T-cell-immunity-based inhibitory effects of orally administered herbal medicine juzen-taiho-to on the growth of primarily developed melanocytic tumors in RET-transgenic mice.

We examined the effect of oral administration of juzen-taiho-to, one of the most popular herbal medicines in Japan, on primary melanocytic tumor growth in RET-transgenic mice. There was virtually no difference between the lengths of tumor-free stages in the juzen-taiho-to-treated mice and the untreated littermate control mice. The rate of tumor growth in the juzen-taiho-to-treated mice, however, was greatly suppressed during the entire period after the initial tumor development. Correspondingly, the life span of juzen-taiho-to-treated transgenic mice was longer (over 6 mo in mean value) than that of control mice. We partially elucidated the mechanism of the antitumor effect of juzen-taiho-to. The addition of juzen-taiho-to at any of a wide range (50-1600 microg per ml) of concentrations to in vitro cultures of Mel-Ret cells, a malignant melanoma cell line derived from a RET-transgenic mouse, caused neither cell death nor cell cycle arrest directly. The addition of 50-400 microg per ml of juzen-taiho-to to cultures of murine spleen cells, however, promoted their DNA synthesis. More importantly, peritoneal exudate cells from the juzen-taiho-to-treated transgenic mice, in which the ratio and number of T cells were increased, displayed an antitumor immunity against Mel-Ret cells in vitro. Interestingly, the peritoneal-exudate-cell-associated antitumor immunity was further augmented by the addition of 200-400 microg per ml of juzen-taiho-to in vitro. This immunity, which was primarily conveyed by Thy-1+ T cells, was antigen (RET/melanoma) specific and cytotoxic. Amongst various chemical ingredients of juzen-taiho-to examined in this study, glycirrhizin displayed an action, partially replacing that of juzen-taiho-to, in promoting anti-Mel-Ret immunity when supplementarily added in vitro. These results suggest that juzen-taiho-to suppresses once-developed primary melanocytic tumors through potentiation of T-cell-mediated antitumor cytotoxic immunity in vivo.

Local levels of soluble tumor necrosis factor receptors in patients with allergic rhinitis are regulated by amount of antigen.

OBJECTIVES: To examine the possible correlation between the amount of antigen and the level of soluble tumor necrosis factor receptor (sTNFR), and to assess its biologic significance in allergic reactions. DESIGN: Randomized control trial. SUBJECTS: Twelve volunteers with Japanese cedar pollinosis and 10 healthy volunteers. INTERVENTIONS: The levels of p55 sTNFR (sTNFR1) and p75 sTNFR (sTNFR2) in samples of serum and nasal epithelial lining fluid (ELF) from 12 subjects with pollinosis and 10 healthy subjects were measured 4 times (preseason, early season, midseason, and postseason) in low (total, 415/cm( 2) per season) and high (total, 19,935/cm(2) per season) pollen-count periods, and the results were compared among the 4 groups. RESULTS: In the low-pollen-count period, increased levels of sTNFR1 and sTNFR2 were observed in ELF samples from the allergic subjects during the midseason. In contrast, in the high-pollen-count period, those levels were already elevated during the preseason and reduced during the midseason. Especially, the levels of sTNFR2 in ELF samples from the allergic subjects during the midseason in the high-pollen-count period were significantly lower than those in the low-pollen-count period. Moreover, a significant negative correlation (sTNFR1, R = -0.82; sTNFR2, R = -0. 73) was found between the levels of sTNFR1 and sTNFR2 in ELF samples and the scores of symptoms in the allergic subjects in the high-pollen-count period, but not in the low-pollen-count period. CONCLUSION: In patients with pollinosis, the amounts of antigen regulate the local levels of sTNFRs, possibly inhibiting nasal allergic inflammation.

Induction of T lymphocyte apoptosis by treatment with glycyrrhizin.

