High positive frequency of antibodies to metallothionein and heat shock protein 70 in sera of patients with metal allergy.

Two principal types of stress protein, heat shock proteins (hsps) and metallothionein (MT), are induced in cells responding to a variety of stresses. They play an important role in protecting cells from these stresses. However, many reports indicate that antibodies to hsps are present in human serum and are associated with several autoimmunity diseases. Metals, which are commonly allergenic to humans, induce both MT and hsp70 (one of the hsps family). Until now, there has been no report of any antibody to MT in human serum. In the present study, serum samples from healthy controls (Group I), and patients suffering from atopic dermatitis without (Group II) or with (Group III) metal allergy, were measured for antibodies to MT and hsp70, using an enzyme-linked immunosorbent assay (ELISA). Metal allergy was confirmed by patch testing. We first found that antibody to MT exists in human serum. We also found a high positive frequency of antibody to MT (51.3%) and to hsp70 (43.6%) in the sera of Group III, compared to those of Group I (3.8% and 5.1%) or Group II (6.4% and 5.1%). Furthermore, there was a strong positive correlation between antibody to MT and antibody to hsp70 in Group III (P = 0.0013), but not in Group I and Group II. Our results indicate that antibody to MT exists in human serum, as do antibodies to hsps, and suggest that elevated levels of MT and hsp70 antibodies are associated with metal allergy in atopic patients.

Long term effects of Toki-shakuyaku-san on brain dopamine and nerve growth factor in olfactory-bulb-lesioned mice.

We used olfactory-bulb-lesioned mice induced by intranasal irrigation with zinc sulfate as a model of dementia, to investigate the effects of Toki-shakuyaku-san (TSS) on monoamines and nerve growth factor (NGF) in brain regions. TSS was given daily through the drinking water for either 1, 2, 3, 4 or 8 weeks from the day after olfactory lesion. The administration of TSS significantly suppressed the decrease of 3,4-dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) in olfactory bulb of olfactory-lesioned mice at 1 week, and tended to suppress the decrease of DOPAC and HVA during the experimental session. However, the administration of TSS had no influence on dopamine contents. NGF contents in the olfactory bulb were increased after the irrigation, and the value returned to the same level as the control at 8 weeks after. Although the NGF contents in the olfactory bulb of TSS-treated mice were immediately increased at 1 and 2 weeks, the value returned to normal level within 3 weeks. These findings indicate that oral administration of TSS prevents the reduction of dopamine metabolites, DOPAC and HVA, and immediately increased NGF contents in the olfactory bulb. This suggested that TSS treatment promotes the NGF contents in olfactory nerves and rescue the neurons from damage.

Inhibitory effect of baicalein, a flavonoid in Scutellaria Root, on eotaxin production by human dermal fibroblasts.

Eotaxin is an eosinophil-specific chemokine associated with the recruitment of eosinophils to sites of allergic inflammation. "Saiboku-to" (Formula magnoliae et bupleuri) is a kampo herbal medicine used for the treatment of bronchial asthma in Japan. In this study, we investigated the effects of Scutellaria Root, a major herb in Saiboku-to and its components such as baicalein and baicalin on eotaxin production by IL-4 plus TNF-alpha-stimulated human fibroblasts. An extract of Scutellaria Root markedly inhibited eotaxin production. Four major flavonoids from Scutellaria Root were found to show inhibitory activity on eotaxin production at a concentration of 10 micrograms/ml in the order of baicalein > oroxylin A > baicalin > skullcapflavon II. The inhibitory effect of baicalein was expressed in a dose-dependent manner, and almost 50% inhibition was observed at 1.8 micrograms/ml. Furthermore, baicalein prevented human eotaxin mRNA expression in IL-4 plus TNF-alpha-stimulated human fibroblasts. These results help explain the pharmacological efficacy of Scutellaria Root in the treatment of bronchial asthma since it would suppress eotaxin associated recruitment of eosinophils.

Effects of hokoei-to (pugongying-tang), a Kampo formula, on monoamine content in brain regions and mitogenic activity of splenic lymphocytes in ovariectomized mice.

