Correlation between expression of antibodies to histone H2B and clinical activity in HIV-infected individuals.

Levels of autoantibodies specific for the histone, H2B, were measured in individuals with HIV infection. In comparison with normal (uninfected) controls, infected patients, particularly those with symptomatic disease, had significantly elevated titres of anti-H2B antibodies. Longitudinal studies confirmed that levels of these antibodies were highest in patients with lymphadenopathy and declined with the development of AIDS. In preliminary experiments designed to determine the biological significance of the anti-histone antibodies, H2B was shown to be immunologically cross-reactive with an 18-kD antigen on the surface of HIV-infected or mitogen-activated CD4+ cells. Protein sequencing of the 18-kD antigen has since shown complete homology with histone H2B. Because the titres of H2B autoantibodies were found to parallel the numbers of circulating CD4 cells, it is possible that these antibodies are involved in the destruction of the helper/inducer T lymphocyte population.

Further evidence linking urolithiasis and blood coagulation: urinary prothrombin fragment 1 is present in stone matrix.

The fact that organic material is always present and distributed throughout each renal calculus suggests that it may play a role in stone formation. The organic matrix of calcium oxalate (CaOx) crystals freshly generated in urine in vitro contains urinary prothrombin fragment 1 (UPTF1) as the principal protein. In this initial study, matrix was extracted from 12 renal calculi and evaluated for the presence of UPTF1 using Western blotting. UPTF1 was present in all eight stones whose principal component was CaOx, and in one of two stones which consisted mainly of calcium phosphate (CaP). UPTF1 was absent from the two struvite calculi examined. The relationship between CaP and UPTF1 was explored further. Matrix harvested from CaP crystals freshly generated in urine in vitro was also shown to contain UPTF1 as its principal component. Our inability to detect UPTF1 in one mixed CaOx/CaP stone may be related to our methods of matrix retrieval, while its absence from two struvite stones argues against it being present in the other stones merely as a consequence of passive inclusion. This absence may be related to the alkaline environment typical of struvite stone growth. The finding that UPTF1 is present in some renal stones provides the first direct evidence that links blood coagulation proteins with urolithiasis.

A comparison of the anti-idiotypic responses generated by antibodies to a protein and a hapten: a common interspecies idiotype on antibodies against human albumin induces an idiotypic network in rabbits.

Idiotypic networks have the capacity to exert significant influences on immune responses and an understanding of the ways to manipulate these networks may lead to new modalities in immunotherapy. In order to gain further insights into the nature of the immune responses stimulated by immunoglobulin idiotypes, rabbits were immunized with a mAb (Ab1) against a large globular protein, human albumin, or a mAb against a hapten, TNP. All rabbits developed anti-idiotypic antibodies (Ab2) and the rabbits immunized with anti-human albumin concomitantly developed antibodies to human albumin (Ab3). Ab2 prepared from these rabbits blocked binding of Ab1 to antigen and the anti-human albumin Ab2 reacted with all species of anti-human albumin including sheep, rabbit, rat and goat. The anti-TNP Ab2 reacted only with the mouse anti-TNP Ab1. This TNP Ab2 bound only to intact Ab1 whereas the human albumin Ab2 reacted with the Ab1 heavy chain. To compare the relative efficiencies of anti-idiotypic antibodies and antigen in inducing antibody, mice were immunized with rabbit Ab2 or antigen. All mice immunized with Ab2 developed anti-idiotypic Ab3, but only the human albumin Ab2 preparations elicited antigen specific Ab3; the amount of antibody produced was less than 1% of that found by immunization with antigen. The type of antibody induced in the Ab2-immunized mice was compared with that found in the antigen-immunized mice and in the Ab1-immunized rabbits. The mouse anti-albumin Ab3 was comparable to mouse Ab1 in terms of affinity and specificity for proteolytic fragments of human albumin. The Ab3 which arose in Ab1-immunized rabbits had a higher affinity and broader epitope specificity and was similar to antibodies raised against antigen. These results show considerable differences in the ability of similar anti-idiotypic antibodies to induce immune responses as well as considerable differences in the nature of a response seen within an intact network compared to an artificially induced network.

