Positively charged cholesterol-recombinant human gelatins foster the cellular uptake of proteins and murine immune reactions.

PURPOSE: Recombinant human gelatins with defined molecular weights were modified with cholesterol to make them amphiphilic in nature. We investigated the feasibility of these modified human gelatins acting as a carrier of antigenic proteins for inducing cellular immunity. The aim of this study was to synthesize novel and effective compounds for vaccine delivery in vivo. METHODS: Two types of cholesterol-modified gelatin micelles, anionic cholesterol-modified gelatin (aCMG) and cationic-cholesterol modified gelatin (cCMG), were synthesized using different cholesterol derivatives such as the cholesterol-isocyanate (Ch-I) for aCMG and amino-modified cholesterol for cCMG. One was anionic and the other cationic, and therefore they differed in terms of their zeta potential. The aCMG and cCMG were characterized for their size, zeta potential, and in their ability to form micelles. Cytotoxicity was also evaluated. The modified human gelatins were then investigated as a carrier of antigenic proteins for inducing cellular immunity both in vitro in DC 2.4 cells, a murine dendritic cell line, as well as in vivo. The mechanism of entry of the polymeric micelles into the cells was also evaluated. RESULTS: It was found that only cCMG successfully complexed with the model antigenic protein, fluorescein-isothiocyanate ovalbumin (OVA) and efficiently delivered and processed proteins in DC 2.4 cells. It was hypothesized that cCMG enter the cells predominantly by a caveolae-mediated pathway that required tyrosine kinase receptors on the cell surface. Animal testing using mice showed that the cationic cholesterol-modified gelatin complexed with OVA produced significantly high antibody titers against OVA: 2580-fold higher than in mice immunized with free OVA. CONCLUSION: Conclusively, cCMG has shown to be very effective in stimulating an immune response due to its high efficiency, stability, and negligible cytotoxicity.

The pH-sensitive fusogenic 3-methyl-glutarylated hyperbranched poly(glycidol)-conjugated liposome induces antigen-specific cellular and humoral immunity.

We examined the ability of a novel liposome, surface modified by 3-methyl-glutarylated hyperbranched poly(glycidol) (MGlu-HPG), to enhance antigen-specific immunity in vitro and in vivo and to function as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) encapsulated in MGlu-HPG-modified liposomes more effectively than free OVA or OVA encapsulated in unmodified liposomes. Immunization of mice with OVA-containing MGlu-HPG-modified liposomes induced antigen-specific splenocyte proliferation and production of gamma interferon (IFN-γ) more strongly than did immunization with free OVA or OVA encapsulated in unmodified liposomes. The immune responses induced by OVA encapsulated in MGlu-HPG-modified liposomes were significantly suppressed by addition of anti-major histocompatibility complex (MHC) class I and class II monoclonal antibodies, indicating the involvement of antigen presentation via MHC class I and II. Furthermore, delayed-type hypersensitivity responses and OVA-specific antibodies were induced more effectively in mice immunized with OVA encapsulated by MGlu-HPG-modified liposomes than with unencapsulated OVA or OVA encapsulated in unmodified liposomes. These results suggested that MGlu-HPG-modified liposomes effectively induced both cell-mediated and humoral immune responses. Collectively, this study is the first to demonstrate the induction of both cell-mediated and humoral immune responses in vivo by MGlu-HPG-modified liposomes.

Induction of antigen-specific immunity by pH-sensitive carbonate apatite as a potent vaccine carrier.

The ability of carbonate apatite (CO(3)Ap) to enhance antigen-specific immunity was examined in vitro and in vivo to investigate its utility as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) containing CO(3)Ap more effectively than free OVA. Interestingly, mice immunized with OVA-containing CO(3)Ap produced OVA-specific antibodies more effectively than mice immunized with free OVA. Furthermore, immunization of C57BL/6 mice with OVA-containing CO(3)Ap induced the proliferation and antigen-specific production of IFN-γ by splenocytes more strongly than immunization with free OVA. Moreover, no significant differences were detected in the induction of delayed-type hypersensitivity responses, an immune reaction involving an antigen-specific, cell-mediated immune response between OVA-containing CO(3)Ap and OVA-containing alumina salt (Alum), suggesting that CO(3)Ap induced cell-mediated immune response to the same degree as Alum, which is commonly used for clinical applications. This study is the first to demonstrate the induction of antigen-specific immune responses in vivo by CO(3)Ap.

Oral administration of immunopotentiator from Pantoea agglomerans 1 (IP-PA1) improves the survival of B16 melanoma-inoculated model mice.

