3.1 Characterization of the synthesized Indo and Pam-Indo
Indo synthesized using the FMOC chemistry was purified by reverse phase HPLC on a C18 column. Figure 5A showed that Indolicidin was eluted as a single peak at 18.8 min. These results showed that synthesis of Indo had proceeded smoothly, and a highly purified grade of the peptide was obtained. Indo was found to have a mass of 1908 Da when it was analyzed in a LC/MS mass spectrometric (Fig 5B ). This is the expected mass for Indo.
Pam-Indo synthesized by coupling Pam to Indo using Fmoc chemistry was also analysed using the reverse phase HPLC on a C18 column. Figure 6A showed Pam-Indo was eluted as a single peak at 27.3 min. LC/MS mass spectrometric analysis of Pam-Indo found that it had a mass of 2449 Da (Figure 6B), and this was its expected mass.
3.2 Immunomodulatory property studies of Pam-Indo, and Indo in BALB/c (H-2d) mice
To investigate the immunomodulatory properties of Pam-Indo and Indo, these compounds were individually mixed with FI-H5N1, and each of the formulations was injected into BALB/c (H-2d) mice. Blood samples were collected from each of the experimental mice, and assayed for the presence of antibodies binding to FI-H5N1immobilized on ELISA wells. Results obtained from these experiments showed that antibodies reacting against FI-H5N1 were not detected in the prebled serum samples of the unimmunized mice. High titers of serum anti-FI-H5N1
antibodies were meaured in mice injected intramuscularly with 3.07 x 107 pfu of FI-H5N1. The antibody titers varied between 1.0 x 105 and 1.1 x 103 for the 6 mice in this group (Fig 7). Mice injected with FI-H5N1-Indo formulation, or FI-H5N1-Pam formulation mounted about the same level of anti-FI-H5N1 antibody responses. However, antibodies reacted against FI-H5N1 were detected at much lower level in mice immunized with FI-H5N1 formulated in Pam-Indo. The antibody titers in this group of mice range from 2 x 103 to 4 x 103. So, they were about forty-fold lower than the levels in mice immunized with FI-H5N1, FI-H5N1 formulated in Indo, or FI-H5N1 formulated in Pam. Another group of mice injected with FI-H5N1 formulated in a mixture of Indo, and Pam also produced much less FI-H5N1-reactive antibodies. The mean anti-FI-H5N1-specific antibody titer measured in this group of mice was 6.35 x 103, which was slightly higher than the values detected in the FI-H5N1/Pam-Indo-formulation imunized group. The results obtained from these studies showed that immunomodulatory property of Indo requires Pam to be present.
Conjugating Pam to Indo to create a composite structure is a more potent immunomodulatory agent than a mixture of Indo and Pam.
A well known immunological phenomenon is that B cells responding to antigens require T helper cells. When they receive “help”, B cells activated by antigens can differentiate to become antibody-producing (plasma) cells [22]. To see how Pam-indo influenced this mechanism, T cell responses to FI-H5N1 were analyzed. The experiments were done by removing spleens from each of the experimental mice, and the splenocytes were cultured with FI-H5N1 to evaluate the numbers of T cells that are stimulated to produce IFN-, and IL-4. These cytokines are produced by antigen specific T-helper cells called Th1 and Th2 lymphocytes, respectively. The results obtained from these experiments were shown in Figure 8. The numbers of
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“immuno-spots” representing lymphocytes secreting IFN- scored in mice immunized with FI-H5N1 were between 76, and 179. IL-4-secreting cells in this group of mice varied between 69, and 174. For mice immunized with FI-H5N1 formulated in Pam-Indo, the numbers of IFN--, and IL-4-secreting cells detected were between 15 and 56, and 10 and 31, respectively. These numbers are significantly lower than those scored in the FI-H5N1-immunized mice. The numbers of T cells secreting these cytokines between mice immunized with Pam-Indo formulated FI-H5N1 and Indo formulated FI-H5N1 are about the same. Mice immunized FI-H5N1 formulated in Pam generated about equal numbers of IFN--, and IL-4-secreting cells as compared to FI-H5N1 immunized mice. These results suggest Pam-Indo, or a mixture of Pam and Indo immunomodulated to generate less number of FI-H5N1-specific CD4+ T cells. Fewer numbers of FI-H5N1-specific Th2 T cells generated had caused lower level of antibody responses elicited against FI-H5N1.
3.3 Effect of Pam-Indo, and Indo on the maturation of dendritic cells.
FI-H5N1-specific CD4+ T cells are generated from naïve T cells (those that have not contacted with antigen before) following their interaction with APCs expressing the MHC class 2 molecules associated with H5N1 peptides. In the presence of IL-12 secreted by APCs, the activated CD4+ could develope into Th1 cells. If IL-4 is present at a high level, the activated CD4+ T cells would differentiate into Th2 cells. With these immunological concepts in mind, the effect of Pam-Indo on the activation of APCs was investigtaed. The experiments were done by flushing out bone marrow cells from the femur and tibiae bone of BALB/c (H-2d) mice. The bone marrow cells were then cultured in the presence of murine GM-CSF to generate immature myeloid
dendritic cells (imDCs). Lipopolysaccharide (LPS) stimulation would cause the activation of imDCs to express higher level of the costimulatory molecules. It was found that Pam-Indo affected LPS-activation of imDCs since the expression of CD40 and CD86 molecules are significantly downregulated. The effect was depndent on the amount of Pam-Indo added to the LPS-stimulated imDC cultures. Pam-Indo at greater than 10.0 g/mL was required to suppress CD40. Significant suppression of CD86 could be achieved at much lower Pam-Indo concentration, at 2.5 g/mL.