The regulatory role of interferon-γ producing gamma delta T cells via the suppression of T helper 17 cell activity in bleomycin-induced pulmonary fibrosis.

Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with poor prognosis and high mortality. However, the pathogenesis of IP remains to be elucidated. The aim of this study was to clarify the role of pulmonary γδT cells in IP. In wild-type (WT) mice exposed to bleomycin, pulmonary γδT cells were expanded and produced large amounts of interferon (IFN)-γ and interleukin (IL)-17A. Histological and biochemical analyses showed that bleomycin-induced IP was more severe in T cell receptor (TCR-δ-deficient (TCRδ(-/-) ) mice than WT mice. In TCRδ(-/-) mice, pulmonary IL-17A(+) CD4(+) Τ cells expanded at days 7 and 14 after bleomycin exposure. In TCRδ(-/-) mice infused with γδT cells from WT mice, the number of pulmonary IL-17A(+) CD4(+) T cells was lower than in TCRδ(-/-) mice. The examination of IL-17A(-/-) TCRδ(-/-) mice indicated that γδT cells suppressed pulmonary fibrosis through the suppression of IL-17A(+) CD4(+) T cells. The differentiation of T helper (Th)17 cells was determined in vitro, and CD4(+) cells isolated from TCRδ(-/-) mice showed normal differentiation of Th17 cells compared with WT mice. Th17 cell differentiation was suppressed in the presence of IFN-γ producing γδT cells in vitro. Pulmonary fibrosis was attenuated by IFN-γ-producing γδT cells through the suppression of pulmonary IL-17A(+) CD4(+) T cells. These results suggested that pulmonary γδT cells seem to play a regulatory role in the development of bleomycin-induced IP mouse model via the suppression of IL-17A production.

One-year follow-up of transgene expression by integrase-defective lentiviral vectors and their therapeutic potential in spinocerebellar ataxia model mice.

We examined integrase-defective lentiviral vectors (IDLVs) with a mutant (D64V) integrase in terms of their residual integration capability, the levels and duration of transgene expression and their therapeutic potential in comparison to wild-type lentiviral vectors (WTLVs) with a wild-type integrase gene. Compared with WTLVs, the IDLV-mediated proviral integration into host-cell chromosomes was approximately 1/3850 in HeLa cells and approximately 1/111 in mouse cerebellar neurons in vivo. At 2 months, transgene expression by IDLVs in the mouse cerebellum was comparable to that by WTLVs, but then significantly decreased. The mRNA levels at 6 and 12 months after injection in IDLV-infected cerebella were approximately 26% and 5%, respectively, of the mRNA levels in WTLV-injected cerebella. To examine the therapeutic potential, IDLVs or WTLVs expressing a molecule that enhances the ubiquitin-proteasome pathway were injected into the cerebella of spinocerebellar ataxia type 3 model mice (SCA3 mice). IDLV-injected SCA3 mice showed a significantly improved rotarod performance even at 1 year after-injection. Immunohistochemistry at 1 year after injection showed a drastic reduction of mutant aggregates in Purkinje cellsfrom IDLV-injected, as well as WTLV-injected, SCA3 mice. Our results suggest that because of the substantially reduced risk of insertional mutagenesis, IDLVs are safer and potentially effective as gene therapy vectors.

Insufficient ex vivo expansion of Valpha24(+) natural killer T cells in malignant lymphoma patients related to the suppressed expression of CD1d molecules on CD14(+) cells.

