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Materials and Methods For Rodent Diabetes Experiemts

Animals

        Pregnant NOD/MrkTacfBR mice were purchased from Taconic (Germantown, NY) and their offspring received the immunization schedules described below. Diabetic prone and diabetic resistant BB/Wor rats, were purchased from the University of Massachusetts (Worchester, MA). Spleen cells from each diabetic resistant rat were suspended in PBS and injected into two diabetic prone female rats. The latter were then mated with diabetic prone male rats and the offspring were used in the trial.

Vaccines

The following vaccines were used: anthrax vaccine (Michigan Department of Health, Lansing, Michigan); pediatric combined diphtheria and tetanus vaccine, DT, (Connaught, Swiftwater, PA); and combined diphtheria, tetanus and whole cell pertussis vaccine, DTP,(Connaught) and plague vaccine (Miles, West Haven, CT). All vaccines were produced from killed microorganisms, and all have an aluminum based adjuvants except the plague vaccine. 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 and 100 parts of 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.

Blood Glucose Determination

        Tail blood was removed and checked for glucose using glucose sensitive chemstrips (Boehringer Mannheim, Indianapolis, IN). In NOD mice experiments the chemstrips were read manually and blood glucose levels over 300 mg/dl were considered positive. In BB rat experiments chemstrips were read using an Accu-Chek III monitor (Boehringer Mannheim) and glucose levels above 250 mg/dl were considered positive.

 

Statistics

P values were calculated from differences in population proportions using a one tail Student's t test. Actual calculations were made using the probability calculator in the program Statistica (Statsoft, Tulsa, OK).

 

Administration of the Anthrax or Plague Vaccine Prevents Diabetes in Female NOD Mice

The first experiment involved injecting the anthrax vaccine diluted in PBS into female NOD mice (n=19) on day 8 (.1 ml, 1:100), day 15 (.15 ml, 1:50), and day 29 (.2 ml, 1:50) of life. A second group of NOD (n=20) received plaque vaccine diluted in PBS on the same days but at a slightly lower dilution, day 8 (.1 ml, 1:50), day 15 (.15 ml, 1:50), and day 29 (.2 ml, 1:25). A third group of NOD mice (n=20) received a similar volume of PBS on the same days as the mice in the first two groups. Starting at approximately 16 weeks and continuing every 2 weeks until 28 weeks, tail blood was removed and checked for glucose. The cumulative incidence of diabetes in the anthrax vaccine treated group flattened out at 42.1% with no new cases detected after 24 weeks. The group receiving the plague vaccine appeared to begin flattening out and reached a cumulative incidence of 57.9% diabetic at 28 weeks. The PBS control group, showed a continual increase in the cumulative incidence of diabetes from 30% at 16 weeks to 65% at 28 weeks . The remaining anthrax vaccine treated animals and PBS controls were bled at 36 weeks. The net result was that no new cases of diabetes were detected in the anthrax vaccine treated group from 24 to 36 weeks and the cumulative incidence of diabetes flattened out at 42.1% compared to an incidence of 75% at 36 weeks (P=0.0219) in the PBS treated mice.

 

Pediatric Vaccines Act Synergistically with the Anthrax Vaccine to Prevent Diabetes in Female NOD Mice

       A second set of experiments was designed to see if the therapeutic potential of the anthrax vaccine could be improved by adding additional agents and changing the dosing schedule. Anthrax vaccine was given alone, with the DT vaccine, or with the DTP vaccine. A series of nine intraperitoneal injections of the vaccines were given to newborn NOD female mice using the following protocol: day 1 (.1 ml, 1:100), day 3 (.1 ml, 1:100), day 10 (.15 ml, 1:100), week 4 and every 2 weeks through week 14 (.2 ml, 1:50). A control group received injections of PBS using the same dosing protocol.

 

    The prolonged dosing schedules greatly reduced the development of diabetes in the NOD mice. The group receiving the anthrax vaccine alone (n=29) had a cumulative incidence of diabetes that peaked at 24 weeks and remained constant at 13.8% through the 32nd week . These results compare to a cumulative incidence of 42.1% in the earlier experiment in the group that received only 3 injections of the anthrax vaccine. The administration of the DT vaccine mixed with the anthrax vaccine inhibited diabetes to a greater extent than the anthrax vaccine alone. Only 7.7% of NOD mice receiving the combination of the anthrax and the DT vaccines (n=26) developed diabetes by 32 weeks. Mice treated with the anthrax vaccine combined with the DTP vaccine had an even more favorable response. None of 29 NOD mice developed diabetes by 32 weeks of age. The injections of PBS slowed the progression of diabetes but the cumulative incidence curve did not flatten out during the 32 week study. The cumulative incidence of diabetes in the PBS control was 48.1% at 32 weeks compared to 65% at 28 weeks in the earlier PBS control using only three injections.

 

Administration of the DTP Vaccine Starting at 2 Months of Age May Enhance the Development of Diabetes in Female NOD Mice

     

        Several experimental groups were designed to study the effect of administering a single injection of DTP vaccine before 4 months of age, as is common in the United States. A group that received 1 injection of the DTP vaccine at 8 weeks (.2 ml, 1:50) developed a cumulative incidence of diabetes of 86.7% by 32 weeks of life. This incidence was higher than in either control groups receiving PBS.

        A second experiment was designed to see the effect of administering a single dose of the whole cell pertussis vaccine at 8 weeks of life to a group already receiving the anthrax and DT vaccines. Only 7.7% of animals receiving the combination of the anthrax and DT vaccines (n=26) developed diabetes by 32 weeks while 23% developed diabetes (P=0.065) in the group receiving the DTP vaccine in place of the DT vaccine at week 8 (n=26). The data from the two experiments above provides evidence that immunization with the DTP vaccine starting at 8 weeks of life does not prevent diabetes and is likely to enhance the disease.

 

Administration of High Doses of the Anthrax and DTP Vaccines to BB/Wor Rats Prevents the Development of Diabetes

A study was performed to determine if the combination of the anthrax and DTP vaccines could prevent the development of diabetes in BB/Wor rats as it did in the NOD mice. Two doses of the combined vaccines were used and a group of untreated rats (n=28) was used as a control. Groups contained approximately equal number of male and female rats. The low dose group (n=28) received a series of 8 intraperitoneal injections of the anthrax and DTP vaccines. The dosing schedule was as follows: day 1 (.1 ml 1:20); day 4 (.1 ml, 1:20); day 11 (.15 ml, 1:20); and every 14 days at (.2 ml, 1:20) through week 12. The high dose group (n=20) received the following dosing schedule: day 1 (.1 ml, 1:5); day 4 (.15 ml, 1:5), day 11 (.15 ml, 1:5), day 25 (.2 ml, 1:5), day 39 (.2 ml, 1:5), day 53 (.2 ml, 1:5), day 61 (.2 ml 1:2.5), and every 14 days for 3 more injections at approximately (.2 ml, 1:2.5). At 20 weeks of age 54% of the untreated rats and approximately 57% of the low dose group of rats had developed diabetes and or died compared to 25% in the high dose group (P=.0269).