The effect of glycyrrhizin (GL), a Chinese herbal drug extracted from licolice roots, on murine lymphocytes for inducing apoptotic cell death was studied. Addition of GL (25-400 micrograms/ml) to cultured splenocytes and thymocytes from BALB/c mice definitely promoted DNA fragmentation. A single injection of GL (100 micrograms/mouse) into BALB/c mice did not cause any detectable DNA fragmentation or cell death of splenocytes and thymocytes. Cytofluorometric analysis of these cells, however, demonstrated a reduction in mitochondrial transmembrane potentials (delta psi m). Repeated injections of GL (100 micrograms/mouse/day) into mice for 7 days actually resulted in induction of low grade DNA fragmentation selectively in splenocytes. Cell population analysis of viable lymphocytes suggested that both CD4+ Th lymphocytes and CD8+ Tc lymphocytes may have been relatively more sensitive than B220+ B lymphocytes for the apoptotic cell death. We concluded from these results that GL acts as a rather selective inducer of mature T lymphocyte apoptosis with a reduction in delta psi m potentially preceding lymphocyte death.

Elevated soluble tumor necrosis factor receptor levels in seasonal allergic rhinitis patients.

In this study, we examined the symptom scores and tumor necrosis factor-alpha (TNF-alpha), p55 soluble tumor necrosis factor receptor (sTNFR1), and p75 soluble tumor necrosis factor receptor (sTNFR2) levels in the sera and nasal epithelial lining fluids (ELF) of 20 patients with Japanese cedar pollinosis from the pre- to the postseason period, and compared the results with those of 10 nonallergic control subjects. The symptom scores of the allergic subjects were significantly (P<0.01) higher than those of the nonallergic subjects during the early stage and mid-stage of the season. There were no statistical differences between the allergic and nonallergic subjects in the TNF-alpha levels in sera and ELF from the pre- to the postseason. In the allergic subjects, however, the levels of sTNFR1 and sTNFR2 in ELF were significantly elevated during the early stage (P<0.05) and mid-stage (P<0.01) of the season, whereas those in sera did not change from the pre- to the post-season period. The levels of TNF-alpha in ELF were more than 10 times higher than those in sera, whereas the levels of sTNFR1 and sTNFR2 in ELF were less than half of those in sera in the allergic and nonallergic subjects. These results suggest that sTNFR1 and sTNFR2 may play a role in the pathogenesis of nasal allergic reaction.

The herbal medicine Sho-saiko-to inhibits growth and metastasis of malignant melanoma primarily developed in ret-transgenic mice.

Sho-saiko-to is the most popular herbal medicine in Japan. We investigated the anti-tumor and anti-metastatic effects of Sho-saiko-to and its chemically defined ingredients on the primary skin melanoma that developed in a metallothionein-I (MT)/ret transgenic mouse line and on a melanoma cell line (Mel-ret), which was derived from a primary tumor developed in a MT/ret transgenic mouse. In vitro, Sho-saiko-to suppressed the growth of Mel-ret cells more strongly than any single ingredient of Sho-saiko-to, although baicalin as one of several ingredients tested also suppressed it significantly. In vivo, Sho-saiko-to (i) significantly (p < 0.02) prolonged the onset of tumor development (1.5 mo), (ii) definitely retarded the transition to malignancy, (iii) significantly decreased the incidence of distant metastasis to brain (p < 0.002), kidney (p < 0.05), and liver (p < 0.05) at the malignant stage, and (iv) significantly (p < 0.02) prolonged life span (2.6 mo). Moreover, Sho-saiko-to and baicalin down-regulated the matrix metalloproteinase-2 and -9 expression levels, and upregulated their inhibitor expression level in both the primary tumors and Mel-ret cells. In conclusion, Sho-saiko-to displayed anti-tumor and anti-metastatic effects on melanoma with regulation of the balance of matrix metalloproteinase and tissue inhibitor of the matrix metalloproteinase levels.

The herbal medicine sho-saiko-to inhibits the growth of malignant melanoma cells by upregulating Fas-mediated apoptosis and arresting cell cycle through downregulation of cyclin dependent kinases.

