Akio Shiraishi

Oral administration of a dominant T-cell determinant peptide inhibits allergen-specific TH1 and TH2 cell responses in Cry j 2–primed mice

BACKGROUND Oral immunotherapy with a peptide for allergic immune responses is theoretically a promising therapy but has not been established yet. OBJECTIVE To evaluate immune suppressive efficacy of oral administration of an immunodominant peptide, we investigated changes in T-cell proliferation, TH1 - and TH2 -cytokine production, and TH1 - and TH2 -mediated antibody production in mice after oral administration of a peptide. METHODS Peptide p246-259, containing a dominant T-cell determinant of Cry j 2, which is the major allergen in Japanese cedar pollen, was used in this study. Groups of mice received p246-259 or PBS alone before or after they were primed intranasally with Cry j 2 and cholera toxin. In another experiment mice were primed intraperitoneally with Cry j 2 and alum. Proliferative response and cytokine production by nasal-associated lymph node cells against Cry j 2 were investigated. Amounts of systemic anti-Cry j 2 IgE and IgG antibodies were also measured. RESULTS Oral administration of the peptide to mice before, or even after, the sensitization induced oral tolerance in T-cell responses against the allergen; the tolerance was associated with decreased production of TH1 (IFN-gamma and IL-2) and TH2 (IL-4) cytokines. Allergen-specific TH1 -mediated (IgG2a and IgG2b) and TH2 -mediated (IgG1 and IgE) antibody responses were also inhibited. CONCLUSIONS Oral administration of a dominant T-cell determinant peptide induces immunologic tolerance in both TH1 and TH2 cell responses against the whole protein allergen. Our study is the first, to our knowledge, to demonstrate the potential for peptide-based oral immunotherapy in order to treat allergic immune responses.

Three T‐cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen antigens, retain their immunogenicity and tolerogenicity in a linked peptide

It has been demonstrated in detail that administration of a dominant T‐cell determinant to animals induces activation or immunological tolerance of T cells. However, it has not been determined whether multiple T‐cell determinants, when integrated into a single peptide, retain their potential to induce T‐cell activation and tolerance. We prepared a synthetic peptide comprising three T‐cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen antigens, and investigated the immunogenicity and tolerogenicity of each T‐cell determinant in the linked peptide by means of lymph node cell proliferation assays using mice. Lymph node cells from mice immunized with each of the three T‐cell determinants proliferated against the linked peptide in a dose‐dependent manner, similar to that of the immunized peptide. Lymph node cells from mice immunized with the linked peptide proliferated against all of the three T‐cell determinants. In addition, the degree of proliferation against the three T‐cell determinants occurred according to their original immunogenicity, as observed in the native protein antigens. Oral administration of the linked peptide to mice before they were immunized with Cry j 1 and Cry j 2 inhibited lymph node cell proliferation against the three T‐cell determinants, depending on the dose of the linked peptide administered. In conclusion, it was demonstrated that three T‐cell determinants retain their original immunogenicity and tolerogenicity in a linked peptide comprising them.

Delivery and cytotoxicity of RS-1541 in St-4 human gastric cancer cells in vitro by the low-density-lipoprotein pathway

RS-1541 is a 13-O-palmitoyl derivative of rhizoxin, an inhibitor of tubulin polymerization. RS-1541 has been shown to bind preferentially to plasma lipoproteins and to exhibit selective and sustained uptake by tumors in mice. To elucidate a mechanism of RS-1541 cytotoxicity, the cellular uptake and the cytotoxicity of a complex of RS-1541 with human low-density lipoprotein (RS-1541/LDL complex) were investigated in cultured St-4 human gastric cancer cells. Both the cellular uptake and the cytotoxicity of the RS-1541/LDL complex were greater in cells with higher LDL-receptor activities than in control cells. Excess amounts of LDL or 1 μM of monensin, a proton ionophore, significantly inhibited both the uptake and the cytotoxicity of the complex. Chloroquine, an inhibitor of lysosomal enzymes, decreased the intracellular level of rhizoxin liberated from RS-1541 and suppressed the cytotoxicity of the RS-1541/LDL complex. However, a detergent-aided solution of RS-1541 showed very low cellular uptake and cytotoxicity, irrespective of the LDL-receptor activities of these cells. These results demonstrate that the RS-1541/LDL complex is incorporated into the cells via the LDL receptor and that it manifests its cytotoxic activity after forming rhizoxin, the original antitumor agent, in the lysosomes.