Hokoei-to (pugongying-tang) is one of the Kampo formulae clinically used for gynecological disturbances such as lack of lactation and mammary swelling. We investigated the effect of hokoei-to on the nervous and immune systems in ovariectomized mice as a climacteric disorder model. Hokoei-to suppressed the decrease of monoamines in the ventral hippocampus and dorsal hippocampus of ovariectomized mice. It was shown that the hokoei-to could improve the metabolic turnover of dopamine. The mitogenic activity of lymphocytes in the spleen was reduced after ovariectomy; a suppression of this reduced activity was observed in the group given hokoei-to.

Pancreatic islet regeneration by ephedrine in mice with streptozotocin-induced diabetes.

In this experiment, we investigated the effects of crude Ephedrae herba, alkaloid extract of Ephedrae herba and 1-ephedrine, a major alkaloid component, on diabetic mice induced by streptozotocin (STZ). The alkaloid extract and 1-ephedrine showed suppression on the hyperglycemia. The suppression by Ephedrae herba of hyperglycemia may therefore be due to 1-ephedrine. Furthermore, we found that Ephedrae herba, alkaloid and 1-ephedrine promoted the regeneration of pancreas islets following atrophy induced by STZ. It is therefore suggested that Ephedrae herba may regenerate atrophied pancreatic islets, restore the secretion of insulin, and thus correct hyperglycemia.

Population pharmacokinetic analysis of ephedrine in Kampo prescriptions: a study in healthy volunteers and clinical use of the pharmacokinetic results.

We conducted a population pharmacokinetic analysis of ephedrine, the main effective constituent of the two Kampo prescriptions, Sho-seiryu-to (Xiao-Qing-Long-Tang, Formula divinitatis caeruleae minor) and Kakkon-to (Ge-Gen-Tang, Formula puerariae), which are routinely used in the treatment of allergic rhinitis. Major pharmacokinetic differences were found in AUC(0-infinity), Cmax and the averages of the distribution volume between the two prescriptions. Using the pharmacokinetic results, we also conducted a simulation-based study comparing the pharmacokinetic profiles of ephedrine for the usual dosage regimen, a constant dose D three times a day before meals, and two alternative dosage regimens which are often used when patients' symptoms are not improved in the morning using the usual dosage regimen. The results suggested that one of the two alternative dosage regimens, two times D before breakfast and D in the afternoon, was the most effective from the pharmacokinetic viewpoint. This result was in accordance with our clinical experience.

Effects of Astragali root and Hedysari root on the murine B and T cell differentiation.

It has traditionally been known in Asian countries that the Astragali and Hedysari roots have similar beneficial effects. They have been applied in clinical practice for immunological diseases, we investigated their effects on specific antibody production, B cells and T cells in BALB/c mice immunized with ovalbumin (OVA). Both the Astragali and Hedysari roots reduced the production of IgG2a in the primary response to OVA. Hedysari root reduced the production of IgG2a in the secondary response. Both reduced the production of IgM, suppressed IL-6 production in spleen cells. It was presumed that the suppression of IgG2a production was induced by the suppression of IL-6, which is an antibody production inducer. It was found that the Hedysari root increased the B cells and activated them, and the Astragali root also tended to increase them. In addition, Hedysari root decreased the proportions of IFN-gamma-producing cells in splenic CD4(+) T lymphocytes. It was shown that the Astragali and Hedysari roots could act as a growth factor of B cells by increasing the proportion of the total B cells and activated B cells. In conclusion, both the Astragali and Hedysari roots showed the same actions, but the latter was more effective.

A new murine model for atherosclerosis with inflammation in the periodontal tissue induced by immunization with heat shock protein 60.

