In vitro correlates of hapten-specific delayed hypersensitivity

Azobenzenearsonate conjugates of l and d-amino acid polymers were studied in respect to their ability to stimulate lymphocytes from guinea pigs sensitized to the hapten. Using the two in vitro techniques of thymidine incorporation and inhibition of macrophage migration, it was shown that only conjugates of the l-amino acid polymers were active.The results confirm previous in vivo studies and suggest as one possibility that a preliminary processing event by macrophages may be obligatory for all associated phenomena of hapten-specific delayed hypersensitivity.     

Requirements for inducing tolerance of hapten-specific delayed hypersensitivity: epitope density.

Tolerance to hapten-specific antibody formation and delayed hypersensitivity was examined in adult rabbits. The azobenzenearsonate (ABA) or sulfonate-specific antibody response to hapten-hemocyanin immunogens was suppressed by deaggregated hapten-rabbit IgG conjugates given 21 and 14 days before challenge. High affinity antibody was preferentially suppressed. Delayed hypersensitivity to ABA-tyrosine was suppressed by deaggregated ABA-rabbit IgG conjugates injected 17 and 10 days before challenge. Conjugates with a high hapten density, ABA15-23-rabbit IgG were effective tolerogens. Conjugates with four to six ABA groups per carrier molecule were very poor tolerogens. Increasing the amount of low substituted conjugate injected did not improve tolerogenicity. It appears that a high epitope density is required for effective induction of tolerance to ABA-specific delayed hypersensitivity in the rabbit.     

Comparison of bcg strains for delayed hypersensitivity induction tested in vivo in guinea pigs and in vitro

Previous studies performed on guinea pigs demonstrated a direct dependence of tuberculin reaction size (in vivo hypersensitivity) on immunogenicity in a number of BCG strains. The present work used an in vitro method, MIF detection, for assessing hypersensitivity and compared the results obtained with tuberculin hypersensitivity tests, correlating the data with the immunogenicity of the individual BCG strains employed. The following strains were used: the Czechoslovak BCG strain No. 725, Japanese BCG strain Tokyo, Danish BCG strain Copenhagen and Soviet BCG strain Moscow. The results obtained by the two hypersensitivity testing methods, in vivo and in vitro were in a direct correlation; a direct relationship was also demonstrated between hypersensitivity tested by the in vitro method and immunogenicity. The in vitro method of MIF detection is reproducible and comparable with the other two methods employed and may be used as an alternative approach to BCG vaccine efficacy testing. It might probably also be applicable to estimation of the status of cell-mediated immunity against intracellularly parasitizing bacteria in general.     

Structural requirements for steroid inhibition of sheep lymphocyte mitogenesis in vitro

The inhibitory effects of different steroids and related compounds on sheep peripheral blood lymphocytes (PBL) during exposure to the mitogen, phytohemagglutinin (PHA), have been measured by the reduction of [3H]thymidine incorporation into DNA. Dose-response curves showed that a maximum (or near maximum effect) was achieved at a steroid concentration of 12.5 microM. At this dose 19 of 41 compounds significantly reduced thymidine incorporation by activated PBL (P less than 0.01 to P less than 0.001). The greatest reduction was observed with 17-hydroxyprogesterone (-59%, i.e. reduced by 59% compared with vehicle control, 100%) greater than androstenedione greater than epitestosterone greater than estradiol-3-methyl ether greater than 20 alpha-dihydroprogesterone greater than medroxyprogesterone acetate greater than 5 beta-pregnane-3,20-dione greater than 5 alpha-pregnane-3,20-dione (-24%). Among the steroids which showed the greatest inhibitory effect, 6 had a 4-en-3-one group in ring A, 4 had a saturated ring A (pregnane or androstane) and one had a 3-methyl ether group and a phenolic ring A. The wide range of structures represented by these inhibitory steroids suggests that inhibition of lymphocyte mitogenesis involves more than one mechanism.     

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.