The anti-tumor effect and its mechanism of the herbal medicine sho-saiko-to were investigated on a murine malignant melanoma cell line (Mel-ret). Sho-saiko-to induced apoptotic cell death of Mel-ret cells with a definite increase of cell surface Fas antigen and Fas ligand (FasL). Sho-saiko-to arrested Mel-ret cells in G1 phase by decreasing the expression of cyclin-dependent kinase (cdk) 4 and its homologue cdk6. Kinase activities of cdk4 and cdk6 were identified to be downregulated by sho-saiko-to. Ingredient analysis revealed that baicalin is likely the main active constituent in the upregulation of Fas antigen and Fas ligand, while glycyrrhizin is the main constituent in the inhibition of cdks.

Evidence of potential regulation by interleukin-4 of the soluble intercellular adhesion molecule 1 level in patients with seasonal allergic rhinitis under provocation by a small amount of natural allergen.

Interleukin-4 (IL-4) and intercellular adhesion molecule 1 (ICAM-1) are assumed to be involved in the pathogenesis of allergic disease. In this study, we examined the potential link between IL-4 and soluble ICAM-1 (sICAM-1) levels in patients with allergic rhinitis. The levels of sICAM-1 and IL-4 in sera and in nasal epithelial lining fluids (ELF) from 12 patients with Japanese cedar pollinosis were measured preseason through postseason, and the results were compared with those from 7 healthy subjects. In sera from the allergic subjects, the levels of sICAM- 1 were upregulated during the early part of the season and downregulated during the middle of the season, with upregulation of the IL-4 levels. Moreover, a negative correlation was found between the serum sICAM-1 levels and serum IL-4 levels during the middle (r = -.80) and late (r = -.73) parts of the season. In ELF from allergic subjects, the levels of sICAM-1 were significantly upregulated during the early and middle parts of the season, and began to be downregulated during the late part of the season, with upregulation of the levels of IL-4. In conclusion, IL-4 possibly acts as a potential suppressor of sICAM-1 in the pathogenesis of seasonal allergic rhinitis, at least under provocation by a small amount of natural allergen.

Differential sensitivities to hyperbaric oxygen of lymphocyte subpopulations of normal and autoimmune mice.

We studied the effect of exposure to hyperbaric oxygen (HBO): 2.8 atm absolute 100% oxygen for 4 h daily over 3-7 days, on the immune system of normal (BALB/c and MRL- +/+) and autoimmune MRL-lpr/lpr) mice. In HBO exposed BALB/c mice, we observed a remarkable decrease in the cell population of the spleen and thymus. We found that the sensitivity to HBO varied among subpopulations of lymphocytes. For example, CD4+ CD8+ double positive cells in the thymus and B220+ B cells in the spleen were more sensitive than CD4+ or CD8+ single positive T cells in the thymus, and Thy-1+ T cells in the spleen, respectively. Accordingly, despite the decrease in total cell number in the spleen, the proliferative response of T cells from the spleen to Con A was not impaired in the HBO exposed mice. Exposure of MRL-lpr/lpr mice to HBO caused a marked reduction of weight and cell population of the otherwise enlarged spleen and lymph nodes, and amongst others of percentages of B220+Thy-1+ double positive abnormal cells. These results suggest the HBO therapy may be applicable for the treatment of some autoimmune diseases.

Dynamics of soluble adhesion molecule levels in patients with pollinosis.

OBJECTIVES: To define the dynamics of systemic and local soluble adhesion molecule levels and to discuss the role of these molecules in the pathogenesis of allergic rhinitis. DESIGN: Randomized control trial. SUBJECTS: Twelve volunteers with Japanese cedar pollinosis and 7 healthy volunteers. INTERVENTIONS: The levels of soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble E-selectin, and soluble L-selectin in serum samples and nasal epithelial lining fluids (ELF) from 12 patients with pollinosis were measured 5 times throughout the allergy preseason to postseason, and the results were compared with those from 7 healthy subjects. RESULTS: The levels of sICAM-1 (P < .05) and sVCAM-1 (P < .05) in sera were up-regulated, and the levels of soluble L-selectin (P < .01) in sera were down-regulated during the early stage of the season in the allergic subjects. The difference between the levels of sICAM-1 and sVCAM-1 in sera in the early and mid-season was statistically significant in the allergic subjects (P < .05). The levels of sICAM-1 in ELF were up-regulated during the early and mid-season. The levels of sVCAM-1, soluble E-selectin, and soluble L-selectin in ELF were undetectably low throughout the preseason to postseason. CONCLUSION: There is evidence of the unique stage-dependent differential contributions of various soluble adhesion molecules in the pathogenesis of seasonal allergic rhinitis with a small amount of natural allergen provocation.