Common epitope on HIV p24 and human platelets.

Mimicry of human antigen by HIV may underlie the autoreactivity seen in AIDS. A mouse monoclonal antibody (VIC8) raised against HIV p24 cross-reacted with human platelets; binding could be abolished by recombinant p24 antigen. VIC8 bound less well to platelets from patients with HIV than to those from healthy individuals. In the HIV group, binding was not related to p24 antigenaemia, disease stage, or platelet counts. This cross-reactivity is another example of antigenic mimicry by HIV and may be mechanistically important in HIV-associated autoimmune-like thrombocytopenia.

Mouse monoclonal anti-idiotypic antibodies to HIV p24: immunochemical properties and internal imagery.

Two mouse monoclonal antibodies (VIC5 and VIC6; referred to as Ab1) reacting with the p24 core antigen of HIV-1 were used to produce mouse monoclonal anti-idiotypic antibodies (Ab2). Six anti-idiotypic antibodies were characterized. The five anti-idiotypic antibodies directed against VIC6 partly competed which each other and thus defined a set of overlapping idiotypes on Ab1. All 6 Ab2s inhibited the binding of the corresponding anti-p24 antibody to antigen, although four (W1, Y16, Y6, X14) were markedly more inhibitory than the remaining two (G6, Y11). All six Ab2s were antigen-inhibitable; however the interaction of G6 and Y11 with Ab1 was blocked with considerably less soluble p24 antigen than the remaining four. Correspondingly, G6 and Y11 had lower affinities for Ab1 than did W1, Y6 and X14; the affinity index of Y16 was equivalent to that of Y11. None of the Ab2s reacted with H or L chains of Ab1 after reduction on SDS-gels. Similarly, both Ab1s failed to react with the H or L chains of Ab2. These criteria appeared to define at least four of these Ab2s as internal image antibodies whose image is composed of both H and L chains. The anti-idiotypic antibodies were injected either individually or as a combined preparation of all 6 into syngeneic mice and Porton rats. Despite the presence of anti-anti-idiotypic antibodies (Ab3) in these animals, when used individually no antigen-specific antibodies were found. A small response to p24 antigen was induced in 3 of 6 mice using preparations containing all 6 anti-idiotypes.

Patterns of corneal graft rejection in the rabbit and reversal of rejection with monoclonal antibodies.

The purpose of this study was to determine whether the local administration of monoclonal antibodies could reverse rabbit corneal graft rejection. To provide a rational basis for the choice of monoclonal antibodies as potential immunosuppressive agents, the phenotypes of cells infiltrating rejecting rabbit corneal allografts were examined by immunohistochemistry. About half the leukocytes accumulating in these grafts bore an immunodominant T cell marker, over two-thirds carried MHC class II antigens, and about one-fifth carried myeloid cell markers. A kinetic study of the cell population appearing in rabbit aqueous during corneal graft rejection was performed by examination of repetitive anterior chamber taps taken over a ten-day period; again, the major components were T cells, MHC class II antigen-positive cells and myeloid cells. Monoclonal antibodies L11/135 (directed against a peripheral T cell determinant), 2C4 (directed against a monomorphic MHC class II antigen), and LION 2 (directed against a myeloid antigen) were chosen for intracameral injection into rabbits with rejecting corneal grafts. Each animal received a total of 50-100 micrograms of antibody in two injections at 3-4-day intervals. L11/135 and LION 2 reversed rejection in 5/9 and 8/12 animals, respectively, in the absence of any other immunosuppression; 2C4 was without effect. We suggest that monoclonal antibody therapy in corneal transplantation deserves further attention.

The H3 anti-phospholipid idiotype is found in patients with systemic lupus erythematosus (SLE) but not in patients with syphilis.

