Relative importance of Salmonella-specific antibody isotypes in phagocytosis of Salmonella typhimurium by ovine mammary neutrophils.

The comparative opsonic efficiency of ovine salmonella-specific antibody isotypes was determined by measurement of specific phagocytic uptake of opsonised virulent Salmonella typhimurium by ovine mammary neutrophils. An in vitro phagocytosis assay revealed that IgM was superior to IgG2 in promoting the phagocytosis of opsonised virulent organisms. IgG1, on the other hand, was non-opsonic. Superiority of the IgM isotype over IgG2 as an opsonin was also evident in studies on the viability of opsonised S typhimurium upon phagocytosis. It was revealed that the percentage of organisms killed was appreciably greater when opsonisation was carried out with the IgM than with the IgG2 isotype, although after ingestion by neutrophils there was essentially no difference in the efficiency with which the ingested organisms were killed.

Amphotericin B modifications of peripheral blood lymphocyte and tonsil lymphocyte responses to concanavalin A.

Peripheral blood lymphocytes (PBL) were shown previously to be activated to incorporate 3H-thymidine by concanavalin A (con A). Amphotericin B (Am B) was reported to be both immuno-enhancing and suppressive. We investigated the response of PBL and tonsil lymphocytes (TL) to con A in culture, and the effect of Am B on these responses. PBL were activated by con A as expected, and the magnitude of the response diminished as cell density increased. The responses of PBL usually were significantly enhanced by Am B at 2.5 or 5 micrograms/ml, but 10 micrograms Am B/ml significantly inhibited the con A response. TL had relatively high rates of spontaneous proliferation, but Am B was a potent inhibitor of this. TL responded to con A just as well as PBL, and Am B strongly inhibited these responses too. When TL were cultured with various doses of con A in the presence of 5 or 10 micrograms Am B/ml, the shape of the dose curves resembled those of PBL stimulated in the absence of Am B except that the incorporation of 3H-thymidine increased with increasing cell density. It was concluded that Am B at 2.5-5.0 micrograms/ml enhanced the response of PBL to con A and suppressed the response at 10 micrograms/ml. All doses of Am B inhibited both spontaneous proliferation and con A-induced proliferation in TL. In spite of this inhibition, the TL population responded to various doses of con A in the presence of Am B.

The molecular weight of J chains derived from human immunoglobulin M.

J chain was isolated from sulphonated human immunoglobulin M molecules by electrophoresis on polyacrylamide gels. When determined by electrophoresis in sodium dodecyl sulphate-polyacrylamide gels, the molecular weight of the protein was about 27000. After suspension in 5m-guanidine hydrochloride solution for 21 days, two groups of three bands appeared on the gels. Most of the protein dissociated to components of molecular weight 15000. The molecular weight of purified J chain was also determined by ultracentrifugation. In borate-saline solution the average weight-average molecular weight was about 29000. The molecular weight slowly decreased upon prolonged exposure to guanidine hydrochloride, and after 14 days the minimum molecular weight was about 15000. Some association between chains still existed. These data suggest that J chain derived from the paraprotein exists in borate-saline solution as dimers held by strong non-covalent forces.

The incorporation of J chain into reassembled human immunoglobulin M.

Human IgM (immunoglobulin M) was reduced with 24mm-mercaptoethylamine. This atreatment resulted in complete dissociation to IgMs subunits and free J chain. Intr-subunit interchain disulphide bonds remained intact. The mixture then was encouraged to reoxidize. The schlieren pattern of the reoxidized mixture showed the presence of a considerable quantity of IgM in addition to residual IgMs. The isolated reassembled IgM did not dissociate in 5m-guanidinium hydrochloride. It apparently contained the same amount of covalently attached J chain as did native IgM. The J chain was a part of the high-molecular-weight Fc fragment obtained from the reassembled IgM.

Investigations of the structural function of the J chain in human immunoglobulin M.

Human IgM molecules were treated with Na(2)SO(3) or mercaptoethylamine in concentrations ranging from 2 to 14mm or 2 to 22mm respectively. The dissociation of IgM to IgM(s) varied from 0% to 100%. At the intermediate concentrations of either reagent the amount of freed J chains was less than expected. In an attempt to find an explanation for this, IgM was partially dissociated to IgM(s) with mercaptoethylamine. The IgM(s) isolated by gel filtration was divided according to the ascending and descending portions of the elution curve. These portions were treated with 24mm-mercaptoethylamine and analysed for the presence of J chains. Only the ascending portion contained free J chains. Thus, after mild reduction where not all the IgM molecules are dissociated to IgM(s), some J chains remain covalently attached to some IgM(s) molecules although most of the J chains are freed. It was concluded that the J chain could serve as a ;hitch' for IgM(s) molecules forming intact IgM.

Mercaptan-induced fragmentation of a subunit-like proteolytic fragment of immunoglobulin M.

Limited papain hydrolysis of immunoglobulin M (IgM) produces a subunit-like proteolytic fragment designated IgM(p) (Inman & Hazen, 1968). In the presence of mercaptans, IgM(p) partially dissociated into Fc(mu)-like and Fab(mu) fragments. Treatment of residual IgM (that remaining after a papain digestion) with 2mm-mercaptoethylamine resulted in fragmentation of the same type that occurs in a routine limited digestion of IgM with papain, although exogenous enzyme was not added to the mixture. When IgM was hydrolysed with (14)C-labelled papain, a small quantity of the enzyme was found to be associated with the residual IgM and IgM(p) fractions. IgM and IgM 7S subunit (IgM(s)) that had been exposed to papain in the absence of activating mercaptan and separated from the enzyme by gel filtration also fragmented when subsequently treated with 2mm-mercaptoethylamine. The fragments resembled those produced during a typical limited papain digestion of IgM. It was concluded that mercaptoethylamine induced fragmentation of IgM(p) by activating adsorbed papain.