Amount of pollen has an effect on the systemic and local levels of soluble ICAM-1 in patients with seasonal allergic rhinitis.

We investigated whether the amount of antigen has an effect on the systemic and local levels of soluble ICAM-1 (sICAM-1) in patients with pollinosis, and assessed its biologic significance. The levels of subjective symptoms and sICAM-1 in sera and nasal epithelial lining fluids (ELF) from 14 subjects with pollinosis (allergic group) and eight healthy subjects (control group) were measured from pre- to postseason in 1993 (total pollen count: 10,854/cm2) and 1994 (total pollen count: 415/cm2), and the results were compared with each other among the four groups. The levels of subjective symptoms and sICAM-1 in ELF from the allergic group significantly increased during the season in both 1993 and 1994. However, there was a significant difference (P < 0.01) between the levels of those in 1993 and those in 1994 during the season. The levels of sICAM-1 in sera from the allergic group were significantly upregulated during the seasons and postseasons only in 1993, and there was a significant difference (P < 0.05) between the levels in 1993 and those in 1994 during the postseason. We conclude that amount of pollen has an influence on the local and systemic levels of sICAM-1, as well as the scores of subjective symptoms, in patients with seasonal allergic rhinitis.

Enhancement of nitric oxide production from activated macrophages by glycyrrhizin.

We studied the actions of glycyrrhizin on nitric oxide production from macrophages and a macrophage cell line RAW264-7. Although glycyrrhizin did not induce nitric oxide from resting macrophages, it enhanced the production of nitric oxide from IFN-gamma activated-macrophages or RAW cells. Glycyrrhizin also enhanced the production of nitric oxide from macrophages stimulated with the supernatant of con A-activated spleen cells. Further, glycyrrhizin enhanced tumor cell killing by macrophages activated with IFN-gamma. This tumor cell killing was mainly by nitric oxide.

Major basic protein and topical administration of ketotifen in pollinosis under natural allergen provocation.

In this study we investigated the potential relation among subjective symptoms, blood eosinophil counts and the levels of major basic protein (MBP) in serum throughout the pre- to postpollen season. In addition, we compared the effects of topical administration of ketotifen on these parameters between the prophylactic treatment group (n = 10) and the postsymptomatic treatment group (n = 10). We found that (1) the levels of MBP in serum during the season were significantly higher than those before the season and (2) the levels of the above three parameters in the prophylactic treatment group were significantly lower than those in the postsymptomatic treatment group during the season. It was concluded from these results that the action of MBP may be involved in the pathogenesis of allergic rhinitis. Furthermore, for the first time we provided evidence that topical ketotifen administration could suppress the systemic upregulation of the blood eosinophil count and MBP level in subjects with pollinosis.

Evidence of redox-linked signaling for producing a giant signal complex.

Previously we showed that a thiol-reactive heavy metal, HgCl2, crosslinked multiple cell surface receptors through a ligand-independent pathway, which produced massive aggregates of phosphotyrosine (PTYR)-containing proteins beneath plasma membrane [Nakashima et al. (1994): J Immunol 152: 1064-1071]. In this study we characterized these unique aggregates at the molecular level. The lysates in Brij 96 of thymocytes treated with HgCl2 were separated into the supernatant and pellet fractions by simple centrifugation. Selected PTYR-containing proteins and p56lck appeared in the pellet fraction as quickly as 5 s after exposure to HgCl2, and were further increased in amount by 5 min. Although the mechanism of triggering these events was redox-linked, the majority of proteins in the Brij 96-insoluble aggregates were dissociated in SDS-PAGE under nonreducing condition. This suggested that PTYR-containing proteins and p56lck themselves do not form dimer or polymer directly by thiol-mediated bond. The pellet fraction was further found to include some other signal delivery elements, such as GTPase activating protein, phosphatidylinositol 3 kinase, and mitogen-activated protein kinase. Finally, all of these signal elements and selected PTYR-containing proteins were collected in the same fraction by the sucrose density gradient centrifugation. These results suggest a unique redox-linked pathway of formation of a giant signal complex.