The human H3 idiotype, defined by a mouse monoclonal antibody S2.9, is commonly found in patients with SLE where it is correlated with the amount of anti-cardiolipin antibodies. No correlation between the amount of anti-cardiolipin antibody and the H3 idiotype is found in patients with syphilis. Using the S2.9 antibody, serum from each of 10 patients with SLE and eight patients with syphilis was separated into H3-bearing and H3-negative fractions. Comparison of the partition of anti-cardiolipin antibody in these two groups of patients revealed that much of the anti-cardiolipin antibody (44-91%) was found in the H3+ fraction in patients with SLE; in patients with syphilis, virtually none of the anti-cardiolipin antibody was H3+. In patients with SLE, the H3+ fraction contained both IgG and IgM and antibodies of both kappa and lambda light chains. The H3+ fraction was polyspecific and frequently reacted with dsDNA.

A common anti-cardiolipin antibody idiotype in autoimmune disease: identification using a mouse monoclonal antibody directed against a naturally-occurring anti-phospholipid antibody.

We have recently produced a series of human monoclonal antibodies reacting with cardiolipin. One of these, H3, a polyspecific IgM/k derived from a normal individual, was used to raise mouse monoclonal antibody to its idiotype. Two anti-idiotypic antibodies, S2.9 (IgG2b) and S2.10 (IgM) were selected for their specific reaction with H3.S2.9 did not react with five other human monoclonal antibodies of IgM/k class despite the fact that these shared some antigen-binding characteristics with H3.S2.9 was able to block the binding of H3 to all of its cross-reactive antigens including cardiolipin, while S2.10 was not. S2.9 was equally efficient in blocking the binding of H3 to three of its cross-reactive antigens, cardiolipin, diphtheria and tetanus toxoids; greater than 90% inhibition could be achieved at an equimolar ratio of H3 to S2.9. The anti-idiotype S2.9 was used to demonstrate the presence of the H3 idiotype in serum. This idiotype was found in amounts greater than that seen in 42 normal individuals, in 30 of 36 patients with systemic lupus erythematosus (SLE), eight of 20 patients with rheumatoid arthritis (RA), 8 of 20 patients with Felty's syndrome as well as 10 of 23 patients with syphilis. Not one of nine patients with drug-induced lupus syndrome had abnormal levels. In patients with SLE and Felty's syndrome there was a good correlation between the amount of anti-cardiolipin antibodies and the amount of H3 idiotype (rs = 0.70 and 0.69 respectively). No such correlation was found in syphilitics or in patients with RA. In patients with SLE the H3 idiotype was present on IgM and IgG anti-cardiolipin antibodies. In 15 of 16 SLE sera with high levels of cardiolipin antibody, S2.9 blocked binding of serum antibodies to cardiolipin by 13-72%, with a mean value of 49%. One patient had a high level of anti-cardiolipin antibody which could not be blocked by S2.9. These results indicate that a mouse monoclonal antibody which reacts with an idiotope in the antigen-binding region of a naturally-occurring phospholipid antibody also defines a common idiotype of anti-cardiolipin antibodies in patients with autoimmune disease.

Polyspecific human and murine antibodies to diphtheria and tetanus toxoids and phospholipids.

Human-human hybridomas produced from lymphocytes of normal individuals yielded seven clones producing monoclonal antibody reacting with tetanus toxoid. Three of these antibodies cross-reacted with diphtheria toxoid. These three and two others also reacted with cardiolipin and two with other phospholipids. One of the seven antibodies reacted with tetanus and diphtheria toxoids, cardiolipin and single-stranded DNA. All seven antibodies were IgM. To examine further this unusual cross-reactivity serum antibodies from patients with SLE and healthy individuals were affinity-purified to yield diphtheria toxoid antibodies. Six out of nine of these anti-diphtheria preparations contained IgG antibodies which cross-reacted with tetanus toxoid and two of these also reacted with cardiolipin; four preparations cross-reacted with DNA. Anti-cardiolipin and anti-DNA cross-reactivity were found in preparations from both normal and SLE sera. Similar cross-reactivities were demonstrated using four mouse monoclonal IgM antibodies raised against phospholipids. All four of these antibodies reacted with both cardiolipin and tetanus toxoid and two also reacted with diphtheria toxoid and DNA. Using a thiocyanate elution procedure, it was shown that the cross-reactivity of the monoclonal antibodies was not related to their relative affinities. The results clearly indicate that cross-reactive epitopes occur on routinely used toxoid vaccines and self antigens. Antibodies which bind to these cross-reactive epitopes are common and are not restricted in isotype, affinity or species of origin.

