Nonimmune rhesus were readily infected by administration of SV40 by intranasal (i.n.), subcutaneous (s.c.), or intragastric (i.g.) route. Viremia, demonstrable in animals of all three groups, occurred earlier and with higher titers in the s.c. group. Neutralizing and viral CF antibodies appeared in all inoculated rhesus and T antibodies in 14 of 16. Viruria was detected in three of six of the s.c. group, most commonly in the third week after inoculation. Virus neutralizing activity occurred in the urine at or after 9 weeks postinoculation and was not confined to previously viruric animals. Virus was isolated from kidney of one rhesus from biopsy cultures performed 29–31 weeks after inoculation. Three rhesus fetuses which were given SV40 intracerebrally and subcutaneously at about 90 days of gestation had, at or soon after birth, high titers of antibodies and no tumors. In the single surviving infant, antibody titers did not fall in the first 11 weeks of life.
The pharmacokinetics and pharmacodynamics (PK/PD) of chimeric (Ch5c8) and humanized (Hu5c8) 5c8, a monoclonal antibody that binds CD154 (CD40 ligand), thus blocking the interaction between CD40 and CD154, were investigated in cynomolgus monkeys. Single-dose groups (n = 3 animals per dose) received saline, 0.2, 1, 5 or 20 mg/kg i.v. doses of Hu5c8. The repeat-dose groups (n = 4 animals) received 0 or 5 mg/kg i.v. doses of Ch5c8 or Hu5c8 on days 1, 2, 3, 5, 7 and 9. The single-dose PK parameters showed dose proportionality, with a terminal half-life of 300 h, a volume of distribution at steady state of 73 ml/kg and clearance of 0.2 ml.h-1.kg-1. The repeat-dose regimen produced a longer terminal half-life (500 h) and lower clearance (0.13 ml.h-1.kg-1) than in the single-dose groups. The antibody titer to tetanus toxoid (ATT) challenge served as the immunodynamic marker. The primary ATT response consisted of a latent phase of approximately 10 days, during which the immune system was processing antigen but not yet producing antibody, a rise to an antibody maximum titer at approximately 18 days and a decline toward baseline by approximately 40 days in controls. The 5c8 produced a log(dose)-proportional reduction in the area under the curve of ATT. An indirect PK/PD model based on the kinetics of tetanus toxoid exposure and inhibition of ATT production in relation to 5c8 concentrations was developed. A median inhibitory concentration of 0.84 microg/ml and a efficacy of 0.84 reflected marked inhibition of ATT response by 5c8. The model provides quantitation of reduced ATT responses after 5c8 and was applicable to primary and secondary immune responses and to both single-dose and multiple-dose treatments. The monoclonal antibody 5c8 blocks the CD40 and CD154 interaction, producing consistent and substantive reduction in antibody formation after administration of tetanus toxoid, which can be characterized with PK/PD modeling. It is anticipated that 5c8 may have utility in the treatment of antibody-mediated autoimmune disease.
Blockade of the CD154–CD40 co-stimulatory pathway with anti-CD154 mAbs has shown impressive efficacy in models of autoimmunity and allotransplantation. Clinical benefit was also demonstrated in systemic lupus erythematosus (SLE) and idiopathic thrombocytopenia patients with the humanized anti-CD154 mAb, 5C8 (hu5C8). However, thromboembolic complications that occurred during the course of the hu5C8 clinical trials have proven to be a major setback to the field and safe alternative therapeutics targeting the CD154–CD40 pathway are of great interest. Recently, effector mechanisms have been shown to play a part in anti-CD154 mAb-induced transplant acceptance in murine models, while this issue remains unresolved for humoral-mediated models. Herein, aglycosyl anti-CD154 mAbs with reduced binding to FcγR and complement were used as a novel means to test the role of effector mechanisms in non-human primate and murine models not amenable to gene knockout technology. While aglycosyl hu5C8 mAb was relatively ineffective in rhesus renal and islet allotransplantation, it inhibited primary and secondary humoral responses to a protein immunogen in cynomolgus monkeys. Moreover, an aglycosyl, chimeric MR1 mAb (muMR1) prolonged survival and inhibited pathogenic auto-antibody production in a murine model of SLE. Thus, the mechanisms required for efficacy of anti-CD154 mAbs depend on the nature of the immune challenge.
Bovine spermatozoa were separated into different density subpopulations utilizing water insoluble hydrocarbon and silicone gels of defined specific gravity. Sperm density profiles were generated for 13 bulls. The separations were found to be repeatable and characteristic of the bull examined. Considerable density variation among animals was demonstrated. Analysis of the separated spermatozoa, before and after freezing, demonstrated that good motility and acrosomal integrity of spermatozoa were maintained. When the least dense fraction of spermatozoa was used for insemination, conception rates were similar to those obtained routinely by artificial insemination with unfractionated spermatozoa. Therefore, this system may be useful in separating spermatozoa of various densities and for removing extraneous matter from semen. However, the sex ratio, among 51 60-day-old fetuses recovered from heifers inseminated with the lowest density fraction of spermatozoa, was 26 males:25 females.
Hyperthyroid Graves’ disease is a common autoimmune disorder mediated by agonistic antibodies to the TSH receptor, termed thyroid stimulating antibodies (TSAbs). Recently members of the TNF superfamily, B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), have been identified along with their receptors, B cell maturation antigen and transmembrane activator and calcium-modulator and cyclophilin ligand interactor, and the BAFF-specific receptor. BAFF is a fundamental B cell survival/maturation factor, and both BAFF and APRIL have been implicated in antibody production. We investigated the effect of interfering with BAFF- and APRIL-mediated signals in an induced model of Graves’ disease by blockade of these factors using soluble decoy receptors. In a therapeutic setting in mice with established hyperthyroidism, we show that blockade of BAFF or BAFF+APRIL with BAFF-specific receptor-Fc and B cell maturation antigen-Fc, respectively, leads to significant reductions in the induced hyperthyroidism. This was supported by a parallel pattern of declining TSAbs in the responding animals. Histopathological analysis of splenic sections from treated animals revealed marked reductions in the B cell follicle regions, but staining with anti-CD138 revealed the persistence of plasma cells. Thus, the reductions in TSAbs in the treated animals were not related to overall plasma cell numbers in the secondary lymphoid organs. Our results are the first to demonstrate attenuation of established hyperthyroidism by therapeutic intervention aimed at autoreactive B cells and indicate that both BAFF and APRIL appear to play important roles in the development and survival of the autoantibody producing cells in this model.
