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Cellular competition for survival signals offers a cogent and appealing mechanism for the maintenance of cellular homeostasis [Raff, M. C. (1992) Nature (London) 356, 397-400]. We present a theoretical and experimental investigation of the role of competition for resources in the regulation of peripheral B cell numbers. We use formal ecological competition theory, mathematical models of interspecific competition, and competitive repopulation experiments to show that B cells must compete to persist in the periphery and that antigen forms a part of the resources over which B cells compete.

In the present study, the receptor binding potential of transferrin (Tf) was linked to an antibody binding specificity. Human Tf was fused to mouse-human chimeric IgG3 at three positions: at the end of heavy chain constant region 1 (CH1), after the hinge, and after CH3. The resulting Tf-antibody fusion proteins were able to bind antigen and the Tf receptor. The CH3-Tf fusion protein showed no complement-mediated cytolysis but possessed IgG receptor I (Fc gamma RI) binding activity. Most importantly, all of the fusion proteins demonstrated significant uptake into brain parenchyma, with 0.3% of the injected dose of the hinge-Tf fusion protein rapidly targeted to the brain. Recovery of iodinated CH3-Tf fusion protein from the brain parenchyma demonstrated that the fusion proteins can cross the blood-brain barrier intact. The binding specificity of these fusion proteins can be used for brain delivery of noncovalently bound ligands, such as drugs and peptides, or for targeting antigens present within the brain.

We have created four IgG3 mutants without a normal hinge region: (i) m0 without a genetic hinge; (ii) m0/C131S, where Cys-131 in m0 was mutated to Ser; (iii) m0/231C232 (formerly HM-1), where a Cys residue was inserted in m0 between Ala-231 and Pro-232; (iv) m0/C131S/231C232, which is a hybrid of m0/231C232 and m0/C131S. The wild-type IgG3 and all mutants bind 5-iodo-4-hydroxy-3-nitrophenacetyl groups. The wild type and mutants, m15 (with 15 aa in the hinge), m0/231C232, and m0/C131S/231C232, were all positive for complement-mediated lysis, antibody-dependent cellular cytotoxicity mediated by peripheral blood leukocytes, and phagocytosis by U937. m0/C131S/231C232 was only weakly positive and sometimes negative for respiratory burst activity mediated by peripheral blood neutrophils (polymorphonuclear leukocytes), whereas m15, m0/231C232, and wild-type IgG3 were strongly positive. The m0 and m0/C131S mutants were mainly negative for complement-mediated lysis, antibody-dependent cell-mediated cytotoxicity, and phagocytosis by U937 and polymorphonuclear leukocytes. The results indicate that a hinge spacer region is not necessary, but the correct alignment of the two second heavy chain constant regions in the IgG3 molecule by a minimum of one disulfide bond is necessary and sufficient for effector functions.

A hydrolytic catalytic antibody, generated against a nitrophenyl phosphonate transition state analogue, has been cloned and expressed in Escherichia coli for use as a model system to demonstrate the feasibility of using genetic selections to enhance catalytic activity. Conditions were found that permit the secretion of active recombinant antibody into the periplasm of a strain of E. coli deficient in the biotin biosynthetic genes (delta bio-gal). A number of substrates were synthesized that, upon hydrolysis by the antibody, yield free biotin, which is required for cell growth. The substrates and selections can be used to identify mutants of the antibody with altered activities. This approach should be generalizable to a wide number of hydrolytic reactions including the selective cleavage of peptide, polysaccharide, phosphodiester, and ester bonds.

Tumor necrosis factors (TNF) alpha and beta are structurally related cytokines that mediate a wide range of immunological, inflammatory, and cytotoxic effects. During bacterial infection of the bloodstream (sepsis), TNF-alpha induction by bacterial endotoxin is thought to be a major factor contributing to the cardiovascular collapse and critical organ failure that can develop. Despite antibiotic therapy, these consequences of sepsis continue to have a high mortality rate in humans. Here we describe a potent TNF antagonist, a TNF receptor (TNFR) immunoadhesin, constructed by gene fusion of the extracellular portion of human type 1 TNFR with the constant domains of human IgG heavy chain (TNFR-IgG). When expressed in transfected human cells, TNFR-IgG is secreted as a disulfide-bonded homodimer. Purified TNFR-IgG binds to both TNF-alpha and TNF-beta and exhibits 6- to 8-fold higher affinity for TNF-alpha than cell surface or soluble TNF receptors. In vitro, TNFR-IgG blocks completely the cytolytic effect of TNF-alpha or TNF-beta on actinomycin D-treated cells and is markedly more efficient than soluble TNFR (24-fold) or monoclonal anti-TNF-alpha antibodies (4-fold) in inhibiting TNF-alpha. In vitro, TNFR-IgG prevents endotoxin-induced lethality in mice when given 0.5 hr prior to endotoxin and provides significant protection when given up to 1 hr after endotoxin challenge. These results confirm the importance of TNF-alpha in the pathogenesis of septic shock and suggest a clinical potential for TNFR-IgG as a preventive and therapeutic treatment in sepsis.