Production of a polyspecific human monoclonal antibody reacting with an epidermal antigen.

A clone of cells secreting an antibody to an epidermal antigen was generated from a patient with a blistering skin lesion. Although produced by fusion of human lymphocytes to a HAT-sensitive myeloma, this clone of cells did not have characteristics of a hybridoma. A true hybridoma was produced by fusion of this clone to a HATr/ouabain(r) myeloma line. The IgM antibody secreted by this clone reacted with the intercellular region of the epidermis of normal human skin in a manner similar to pemphigus autoantibodies. In addition, in normal human kidney the antibody bound to glomeruli and tubules. It also reacted with an antigen present in the cytoplasm of a wide variety of cell lines including epithelial, lymphoid and myeloid types. No reaction was found with the surface of any of the cell lines, nor with DNA or phospholipid antigens. This monoclonal antibody may define an autoantibody specificity which mediates some autoimmune skin lesions. Its polyspecificity is reminiscent of some other human hybridoma autoantibodies, and its reaction with components of the kidney suggests an alternative pathology for renal disease in such patients.

Mechanism of induction of mouse erythrocyte receptor switch in human B cells.

An early event in phorbol ester-induced maturation of chronic lymphocytic leukemic (CLL) B cells is a membrane change characterized by the inactivation of a mouse erythrocyte receptor (MER). This event, the MER-switch, is quantified by inhibition of rosette formation. By using [3H]phorbol dibutyrate ([3H]PDBu), both to stimulate MER-switch and assay binding of PDBu to CLL cells, it was shown that MER-switch was an irreversible, time-dependent event which occurred some time after maximal binding of [3H]PDBu to cells. Two classes of binding sites, one of high affinity (Kd 1 to 2 nM) at low frequency (1.5 to 5 X 10(4) sites per cell), and a lower affinity site (Kd 33 to 50 nM) of higher frequency (2 to 3.5 X 10(5) sites per cell), were detected. Binding of [3H]PDBu was inhibited by phorbol ester analogs that stimulated MER-switch, but not by inactive analogs. This, and the similarity in shapes of the binding and rosette inhibition curves over a range of concentrations, suggests that stimulation of MER-switch by phorbol esters is due to this specific binding. The phorbol ester receptor and MER are distinct because MER-ve T cells and MER-ve atypical B cells from a patient with CLL had both classes of PDBu receptor. Solubilized MER did not bind [3H]PDBu. Time-course studies, and the irreversibility of the switch, despite removal of most of the bound [3H]PDBu, indicate that inhibition of rosetting is not due to competitive or steric hindrance by phorbol esters. Equivalent activities of soluble MER were released from fresh and phorbol ester-treated CLL cells, indicating a rearrangement of MER, rather than a loss. A supernatant of phytohemagglutinin-stimulated human spleen cells also induced MER-switch in CLL lymphocytes, suggesting that a lymphokine may be a natural inducer of this event.

A subclass of albumin recognized by inhibition of phosphatidylethanolamine-mediated agglutination of mouse erythrocytes.

Agglutination of mouse erythrocytes by non-choline phospholipids is inhibited by a factor in mammalian sera. The inhibitor cochromatographed with albumin on dye-agarose conjugates, was retained by an anti-albumin affinity column, was neutralized by anti-albumin antibody and found in a serum fraction in which only albumin could be detected. A variety of commercial preparations of albumin (fraction V, crystalline) did not inhibit. However, they acquired potent inhibitory activity when treated with low molecular weight thiols. The inhibitory activity of serum was increased 8-fold by treatment with dithiothreitol. Other proteins were not activated in this way. Inhibitory activity increased with average free sulphydryl content of treated albumin, up to six thiol groups per molecule. Alkylation of these sulphydryl groups did not diminish inhibitory activity. Thiols also induced polymerization of albumin. Inhibitory albumin in serum was largely monomeric. We propose that the inhibitor is a type of serum albumin which is lost or inactivated during preparation of commercial albumin, and which shares a structural feature, necessary for inhibition, with thiol-reduced albumin and the ligand on mouse erythrocytes.