It has recently become apparent that the anti-heat shock protein (HSP) antibody plays an important role in the pathogenesis of atherosclerosis. We studied whether immunization with human HSP60 could lead to atherosclerosis in mice. We attempted to induce atherosclerosis in C57BL/6NJcl mice by immunization with human HSP60 under a high-cholesterol diet. The size of fatty streak lesions was significantly enhanced in mice immunized with human HSP60 under a high-cholesterol diet relative to the number in control mice receiving a high-cholesterol diet alone. In addition, these new atherosclerotic model mice showed lesions of inflammation in the periodontal tissue. This new model may thus provide theoretical support for the clinical observation that patients suffering from periodontitis are frequently found to have atherosclerosis. The cytokine ratio of interferon-gamma/interleukin-4 in the high-cholesterol diet group was significantly higher than that in the standard chow group (p<0.05). This suggests the presence of a predominantly Th1-type immune response in atherosclerosis.

Effects of Kampo medicine, Toki-shakuyaku-san (Tang-Kuei-Shao-Yao-San), on choline acetyltransferase activity and norepinephrine contents in brain regions, and mitogenic activity of splenic lymphocytes in ovariectomized mice.

We investigated the effects of Toki-shakuyaku-san (TSS, Tang-Kuei-Shao-Yao-San in Chinese), Japanese traditional herbal medicine, on the nervous and immune systems in ovariectomized mice as a climacteric disorder model. Female C57BL/6 mice were ovariectomized (OVX) and TSS was given daily through the drinking water for either 10 or 20 days from the day after ovariectomy. After completion of experimental sessions, animals were sacrificed and specific brain regions were assayed for choline acetyltransferase (ChAT) activity and norepinephrine contents. The mitogenic activities, alkaline phosphatase activity and 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H terazolium bromide (MTT) activity, in splenic lymphocytes has also measured. Furthermore, the effects of TSS on learning and memory ability were studied by the step-through type passive avoidance test. As the results, the administration of TSS significantly suppressed the decrease of ChAT activity in the cerebral cortex (CC) and the dorsal hippocampus (DH) of ovariectomized mice at 10 days after ovariectomy, however no significant effect was observed at 20 days after ovariectomy. Norepinephrine contents in OVX group were decreased at 10 and 20 days after ovariectomy in the CC and the ventral hippocampus (VH). The administration of TSS significantly suppressed the decrease of norepinephrine contents at 20 days after ovariectomy. The mitogenic activities of lymphocyte in spleen were increased at 10 days after ovariectomy, and decreased at 20 days after ovariectomy. However, the suppression of these changes was observed in the group given TSS. The mean latent period was also shortened in the passive avoidance test in the OVX group, but TSS treated group improved mean latency. From these observations, it is inferred that administration of TSS brings on the synthesis of acetylcholine and norepinephrine in the CC and hippocampus, and may improve the memory related behavior and the abnormalities in lymphocytes in the models of the climacteric disorder.

Preventive effect of Ninjin-to (Ren-Shen-Tang), a Kampo (Japanese traditional) formulation, on spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice.

We previously found that ingestion of an extract of Ninjin-to (NJT; Ren-Shen-Tang) suppressed the development of autoimmune diabetes in C57BL/KsJ mice induced by multiple low doses of streptozotocin. To verify this effects on spontaneous autoimmune diabetes, the effects of NJT on NOD mice were investigated in the present study. NJT, provided in drinking water (0.25%, 450 mg/kg/day) from 6 weeks of age, significantly prevented the incidence of spontaneous diabetes in female NOD mice at 30 weeks of age (2/10) compared with that of the controls (7/10), with no effects on body growth or food intake. Even in non-diabetic mice, the blood glucose levels of the NOD controls gradually increased with age, while such increase in NJT-treated mice was significantly suppressed by preventing any deficiency of glucose tolerance. NJT also significantly suppressed the progression of insulitis, which causes insulin deficiency and diabetes. It is well known that NOD mice develop insulitis and diabetes because of their Th1-dominant autoimmune response. IFN-gamma production from splenic T lymphocytes stimulated with anti-CD3 monoclonal antibodies was increased, whereas IL-4 production was decreased in NOD controls compared to age- and sex-matched normal ICR mice. NJT-treatment reduced these deviations of cytokine production in NOD mice. These data all suggest that NJT can prevent spontaneous insulitis and diabetes by the modification of deviated cytokine production in NOD mice.

Hepatoprotective effects of Inula britannica on hepatic injury in mice.