Enhancement of nitric oxide production from activated macrophages by a purified form of ginsenoside (Rg1).

We studied the actions of purified ginsenosides Rg1 and Rb1 on nitric oxide production from macrophages and a macrophage cell line RAW264-7. Although neither Rg1 nor Rb1 induced nitric oxide from resting macrophages, Rg1 enhanced the production of nitric oxide from IFN-gamma activated-macrophages or RAW cells. Rg1 also enhanced the production of nitric oxide from macrophages cocultured with nonadherent spleen cells stimulated by conA, LPS or anti-CD3. Rb1, however, did not significantly enhance nitric oxide production from stimulated macrophages or RAW cells. Rg1 enhanced the tumor cell killing by nitric oxide produced from IFN-gamma-activated macrophages.

Characterization of the immunoregulatory action of saikosaponin-d.

The immunoregulatory action of saikosaponin-d (SSd), which was isolated from the root of Bupleurum falcatum L. and has a steroid-like structure, was examined on splenic T lymphocytes of C57BL/6 mice. SSd displayed a definite action in vitro to bidirectionally control the growth response of T lymphocytes stimulated by concanavalin A, anti-CD3 monoclonal antibody, and calcium ionophore A23187 plus phorbol 12-myristate 13-acetate. Low concentrations (1-3 micrograms/ml) of SSd upregulated the responses to suboptimum stimuli of agonists, particularly during the relatively late stage of the responses, whereas it downregulated the responses to supraoptimal stimuli. Under appropriate experimental conditions, SSd promoted interleukin-2 (IL-2) production and IL-2 receptor expression. It also accelerated c-fos gene transcription, but it did not modulate the level of tyrosine phosphorylation of cellular proteins. We concluded from these results that SSd uniquely modulates T lymphocyte function and that at least one target of the action of SSd is located at or before the step of c-fos gene transcription and after T-cell receptor/CD3-mediated protein tyrosine kinase activation.

Establishment of drug resistance in human gastric and colon carcinoma xenograft lines.

We established multidrug-resistant human gastric and colon xenograft lines by means of intratumoral injections of four agents, doxorubicin (DXR), cisplatin (CDDP), 5-fluorouracil (5-FU) and mitomycin C (MMC), into subcutaneous SC1NU and SW480 tumors once a week or less. Such intermittent drug exposure is commonly used in clinical chemotherapeutic protocols. All xenograft lines acquired resistance to the injected drugs as evaluated by in vivo drug-resistance tests. Many of the drug-resistant lines showed various patterns of cross resistance to other drugs. In order to analyze the mechanism of resistance in vivo, we investigated the expression of drug resistance gene, which has been extensively studied in vitro. We used four complementary DNAs (cDNAs) for multidrug resistance (MDR1), glutathione S-transferase-pi (GST-pi), thymidylate synthase (TS) and dehydrofolate reductase (DHFR), as probes. We observed GST-pi, DHFR and TS mRNA expression at various levels, but MDR1 mRNA expression was found only in SW480/DXR by the method of poly (A+) RNA selection. Four resistant SW480 lines had higher TS mRNA expressions. Six resistant lines had stronger GST-pi mRNA expression. Five resistant lines had higher DHFR mRNA expression. Drug resistance genes related to the treated drug were also expressed in this in vivo model; MDR1 in SW480/DXR, GST-pi in SW480/CDDP and in SC1NU/CDDP and TS in SW480/5-FU. In contrast to in vitro resistant lines which have been reported as models of drug resistance, the expression of drug resistance genes in vivo was not always correlated to the acquisition of cross resistance. These resistant xenograft lines and the methods developed to induce drug resistance in vivo should be useful for studies on the mechanism of drug resistance in the clinical setting.