To investigate the usefulness of the immunopotentiator from Pantoea agglomerans 1 (IP-PA1) as a supportive drug in melanoma therapy, we analyzed the immunological effects of IP-PA1 on melanoma-inoculated model mice. Oral administration of IP-PA1 increased the serum levels of tumor necrosis factor (TNF)-α at 2 h after the administration and interferon (IFN)-γ and IL-12 at 12 h after the administration in naïve BALB/cCrSlc mice as evaluated by ELISA. IP-PA1 did not affect the proliferation of melanoma cells directly determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Combinatory treatment of IP-PA1 with doxorubicin for 9 days increased the serum levels of IFN-γ and IL-12 by 71.0 and 15.3%, respectively, compared to the treatment of doxorubicin alone in melanoma-bearing C57BL/6NCrSlc mice as evaluated by ELISA. It also increased the proportion of natural killer (NK) cells and the ratio of CD4(+) to CD8(+) T cells in the spleen from 6.1 ± 0.3 to 7.4 ± 0.5% and from 1.25 ± 0.03 to 1.38 ± 0.04, respectively, compared to the treatment of doxorubicin alone as analyzed by flow cytometry. The mean survival period of melanoma-bearing, doxorubicin treated mice was prolonged from 31.4 ± 7.1 to 35.3 ± 8.4, 51.1 ± 5.4, and 45.0 ± 8.4 days by combinatory treatment of IP-PA1 at the daily doses of 0.1, 0.5, and 1 mg/kg, respectively. In conclusion, the results of the present study suggest the usefulness of IP-PA1 as a supportive drug in melanoma therapy.

Recovery from immunosuppression-related disorders in humans and animals by IP-PA1, an edible lipopolysaccharide.

Immunopotentiator from Pantoea agglomerans 1 (IP-PA1), an edible lipopolysaccharide (LPS) derived from symbiotic bacteria in crops, is a promising immunomodulator. It activates macrophages and protects from chemotherapeutic agent-induced growth inhibition in macrophages in vitro. We showed the immune-recovery effects of IP-PA1 in a chicken model of dexamethasone-induced stress in which IP-PA1 inhibited thymic and bursal atrophy and improved antibody production in response to vaccination. Furthermore, we showed IP-PA1 improved survival of melanoma-bearing, doxorubicin-treated mice, although not directly affecting the proliferation of melanoma cells, dominantly through the improvement of host antitumor immunity. These results suggest that IP-PA1 could have other possible applications in the treatment of various immunosuppression-related disorders in humans and animals.

Protective effects of the immunopotentiator from Pantoea agglomerans 1 on chemotherapeutic agent-induced macrophage growth inhibition.

BACKGROUND: The immunopotentiator from Pantoea agglomerans 1 (IP-PA1) is an edible lipopolysaccharide (LPS) derived from symbiotic bacteria found in crops. IP-PA1 is known to ameliorate chemotherapy-induced immunosuppression; therefore, its macrophage-activating effect in the presence of chemotherapeutic agents was evaluated. MATERIALS AND METHODS: Nuclear factor-kappaB (NF-kappaB) activation in IP-PA1-treated RAW264 and J774.1 cells was examined using Western blot analyses; Griess assay and ELISA were used to examine the production of nitric oxide and tumour necrosis factor alpha, respectively. The expression of apoptosis-related proteins was also assessed using Western blot analyses. The effect of IP-PA1 on doxorubicin-induced apoptosis was analyzed by flow cytometry after annexin-V staining. The growth of macrophages treated with chemotherapeutic agents and IP-PA1 was analyzed using an MTT assay. RESULTS: IP-PA1 activated NF-kappaB and ameliorated chemotherapy induced growth inhibition in the cells. CONCLUSION: IP-PA1 is an edible drug that can potentially support chemotherapy by ameliorating chemotherapy-induced immunosuppression.

Immune recovery effects of immunopotentiator from Pantoea agglomerans 1 (IP-PA1) on low antibody productions in response to Salmonella enteritidis vaccine and sheep red blood cells in dexamethasone-treated stressed chicken models.

Considering the usefulness of the immunopotentiator from Pantoea agglomerans 1 (IP-PA1), which is a purified lipopolysaccharide (LPS) derived from symbiotic gram-negative bacteria of food crops, in controlling immunosuppression in poultry husbandry, in this study, we examined its immune-recovery effects in dexamethasone-treated stressed chicken models. Three-week-old chickens daily administered 10 microg/kg of dexamethasone for 35 days to induce stress showed more whole body weight loss; relative thymic, bursal, and splenic weight losses; and decrease in the number of peripheral blood lymphocytes, as compared with the control chickens on day 35; the IP-PA1-pretreated, dexamethasone-treated chickens showed reduced weight losses. Five- to eight-week-old chickens administered 5 mg/kg of dexamethasone showed excessive apoptosis of thymic and bursal lymphocytes 24 hr after a single dexamethasone treatment; apoptosis was inhibited in the IP-PA1-pretreated, dexamethasone-treated chickens. Chickens daily administered 10 microg/kg of dexamethasone for 35 days and injected with commercial Salmonella Enteritidis (SE) vaccine or sheep red blood cells (SRBC) on days 7 and 21 showed about 8- or 2-fold lower antibody production in response to SE or SRBC, respectively, as compared with the control chickens on day 35; the antibody production in response to SE or SRBC was increased in the IP-PA1-pretreated, dexamethasone-treated chickens. These results indicate that IP-PA1 exerts inhibitory effects on dexamethasone-induced immunosuppression and that it may be useful in controlling immunosuppression in poultry husbandry.