Antiphospholipid antibodies, including anti-

In previous studies of infectious mononucleosis, we found IgM autoantibodies which react with hematopoietic cell antigens. Many of these were inhibited by synthetic glycine/ alanine peptides representing the glycine/alanine repeat of Epstein-Barr virus nuclear antigen-i. We have cloned and expressed fragments of genes encoding two of these autoantigens. One gene (p542) encodes a protein containing a glycine-rich 28-mer, which is its chief autoantigenic epitope and which represents a newly identified class of evolutionarily conserved autoepitopes. The other gene (p554) encodes a protein that is not demonstrably cross-reactive with EpsteinBarr virus nuclear antigen-1 or with any other EBV protein, but forms complexes with other proteins. Immunoaffinitypurified anti-p542 and anti-p554 have relatively high binding affinities, as evidenced by inhibition at 106-108 M-', and neither autoantibody showed polyreactivity with other common antigens. The data thus suggest that neither autoantibody is simply an expression of polyclonal B cell activation. We conclude that the two autoantigens stimulate autoantibody synthesis by different mechanisms. One autoantigen shares homology to a viral protein which generates cross-reacting antibodies to the autoantigenic epitope. The other has no recognizable cross-reaction with the infecting pathogen and may become immunogenic through complexing with other proteins. (J. Clin. Invest. 1995Invest. . 95:1306Invest. -1315

UC 729-6, a 6-thioguanine-resistant human lymphoblastoid B-cell line, was fused with normal and malignant human lymphocytes. Parent UC 729-6 cells were diploid with a 21p+ marker chromosome, expressed surface and cytoplasmic IgM Kc, and doubled every 17 hr. The resulting human-human hybridomas were pseudotetraploid containing the 21p+ marker, doubled every 20-30 hr, and secreted 3-9 jig of human Ig per ml per 106 hybrid cells for >9 months in continuous culture. Human Ig-secreting hybridomas were generated in 88% (14/16) of the fusions carried out and were cloned by limiting dilution (one cell per three wells) without the use of feeder layers. The mean fusion frequencies (number of wells, plated at 105 cells per well, showing hybrid growth per 106 lymphocytes fused) of UC 729-6 with normal lymphocytes ranged from 0.45 to 2.9 and with malignant B lymphocytes, from 3 to 10. Analysis of the human-human hybridomas derived from lymphocytes isolated from regional draining lymph nodes of cancer patients revealed several that secreted human monoclonal antibody that reacted by an enzyme immunoassay with some carcinoma cell lines but not with normal fibroblast cell lines. These data suggest that (i) UC 729-6 can be fused with human lymphocytes to generate stable human-human hybridomas, some of which secrete antibody reactive to human cell surface antigens, and (ii) UC 729-6 can be used to rescue Ig from nonsecretory malignant B cells and thereby allow for the production of anti-idiotype antibodies.The use of somatic cell hybridization to produce monoclonal antibodies (mAbs) of predefined specificity, as pioneered by Kohler and Milstein (1), has revolutionized many areas of human biology. However, since most of these mAbs are of rodent origin their application to in vivo use in humans may be limited. Therefore, the production of human mAbs could potentially be of considerable value and in many instances may offer advantages over mAbs of other species.Currently available human mAbs have been obtained by mouse-human hybridizations (2-4), by Epstein-Barr virus (EBV) transformation of human immune lymphocytes (5-7), and by human-human hybridization (8-10) and has recently been reviewed (11). However, each of these approaches suffers from individual difficulties and each has met with limited success. Mouse-human hybrids preferentially lose human chromosomes and, therefore, are genetically unstable (12, 13). EBV transformation yields cell lines that produce small quantities of Ig (5-7). The reproducibility of generating human-human hybridomas is limited by the lack of a suitable fusion partner in that currently available cell lines for fusion (8)(9)(10) 6327The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Background/Aims: To test the safety and efficacy of the Aethlon Hemopurifier®, a lectin affinity cartridge, in clearing hepatitis C virus (HCV) from the blood of HCV-positive end-stage renal disease patients undergoing dialysis. Viral RNA was measured using real-time quantitative reverse transcriptase polymerase chain reaction. Results: HCV clearance from plasma or blood was measured using either direct capture on immobilized Galanthus nivalis agglutinin (GNA) or using miniature plasmapheresis cartridges containing immobilized GNA. HCV in plasma samples was rapidly cleared by direct affinity capture (t_1/2 = approx. 20 min) and HCV in human blood was cleared using the Hemopurifier (t_1/2 = 2–3 h). Institutional-review-board-sanctioned clinical safety studies were conducted at the Apollo and Fortis Hospitals in India. At Apollo, 4 patients were treated 3 times/week for 2 weeks. HCV captured on the Hemopurifier averaged 8.9 × 10⁸ viral copies/cartridge (n = 5), representing approximately 30% of the initial viral body burden. At Fortis, 3 patients treated 3 times/week for 1 week completed the viral load studies. Two patients showed measurable viral load reduction, while the third showed both increases and decreases in viral load. After Hemopurifier treatment, average HCV viral load was reduced by 57%. Surprisingly, average viral load was also 82% lower 7 days after treatment. Control samples also showed a marked transient reduction in HCV viral load as previously reported. Conclusion: The Hemopurifier rapidly cleared HCV from blood treated in vitro. In patients, the combination of the Hemopurifier plus dialysis decreased HCV viral load by 57% in 1 week. Moreover, viral load reduction continued up to 7 days after treatment.

The effect of Fibroblast Growth Factor (FGF) on the initiation of DNA synthesis and on the rate of division of the Y1 adrenal cell line has been investigated. In sparse populations of Y1 cells (4 times 10- minus 3 cells/cm2) maintained in 0.2 percent calf serum, FGF was able to initiate DNA synthesis to the same extent that an optimal concentration of serum could. Cells maintained in 0.2 percent calf serum sustained continuous growth when given 5 ng/ml of FGF daily. Cultures fixed and stained with crystal violet showed FGF colonies to be of equivalent size and quantity as those with serum alone. Insulin had no mitogenic activity of its own at concentrations as high as 500 ng/ml nor did it have any potentiating effect on the mitogenic activity of FGF. Glucocorticoids (0.1 mug/ml to 1 mug/ml) inhibited (25 percent) the initiation of DNA synthesis as well as the rate of division induced by FGF. ACTH (0.75 IU/ml) was clearly inhibitory. Not only did it reduce the rate of division of cells in serum but it also reduced the rate of DNA synthesis and inhibited division in the presence of FGF.