Immunoglobulin- or multiple myeloma-associated amyloidosis has been distinguished by the tissue deposition of Congophilic, fibrillar protein consisting of light chains or light-chain fragments (AL amyloidosis). We now report the isolation and characterization of another form of immunoglobulin-associated amyloid obtained from a patient who had extensive systemic amyloidosis and in whom the amyloid deposits consisted not of light chains but rather of an unusual form of heavy chain. This component, isolated from splenic amyloid extracts, represented an internally deleted IgG1 heavy chain as evidenced by immunochemical, electrophoretic, and amino acid sequence analyses. A comparable immunoglobulin-related monoclonal protein, consisting only of IgG heavy chains, was present in the patient's urine. Based on serologic reactivity with a battery of anti-immunoglobulin antisera, these two immunoglobulin-related components were antigenically identical; however, when compared to normal IgG, both were deficient in Fc-associated gamma-chain determinants. The structural abnormality of the amyloid gamma-chain protein was further evidenced by SDS/PAGE and immuno-blotting analyses: An unusually low molecular mass of approximately 22 kDa was found for this material vs. the expected value of approximately 55 kDa for a normal gamma heavy chain. Despite the lack of certain Fc determinants, the amyloid and urinary heavy-chain proteins expressed the IgG1 subclass allotype marker G1m(a) located on the third constant region (CH3) domain of the internally deleted IgG1 heavy chains. That the amyloid protein contained an intact CH3 domain was established through amino acid sequence analyses of cyanogen bromide fragments and peptides generated by a lysine-specific protease. These studies also revealed that the gamma-chain amyloid protein contained the complete heavy-chain variable (VH) domain [including the diversity (DH) and joining (JH) segments] that was contiguous with the CH3 domain. The low molecular mass of the protein resulted from the total absence of the first (CH1), hinge, and second (CH2) heavy-chain constant regions. Such extensive CH deletions and the presence of a complete VH distinguish this amyloid-associated heavy chain from all other heretofore characterized gamma-heavy-chain disease proteins. This heavy-chain-related form of immunoglobulin-associated amyloidosis is tentatively designated AH amyloidosis.

The papain-solubilized fragment of the heavy chain of HLA-B7, which is the NH2-terminal part of the whole polypeptide chain, can be divided into three regions by mild acid and cyanogen bromide cleavages. The first 100 amino acids terminating in a methionine residue contain the carbohydrate moiety; this segment is followed by two others of molecular weights 9,999 and 13,000, each containing an intrachain disulfide bridge. The two intrachain disulfide bridges are separated by a stretch of amino acids containing an acid-labile aspartyl-prolHLA-2, A28, and AW25 contain this acid-labile peptide bond in their larger subunit. Sequencing from the acid cleavage site of HLA-7 through the third half-cystine revealed consideralbe homology with amino acid sequences around a half-cystine in immunoglobulin variable regions.

Two monoclonal anti-platelet antibodies, 3B2 and 8G11, have been raised that are specific for normal human platelets. 3B2 is unique in that it has decreased reactivity for platelets from 16 patients with autoimmune thrombocytopenic purpura [mean platelet count, 65,000 +/- 6,000 (SEM)]. With 8G11 in an enzyme-linked immunosorbent assay, the mean of the ratios of patient platelet OD to control platelet OD was 0.95 +/- 0.07, whereas with 3B2, the mean of the ratios of patient platelet OD to control OD was 0.24 +/- 0.04, P less than 0.001. With 3B2 the mean of the OD ratios of five patients with autoimmune thrombocytopenic purpura in remission (greater than 150,000 platelets per mm3) compared to controls was 0.80 +/- 0.14. 3B2 did not react with platelets from a patient with Glanzmann's thrombasthenia, in which membranes lack glycoproteins IIb and IIIa (GPIIb and GPIIIa). Platelet membranes were run on crossed immunoelectrophoresis against a rabbit polyclonal anti-human platelet membrane antibody with 125I-labeled purified 3B2 in an intermediate spacer gel. 3B2 reacted with the GPIIb-GPIIIa-Ca2+ complex in the presence of excess Ca2+ and with GPIIb alone in the presence of excess EGTA. When Triton X-100-solubilized platelet membranes were immunoprecipitated with 3B2 plus rabbit anti-mouse IgG, reduced, and run on NaDodSO4/polyacrylamide gel electrophoresis, a single protein band was obtained with a molecular weight of 120,000 (the molecular weight of GPIIb). Thus, the reactivity of monoclonal antibody 3B2 with GPIIb or the GPIIb-GPIIIa-Ca2+ complex appears to be inhibited by the presence of autoantibody on platelets.