Antibody response to haptenic sugar antigen: immunodominancy of protein-bound lactose formed by amino-carbonyl reaction.

Several reducing sugars with structural analogy (glucose, galactose, lactose, melibiose, maltose and cellobiose) were bound to a carrier protein ovalbumin by amino-carbonyl reaction, and the non-enzymatically glycosylated proteins were injected into mice (BALB/c, C57BL/6, DBA/2 and C3H/He strains) with Freund's adjuvant. Antibody responses to the haptenic sugar antigens were analyzed quantitatively by enzyme-linked immunosorbent-assay using each sugar-bovine serum albumin complex. The haptenic sugar antigen from lactose induced a markedly higher response of specific antibody as compared with the haptenic sugar antigens from the other sugars. The antibody raised against the lactose adduct reacted well with lactulose (4-o-beta-D-galactopyranosyl-D-fructose) or alpha-N-acetyl-epsilon-N-deoxylactulosyl-L-lysine. The results suggested that immunogenicity of haptenic sugar antigens was remarkably different in sugar chemical structure, and that the lactose adduct formed by lactose-protein amino-carbonyl reaction could be an immunodominant antigenic determinant.

Augmentation of antibody responses of mice to inhaled protein antigens by simultaneously inhaled bacterial lipopolysaccharides.

Serum antibody responses of mice to repeatedly inhaled protein antigens such as bovine serum albumin and ovalbumin, plus or minus bacterial lipopolysaccharide (LPS) in the form of an aerosol were studied. Results showed that the levels of responses to inhaled protein antigens varied, depending on the mouse strain-antigen combination and that LPS inhaled simultaneously with the antigens definitely augmented the responses which were not otherwise very high. LPS extracted from Klebsiella O3 (LPS-K) but not LPS from Escherichia coli O55 (LPS-E), which was inhaled at the time of initial inhalation of antigen, significantly intensified the priming for the secondary antibody response to the antigen subsequently inhaled. Both LPS-K and LPS-E, however, definitely acted to augment the response when they were inhaled repeatedly together with the antigen. Oral administration of antigen or antigen plus LPS-K did not induce any detectable antibody response in our experiment, ruling out the possibility that the antigen and LPS stimulated the immune system via alimentary canal rather than via lung. Tissue distribution of the radioactivity soon after inhalation of 131I-labeled antigen and decay speed of the radioactivity were not significantly changed by LPS-K inhaled simultaneously. This suggested that the augmentation of responses was not mediated by the action of LPS to modulate the air-blood barrier against the entry of antigen via lung. All the results prove for the first time that inhaled LPS displays a definite adjuvant action on antibody responses to inhaled antigens.

Conversion of thyroxine into tri-iodothyronine in zinc deficient rat liver.

Hepatic thyroxine (T4) to tri-iodothyronine (T3) conversion was measured in six animal groups: Group A was fed a severe zinc-deficient diet (1.98 ppm) for 5 weeks; group B was a pair-fed control group for group A; group C was fed a less severe zinc-deficient diet (6.10 ppm) for 5 weeks; group D was a pair-fed control group for group C; group E was fed a zinc-supplemented control diet (90.4 ppm) for 5 weeks; and group F was first fed the severe zinc-deficient diet for 5 weeks and then placed on the zinc-supplemented control diet until a body weight corresponding to the final weight of group E was obtained. Serum T3 and T4 levels and T4 to T3 conversion were significantly reduced in group A. A significant positive correlation was observed between T4 to T3 conversion and alcohol dehydrogenase (zinc-metalloenzyme) activity in liver tissue from the six groups. Thus, it appeared that an appropriate amount of zinc might be one of the factors in thyroxine to tri-iodothyronine conversion in liver tissue normally.