Inula britannica, a Kampo medicine, is prepared from the heads of Compositae plants such as Inula britannica L., which has been used clinically as a remedy for nausea, hiccup and excessive sputum. Here it is shown that administration of Inula britannica improves the survival rate of mice with hepatic injury induced by LPS/PA. It is also suggested that administration of Inula britannica significantly reduces the fluctuation in the amount of cytokine in the spleen of mice with hepatic injuries, and that the Th1/Th2 control effect is related to the inhibitory action of Inula britannica against hepatic injury. In vitro testing suggests that Inula britannica suppresses Th1 differentiation and induces Th2 differentiation by inhibiting the production of macrophage IL-12 and promoting the production of IL-10, thus showing the immunological effect of hepatic injury inhibition by affecting the balance between Th1 and Th2.

Clinical and pharmacological studies on liver diseases treated with Kampo herbal medicine.

Hepatitis C virus (HCV) infection frequently causes chronic hepatitis, which is linked to the development of liver cirrhosis and hepatocellular carcinoma. Most physicians who practice Kampo medicine in Japan have observed that Kampo medicine can be as effective as interferon therapy in the treatment of chronic hepatitis C. In the present study, to evaluate the effect of Kampo medicine on chronic hepatitis C, clinical treatment was assessed in short-term and long-term study, and it was shown that ninjin-yoei-to (Formula ginseng compositae: TJ-108) was very effective. Therefore, to find the most active herbal component of TJ-108 in the treatment of HCV, Citrus Unshiu Peel, Schisandra Fruit, and Polygala Root, which are specific to TJ-108, were screened using an in vitro HCV infection model. Among the three herbs, Schisandra Fruit was found to be most active. In the next step, Gomisin A, an active component of Schisandra Fruit, was studied using an in vitro model with MOLT-4 cells and an animal model of immunologically induced acute hepatic failures. It is concluded that the therapeutic effect of TJ-108 on chronic hepatitis C is from the inhibitory effect on HCV infection, and also from the protective effect on immunological hepatopathy of Schisandra Fruit and its lignan component, Gomisin A.

Effects of Kampo formulations (Chinese herbal medicine traditionally used in Japan) on event-related electric potentials.

Contingent negative variation (CNV) is a brain function test of the central nervous system in reference to attention, will preparation and motivation. We compared the neuropharmacological effects of 6 main Kampo formulations (1. Mao-to: MA HUANG TANG; 2. Shimbu-to: ZHEN WU TANG; 3. Ninjin-to: REN SHEN TANG; 4. Shigyaku-san: SI NI SAN; 5. Keishi-to: GUI ZHI TANG; and 6. Shimotsu-to: SI WU TANG) on CNV. Mao-to induced significant decrease of CNV in 90 minutes after oral administration. Shimbu-to showed a tendency to increase CNV in 90 minutes after oral administration. Shimotsu-to also induced a transient tendency to increase CNV in 60 minutes after oral administration. Keishi-to showed a decrease of CNV in the early stage of the experiment, while Ninjin-to and Shigyaku-san showed no changes of CNV. From these, it may be concluded that Mao-to and Keishi-to induce sedation of brain function, but Shimbu-to and Shimotsu-to show activation, while Ninjin-to and Shigyaku-san show no changes of brain function regarding CNV related brain function. Furthermore, the time courses of CNV changes were different within the same group (sedation or activation) of Kampo formulations.

Effects of acupuncture on peripheral T lymphocyte subpopulation and amounts of cerebral catecholamines in mice.

