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        1. The graph below shows that  immunization starting in the second week of life can prevent organ specific autoimmunity in animals which have been made prone to autoimmunity by exposure to cyclosporine in the first week of life .

 

cyclosporine01a.wmf (20046 bytes)

 

Methods

CsA 

  Cyclosporine powder, CsA, Sandoz (East Hanover, NJ) was dissolved in a solution containing a 2:1 volume by volume mixture of Cremophor (Sigma, St. Louis, MO) and 95% ethyl alcohol (Warner-Graham, Cockeysville, MD) at a concentration of 50 mg CsA / ml. The mixture was further diluted in PBS and sterile filtered.

Animals

    Pregnant C3H/Hen mice were purchased from Harlan Sprague Dawley (Indianapolis, IN) and the offspring were used in the cyclosporine trial.

Vaccines

The following vaccines were used: anthrax vaccine (Michigan Department of Health, Lansing, Michigan); combined diphtheria and tetanus vaccine, DT, (Connaught, Swiftwater, PA); and combined diphtheria, tetanus and whole cell pertussis vaccine, DTP,(Connaught). All vaccines are produced from killed microorganisms, and all have an aluminum based adjuvant. Vaccines were diluted using sterile technique in sterile phosphate buffer solution, pH 7.4, using the notation 1:100 to indicate 1 part vaccine per 100 parts PBS by volume. In those examples where a combination of two vaccines is given the notation 1:100 refers to 1 part of each vaccine per 100 parts PBS by volume. On occasions the actual day of injection of the vaccines varied by plus or minus one day from the planned immunization schedules discussed below.

ELISAs

Tail blood was drawn from CsA treated mice at the age of about 8 weeks and the resulting sera, diluted 1:80 in a solution containing PBS and 3% fetal calf serum, were screened for autoantibodies using an ELISA assay against gastric antigens %%34(Sakaguchi & Sakaguchi, 1989)%%. The microsomal antigens were plated on Immulon 3 plates (Dynatech Laboratories, Chantilly, VA). An alkaline phosphatase-conjugated anti-IgG Fc fraction (Jackson Immunoresearch, West Grove, PA) was used as the secondary antibody and the substrate was a 2 x 10-4 M 4-Methyumbelliferyl phosphate solution (Classen & Shevach, 1991). Plates were read on a Dynatech MicroFLOUR machine that uses a 365 nm Broadband Filter for the excitation beam and 450 nm narrow band interference filter for the emission beam. Sera was scored positive based on a absorbance that was twice the level of absorbance produced by an a reference standard sera. The later reference was placed on each plate in triplicate allowing samples from different plates to be compared.

 

Administration of Vaccines to Cyclosporine Treated Mice Prevents the Development of Anti- Gastric Antibodies

Newborn C3H/Hen mice were injected with 15mg/kg/day of cyclosporine intraperitoneally for the first 7 days of life to make the animals prone to developing autoimmunity (Sakaguchi & Sakaguchi, 1989). Female mice were then injected with a combination of the anthrax vaccine and the DT vaccine (n=23), or PBS (n=25) starting several days later to see if the vaccine would prevent the development of autoimmunity. The injection schedule was as follows: day 10 (.15 ml, 1:100), day 17 (.2 ml, 1:50) and every 2 weeks for 2 more injections (.2 ml, 1:50). A second injection schedule was performed on a group of male and female mice using the anthrax and DTP vaccines (n=23). Injections were as follows: day 7 (0.15 ml, 1:10); day 15 (0.2 ml, 1:10); and day 28 (0.2 ml, 1:10). Of those receiving the anthrax and DT vaccines 22% were free of anti-gastric antibodies compared to 12% of female mice that received the PBS control. The effect was significantly greater in those mice receiving the anthrax and DTP vaccines, as 61% did not develop anti-gastric antibodies (P=.0005) .