The incidence of Japanese cedar pollinosis and sensitization to the pollen allergens among Japanese monkeys in a troop

The natural occurrence of Japanese cedar (Cryptomeria japonica; CJ) pollinosis has been reported in Japanese monkeys (Macaca fuscata), an appropriate animal model for developing antipollinosis therapies. However, there has been no study on the incidence of Japanese cedar pollinosis in monkeys. To evaluate the incidence of CJ pollinosis in Japanese monkeys, we investigated the presence of pollinosis symptoms among monkeys in a troop, and the response to CJ allergens in pollinosis monkeys. We examined the presence of pollinosis symptoms in 272 monkeys in a troop throughout the CJ pollination season (February to April). Of the 272 monkeys, 21 (7·7%) showed pollinosis symptoms during the CJ pollen season. Blood samples were taken from the 21 monkeys that showed pollinosis symptoms and were tested for the presence of immunoglobulin E (IgE) antibody for CJ allergens. All 21 monkeys with CJ pollinosis had anti‐CJ IgE. Of the 21 monkeys, peripheral blood mononuclear cells (PBMC) could be taken from 12, all of which showed CJ allergen‐specific PBMC proliferation. The incidence of CJ pollinosis in a troop was 7·7%. The monkeys with CJ pollinosis demonstrated specific IgE and PBMC proliferation for CJ allergens.

Intracellular activation and cytotoxic action of RS-1541 against cultured human tumor cells

RS-1541, an acyl-derivative of rhizoxin (Fig. 1), is a potent antitumor compound. This agent showed cytotoxicity in vitro on some cultured human tumor cells, although it was less potent than rhizoxin. Rhizoxin exhibited antitumor effects by inhibiting the polymerization of tubulin, whereas RS-1541 did not inhibit tubulin polymerization in vitro. However, cell cycle analysis in vivo showed that the two agents had the same mode of action. The cytotoxicity of RS-1541 was enhanced when the initial cell density of the cells was increased. The cytotoxicity was also enhanced when the membrane fraction of St-4 cells, which were the most sensitive to RS-1541 among the cell lines tested, was added to the target cells. When St-4 cells were incubated with [14C]-RS-1541, significant amounts of [14C]-rhizoxin were produced within the cells. Further fractionation of the crude membrane showed that the activity that enhanced the cytotoxicity of RS-1541 (RS-1541-enhancing activity) belonged to the mitochondrial-lysosomal fraction, not to the microsomal fraction. Both the enhancing activity and the activity that converting [14C]-RS-1541 to [14C]-rhizoxin (RS-1541-converting activity) were inhibited by treatment with chloroquine, an inhibitor of lysosomal function. Cholesterol esterase derived fromCandida cylindracea had RS-1541-enhancingand-converting activities. These data suggest that RS-1541 exerts its cytotoxic action after being converted to rhizoxin within the cells by a lysosomal enzyme such as cholesterol esterase.

Seasonal changes of humoral and cellular immune responses to Japanese cedar (Cryptomeria japonica) pollen allergens in Japanese monkeys (Macaca fuscata) with pollinosis.

The natural occurrence of Japanese cedar [Cryptomeria japonica (CJ)] pollinosis has been reported in Japanese monkeys (Macaca fuscata). The present study was designed to investigate seasonal changes in immunological reactions to CJ pollen allergens in monkeys with CJ pollinosis. Blood samples were collected from six monkeys with CJ pollinosis before and after CJ pollen season. Seasonal changes in specific IgE and IgG to major allergens (Cry j 1 and Cry j 2) were observed before and after CJ pollen season. The humoral responses decreased significantly before CJ pollen and increased after CJ pollen season. Similar seasonal changes in peripheral blood mononuclear cells proliferative responses to CJ allergens were observed before and after CJ pollen season. These humoral and cellular immune responses might serve as a biomarker for assessing new immunotherapies for monkeys with pollinosis.