chains in a proteoglycan rises, their degree of affinity for antithrombin also increases [Horner (1987) Biochem. J. 244, 693-698]. The antithrombin-binding-site densities of such chains have now been determined, by measuring heparin-induced enhancement of the intrinsic fluorescence of antithrombin and by chemical analysis for the disaccharide sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate), which is unique to this site in heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555]. Antithrombin-binding-site density ranged from one to five sites per chain.

Animals↗
PubMed1987

Heterogeneity of rat skin heparin chains with high affinity for antithrombin.

Subfractions of 35S-labelled rat skin heparin proteoglycans with various degrees of high affinity for antithrombin were obtained by gradient elution from a column of antithrombin-agarose. Heparin chains released from the proteoglycan preparations by beta-elimination with alkali were re-fractionated on the same column. Proportions of chains with high affinity for antithrombin (HA-chains) ranged from 17% to 76%. These separations also revealed three overlapping subfractions of HA-chains. Their proportions varied in a manner consistent with a stepwise increase in the degree of affinity of HA-chains for antithrombin, this presumably being due to the biosynthesis of increasing numbers of antithrombin-binding sites per chain. The anticoagulant activity, with respect to thrombin neutralization, ranged from 32 units/mg to 287 units/mg. It is suggested that HA-chains may have from one to five or six antithrombin-binding sites. Thus the asymmetric distribution of these sites in rat skin heparin proteoglycans is much more marked than was realized from the earlier work of Horner & Young [(1982) J. Biol. Chem. 257, 8749-8754].

Animals↗
PubMed1986

Location of antithrombin-binding regions in rat skin heparin proteoglycans.

Rat skin heparin proteoglycan labelled biosynthetically with 35S was fractionated on a column of antithrombin-Sepharose into fractions with varying degrees of affinity for antithrombin. These were treated with NaOH to release heparin chains (Mr 60,000-100,000), by beta-elimination or incubated with serum to produce fragments of the same order of size as commercial heparin (Mr 5000-30,000), by endoglycosidase cleavage. Chains and fragments were then fractionated on antithrombin-Sepharose. The various fractions were deaminated with HNO2 at pH 1.5 followed by reduction with NaB3H4. Approx 90% of the incorporated 3H was associated with disaccharides. These were fractionated by high-performance ion-exchange chromatography. A unique minor component corresponding to the sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate) in the polysaccharide was found only in fractions with high affinity for antithrombin. The glucosamine residue linked to C-4 of this glucuronosyl unit was predominantly (or exclusively) N-sulphated rather than N-acetylated, pointing to a structural difference between the antithrombin-binding region of rat heparin and that of pig mucosal heparin. Calculations based on the distribution of the glucosaminyl 3-O-sulphate group showed that approximately two-thirds of the total antithrombin-binding regions present in the unfractionated material were accommodated by only 20% of the proteoglycan molecules, and by 10% of the polysaccharide chains. While most of the proteoglycan molecules thus lacked such regions (and hence affinity for antithrombin) a minor proportion of the polysaccharide chains contained on the average three binding regions per molecule. These findings support by direct chemical analysis an earlier proposal, based on anticoagulant activities of similar rat skin heparin fractions, that the distribution of antithrombin-binding sites in intact heparin proteoglycans is markedly non-random.

Animals↗
PubMed1986

Rat heparins. A study of the relative sizes and antithrombin-binding characteristics of heparin proteoglycans, chains and depolymerization products from rat adipose tissue, heart, lungs, peritoneal cavity and skin.

35S-labelled heparins were recovered from adipose tissue, hearts, lungs, peritoneal cavities and skins of rats given H2(35)SO4. Their purification involved incubation with Pronase, precipitation with cetylpyridinium chloride in 1.0 M-NaCl, gradient elution from DEAE-Sephacel and incubation with chondroitinase ABC. Each product was divided into proteoglycan and "depolymerization products' fractions by gel filtration on Bio-Gel A-15m. Heparin chains were released from a portion of each proteoglycan fraction by beta-elimination with NaOH. Proteoglycans, chains and depolymerization products were separated by gradient elution from a column of antithrombin-agarose into fractions with no affinity, low affinity and high affinity for antithrombin. The relative sizes of the products were determined by gel filtration on columns of Bio-Gel A-50m, A-15m, A-1.5m and A-0.5m. Skin was the major source of heparin and contained the largest proteoglycans and the lowest proportion of depolymerization products. Lungs contained the smallest proteoglycans, the smallest depolymerization products and the highest proportion of depolymerization products. The highest proportions of proteoglycans, chains and depolymerization products with high affinity for antithrombin were found in adipose tissue. The lowest proportions of each of these fractions were found in the peritoneal cavity. The data suggest that there was relatively little biosynthesis of sites with high affinity for antithrombin in peritoneal-cavity mast cells and that heparin catabolism was most active in lungs. Each source of heparin was unique with respect to both biosynthesis and subsequent breakdown of its proteoglycans.