The aim of this study was to investigate the effects of acupuncture on peripheral lymphocyte subpopulations and cerebral catecholamines. In order to examine the effects of acupuncture, two experiments were performed. Experiment 1: Eighteen female mice (strain; C57BL/6) at the age of 7 weeks were divided three groups, (a) sham operated (control; n=6), (b) ovariectomized (OVX; n=6), and (c) ovariectomized and stimulated by subcutaneous needles on acupuncture point, Shenshu (BL23) at the both sides of the back for 20 days (OVX+Acu; n=6). These animals were sacrificed at 20 days after needle insertion, and the splenic lymphoid cells were examined by two-color flow cytometry, using monoclonal antibodies (mAb) to the cell surface antigens, CD3, CD4, CD8a and NK1.1 (CD56). In the ovariectomized (OVX) group, the peripheral CD4/CD8 ratio was significantly increased and the ratio of natural killer (NK) cells (CD3-NK1.1+; CD3 negative, NK1.1 positive) to T lymphocytes was decreased compared to the sham control group. In the ovariectomized with needle insertion (OVX+Acu) group, the CD4/CD8 ratio was reduced, but the NK cells ratio was not changed compared to the OVX group. Experiment 2: To investigate the acute effects of subcutaneous needle insertion, male C57BL/6 mice (7 weeks old) were used (n=6, each group). The acupuncture points Shen-shu (BL23) on the backs of the male mice were also stimulated by subcutaneous needles for 3 and 7 days. As a result, the CD4/CD8 ratio was significantly decreased at day 3 and day 7, compared to the control group. On the other hand the NK cells ratio and activated T-cells were increased at day 7. The mitogenic activities in the splenic lymphocytes were also increased by acupuncture stimulation at day 3. Catecholamine contents in the hippocampus were measured by high performance liquid chromatography with the electro-chemical detector (ECD-HPLC) method. No significant change was observed in either dopamine contents or norepinephrine; however, dopamine metabolite, homovanillic acid (HVA) and DOPAC (3,4-dihydroxyphenylacetic acid) were increased at day 3. The study suggests that acupuncture has effects on peripheral lymphocyte subpopulations and may modulate mitogenic activity. In addition, acupuncture may stimulate dopamine turnover.

Juzen-taiho-to, a Japanese herbal medicine, modulates type 1 and type 2 T cell responses in old BALB/c mice.

Aging is associated with skewed type 2 (T2) T cell responses that may be modulated by herbal medicines. A group of Japanese herbal medicines, so-called "Hozai," have been used to improve the physical condition of the elderly. One representative "Hozai," Juzen-Taiho-To (JTX) appears to have beneficial effects on cancer patients. In this study we hypothesized that JTX modulated skewed T2 responses in the elderly. T1 and T2 responses against ovalbumin (OVA) were examined in old BALB/c mice fed JTX (0.2% w/w). We measured anti-OVA IgG1, IgG2a, and IgG2b antibody (Ab) levels after the primary and secondary OVA challenges; T1 and T2 responses augment IgG2a/IgG2b Ab and IgG1/IgE Ab production, respectively. We also assessed production of T1 and T2 cytokines (IFN-gamma and IL-5, respectively), and co-stimulatory molecule expression by regional draining lymph node cells. JTX-fed mice had higher IgG2b Ab and IFN-gamma production than controls along with lower IgG1 Ab. JTX did not alter IL-5 production or co-stimulatory molecule expression. Hoelen, an herbal component, induced similar changes. Our results indicate that JTX and Hoelen modulate T cell responses against OVA toward more balanced T1/T2 responses in old BALB/c mice. Such effects of JTX may help prevent the development of diseases associated with immunodisregulation in the elderly.

Acupuncture inhibits the decrease in brain catecholamine contents and the impairment of passive avoidance task in ovariectomized mice.

The effects of acupuncture on the disorders elicited by abnormalities of endocrine system were investigated in ovariectomized mice. Female mice (strain; C57BL/6) were ovariectomized (OVX) and acupuncture points, Shenshu ([Japanese pictograph see text] : BL23) on both side of the back were continuously stimulated by subcutaneous needles for 20 days. After completion of experimental sessions, animals were sacrificed and specific brain regions were assayed for catecholamine contents by high performance liquid chromatography with electro chemical detector (ECD-HPLC). The mitogenic activities of splenic lymphocytes were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTS) assay and alkaline phosphatase (ALP) assay. Furthermore, the effects of needle stimulation on learning and memory ability were studied by the step-through type passive avoidance test. Norepinephrine and dopamine contents in the frontoparietal cerebral cortex, ventral hippocampus and olfactory bulb were decreased in the OVX group, and both MTS activity and ALP activity were decreased 20 days after ovariectomy. The mean latent period was also shortened in the passive avoidance test in the OVX group. However, applying needle stimulation increased norepinephrine and dopamine contents in the brain regions, and enhanced mitogenic activities of splenic lymphocytes. The stimulation also improved memory-related behavior. It was concluded from this study that after mice were stimulated by subcutaneous needle insertion, overall changes were observed in central nervous system (including retention of memory) and immune functions. The study suggests that acupuncture improves the memory loss and decrease of immune responses accompanying aging and/or menopause, and the that it may have an important role in medical care for the elderly.

