T cell stimulation.

<p>Splenocytes isolated from immunized (2–3 months after the final boost) and non-immunized C3H/HeNmice were incubated with increasing concentrations of hTg protein for 72 hrs. Splenocytes from hTg immunized/electroporated mice showed significantly increased proliferation, compared to similarly challenged splenocytes from non-immunized mice. The x axis represents the concentration (µg/ml) of hTg which was used to stimulate the splenocytes. All T-cell proliferation experiments were performed in triplicates. Data, which were initially recorded as cpm, were subsequently expressed relative to splenocytes which did not receive thyroglobulin, are shown as mean + SEM.</p

IgG subtypes or anti-mTg antibodies produced by hTg immunized/electroporated mice.

<p>Three, 8-week old C3H/HeN mice received hTg immunization/electroporation while 4 mice received an empty pcDNA plasmid electroporation (denoted by ‘**’). Anti-mouse Tg antibodies of the IgG2a subclass were the most prevalent isotype, followed by IgG1. There were very low levels of anti-mTg antibodies of the IgG2b class. All ELISA experiments were performed in triplicates. Data are presented as mean + SEM.</p

Effects of different means of cDNA delivery on anti-Tg antibody response.

<p>Shown is an ELISA measuring total IgG (all subclasses), directed against murine thyroglobulin (Tg). The results demonstrate a significant anti-Tg response in mice immunized with hTg cDNA. For this analysis, we compared 2 non-immunized mice, the 2 best responders, out of a group of 6 that were immunized with hTg without electroporation, and 6 hTg cDNA immunized/electroporated mice. Relative to directly immunized mice, the use of an electric current (6 pulses total, 60 V/cm, and 50 ms pulse length, with a 200 ms respite between pulses) caused a significant increase in the level of anti-Tg antibodies (p = 0.037). All readings were performed in triplicate. Data are shown as mean + standard error of the mean (SEM). Animals immunized with empty vector (pCDNA3.1+) exhibited no response (data not shown) to mouse Tg.</p

IgG subtypes or anti-mTg antibodies produced by C3H/HeN mice induced with classical EAT by immunizing with hTg protein and adjuvant.

<p>Three, 8-week old C3H/HeN mice were intravenously immunized with 50 µg of hTg in 100 µl of PBS into the tail vein. 2–3 hours later, 20 µl of LPS was injected into the tail vein. Three immunizations, spaced two weeks apart, were administered. Animals were sacrificed two weeks after the third and final round of immunization. At sacrifice, sera were collected and were analyzed for the presence of anti-mTg and the specific isotype of the antibodies. All ELISA experiments were performed in triplicates. Data are shown as mean + SEM.</p

Th1 nature of the Cytokine Profile.

<p>Splenocytes were isolated from immunized/electroporated (2–3 months after the final boost) and non-immunized mice. 2×10⁵ cells were challenged with 0, 5, or 20 µg/ml of human thyroglobulin. Supernatants were collected and were analyzed for the presence of cytokines. hTg-treated splenocytes from immunized/electroporated animals secreted significantly higher levels of interferon-gamma than controls. Mice immunized with empty vector showed no differences compared to the non-immunized controls (data not shown). All readings were performed in triplicate. Data are shown as mean + SEM.</p