Adipose Tissue↗
PubMed1982

Asymmetric distribution of sites with high affinity for antithrombin III in rat skin heparin proteoglycans.

The distribution of sites with high affinity for antithrombin III in [35S]heparin proteoglycans from rat skin was studied by affinity chromatography of the intact proteoglycans (Mr congruent to 1 x 10(6)) and degradation products. Unfractionated proteoglycan and proteoglycan fractions with low affinity and high affinity separated on antithrombin III-agarose were treated with alkali, releasing heparin chains (Mr congruent to 1 x 10(5)). Each chain preparation was fractionated on antithrombin III-agarose into fractions with low affinity and high affinity. Unfractionated proteoglycan and proteoglycan fractions with low affinity and high affinity were incubated with rat serum at pH 6.0, which gave products of similar size to commercial heparins (Mr congruent to 1 x 10(4)) termed heparin fragments. Each fragment preparation was fractionated on antithrombin III-agarose, yielding fractions with no affinity, low affinity, and high affinity, 40% of the proteoglycan preparation had low affinity, containing 4% high affinity chains and 7% high affinity fragments. The high affinity proteoglycan fraction yielded 40% high affinity chains and 22% high affinity fragments. The data show that the distribution of binding sites with high affinity for antithrombin III in heparin proteoglycans is highly asymmetric. Therefore, the concept that polymer modification reactions occurring during heparin biosynthesis, which must be involved in the formation of high affinity binding sites, occur in a random way must be reappraised.

Animals↗
PubMed1981

Immunoglobulin-sulfated polysaccharide interactions. Binding of agaropectin and heparin by human IgG proteins.

The interaction of immunoglobulins with certain acidic polysaccharides was demonstrated by the binding of the sulfated glycans agaropectin and heparin by certain human IgG proteins. Heparin-binding IgG proteins can distinguish between the molecular forms of heparin derived from porcine intestine, bovine lung, and rat skin. The major specificity of these proteins is for native and certain high molecular weight subunit components of rat skin heparin. The interactions with multi-chain and single chain rat skin heparin are stable under physiological conditions and involve the Fab and, more specifically, the Fv region of the IgG molecule. These reactions occur as a result of an electrostatic interaction between cationic sites on certain IgG proteins and anionic sulfate resides of agaropectin or heparin. The characteristics of heparin-IgG interaction resemble those of heparin with other plasma proteins, the interactions of which have biological significance.

Agar↗
PubMed1979

The assay and partial characterization of macromolecular heparin depolymerase activity in rat small intestine.

Homogenates of rat small intestine can depolymerize macromolecular rat skin heparin (RS heparin) to products similar in size to commercial heparin [Horner (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3469--3473]. This activity is attributed to an enzyme provisionally named 'macromolecular heparin depolymerase'. An assay for macromolecular heparin depolymerase activity in rat small intestine has been developed, based on the action of the enzyme on 35S-labelled macromolecular RS heparin. The depolymerized products are separated into two peaks by gel chromatography through columns of Bio-Gel A-15m. The amount of label in the second peak, expressed as a percentage of the total radioactivity, is the index of enzyme activity. The pH optimum was found to be 6.0 and the temperature optimum 45 degrees C. The enzyme was shown to be most stable in 50mM-Tris/maleate buffer containing 1 mM-EDTA. Macromolecular heparin depolymerase activity measured as a function of time and substrate concentration produced curves typical of an enzymic reaction. Evidence was obtained demonstrating that the activity did not originate from bacteria in the intestine. Macromolecular heparin depolymerase activity was increased by dilution and storage at 7 degrees C for 24 h. This suggests that homogenates of rat small intestine contain an unstable inhibitor of the enzyme.

Animals↗