Effects of coffee cherry on the activation of splenic lymphocytes in mice.

We previously observed that the ingestion by mice of a hot water extract (CC) and the methanol-extracted and water-soluble fraction (CC-W) of coffee cherry, the residue remaining after the removal of coffee beans from the fruit, enhanced the differentiation of thymocytes and the activation of peripheral T-lymphocytes; and the anti-mammary tumour effects of coffee cherry extract was considered to be associated with this immunomodulation. To study further these effects, mitogen response and some immune parameters were examined in a high mammary tumour strain of SHN mice. While the T-lymphocyte response to concanavalin A was not significantly changed by either CC or CC-W, the lipopolysaccharide response was significantly enhanced by both treatments. The proportion of CD45R/B220+ (B) cells in the splenic lymphocytes was significantly increased by CC, and the percentage of activated (CD25+) cells in B-lymphocytes was increased by CC and CC-W. These data indicate that coffee cherry extract can enhance B-lymphocyte response, and suggest that this immunopotentiation contributes to the antitumourigenic role of coffee cherry.

[New BRM from kampo-herbal medicine].

The action mechanisms of Toki-shakuyakusan (TSS), one of Kampo-herbal medicine, on the clearance of immune complexes and macrophage function were investigated. In the in vivo study, oral administration of TSS enhanced the immune complexes clearance from the circulation in MRL Mp-lpr/lpr mice and C3H/He mice, but no effect was observed in the carbon clearance assay. In the in vitro study, TSS increased the binding of immune complexes to macrophages or Kupffer cells, and the digestion of immune complexes by Kupffer cells. By flow cytometric analysis, the expressions of Fc gamma 11/111 receptors and complement receptor 3 (CR3) on macrophages were increased by the treatment with TSS. Besides, it was also reported that the appearance of the activity was owe to the combination of Angelicae Radix and Atractylodis Lanceae Rhizoma, two of six ingredients of TSS. Both outer and inner dialysate of the extract of Angelicae Radix and Atractylodis Lanceae Rhizoma potentiated the binding of immune complexes to macrophages. Low molecular fraction was further fractionated by using colomn chromatography, and the active components were concentrated in fraction 5-C (named LMW5-C). In conclusion, one of the mechanism of enhancement of immune complexes clearance was thought to due to increase the immune complexes binding to macrophage though augment of Fc gamma 11/111 receptors and CR3 expression. And it was revealed that the active components were not only high moleculer substances but low molecular ones.

Effects of coffee cherry on the immune system in SHN mice.

Previously, we found that chronic ingestion of the extract of coffee cherry (CC), the residue after the removal of coffee beans, induced a marked suppression of the development and the growth of spontaneous mammary tumours in a high mammary tumour strain of SHN mice. As a possible step to clarify the mechanism of this effect, the immunomodulating role of CC was examined in this study. CC treatment resulted in significant weight gain in the spleen. CC treated experimental mice showed a significant decrease in the proportion of immature (CD4+8+) thymocytes and an increase in the percentages of mature cells expressing helper/inducer (CD4+8-) or cytotoxic/suppressor (CD4-8+) phenotypes. The proportion of T cells expressing CD25, a lymphocyte activation marker, in the spleen and peripheral blood tended to increase in the CC treated group. The natural killer activity of the spleen cells was not affected by CC ingestion. These results have revealed that CC can enhance the differentiation of thymocytes and the activation of peripheral T lymphocytes.