BACKGROUND: Valpha24(+) natural killer T (NKT) cell is a human counterpart of mice Valpha14(+) NKT cell that has a regulatory role for innate and acquired potential antitumor activity. The efficient expansion of NKT cells is an obstacle to the clinical application of Valpha24(+) NKT cells for immunotherapy. METHODS: We used mononuclear cells (MNC) obtained from the peripheral blood (PB) of normal healthy donor (HD) and malignant lymphoma (ML) patients before and after granulocyte colony-stimulating factor (G-CSF) treatment. MNC were cultured for 12 days with alpha-galactosylceramide (100 ng/mL) and interleukin-2 (IL-2; 100 U/mL). RESULTS: The fold expansion of Valpha24(+) NKT cells was higher in HD than in ML patients (208 versus 0.00), despite comparable numbers of Valpha24(+) NKT cells before culture. G-CSF administration enhanced the predominance of Valpha24(+) NKT cell fold expansion in HD compared with ML patients (1935 versus 1.95). After treatment with G-CSF, the expression of CD1d molecules was up-regulated in CD14(+) cells from HD but not ML patients. The fold expansion of Valpha24(+) NKT cells and CD1d expression on CD14(+) cells was strongly correlated in both HD and ML patients (r(2)=0.84). However, replacement of a patient's CD14(+) cells with HD cells did not increase the efficacy of Valpha24(+) NKT cell expansion. DISCUSSION: G-CSF-mobilized PB from ML patients has inhibitory characteristics for Valpha24(+) NKT cell expansion as a result of both monocytes and Valpha24(+) NKT cells. Multiple procedures would be needed for the expansion of patients' Valpha24(+) NKT cells.

Induction of KAI-1 expression in metastatic cancer cells by phorbol esters.

KAI-1 is a tumor suppressor gene whose down-regulation has been shown to be associated with the development of metastases of cancer cells. Here, we demonstrated that KAI-1 expression was induced by activating protein kinase C even in metastatic prostate cancer cell lines in which its expression was significantly down-regulated. KAI-1 expression was enhanced in a dose-dependent manner by PMA, and its induction is at least in part due to transcriptional activation. Pretreatment with calphostin C abrogated its induction by PMA. Our findings may provide useful information for developing a novel drug capable of inducing KAI-1 expression and thereby inhibiting metastasis.

Antioxidative effects of Choi-oki-to and its ability to inhibit the progression of atheroma in KHC rabbits.

Agents which inhibit the oxidative modification of low density lipoprotein (LDL) have been thought to be helpful in preventing the formation of atherosclerotic lesions; the so called "oxidation hypothesis". To test this hypothesis, we examined the antioxidative activities of 127 Kampo medicines in vitro and their inhibitory effects on the development of atheromatous plaque formation in KHC rabbits, a model of spontaneous familial hypercholesterolemia. Some of the 127 Kampo medicines showed scavenging or antioxidative effects equal to or stronger than those of probucol in vitro. Choi joki to, which had the strongest antioxidative effects on LDL in vitro, was chosen for a study in vivo. After 24 weeks, 1 g/kg of Choi joki to successfully inhibited the progression of atherosclerotic lesions in KHC rabbits (P < 0.01). Further investigations regarding the antioxidative effects of Kampo medicines are expected.

Changes in citrate concentration in the mouse uterus with experimentally-induced adenomyosis.

Components of the uterine fluid in mice with experimentally-induced adenomyosis and in controls were examined by proton nuclear magnetic resonance spectroscopy. One of the components was markedly different in mice with adenomyosis. As this component was estimated to be citrate by comparison with authentic samples (standard spectrum), its levels in uterine fluid, uterine tissue and blood were determined by enzymatic analysis. The fluid obtained from the uterus with adenomyosis showed significantly lower concentration of citrate than that from normal uterus. However, the uterine tissue concentration was significantly higher in the experimental mice with adenomyosis. There was no difference in the blood citrate level between the experimental and the control groups. Since adenomyosis was induced by chronic hyperprolactinemia, the change of citrate level in the uterus with this lesion might imply some effects of prolactin (PRL) on metabolism and/or secretion of citrate. However, in normal mice, no significant change was demonstrated in uterine citrate concentration after short-term experimental modulation of the circulating PRL level. Thus, it is unlikely that PRL can regulate directly citrate metabolism in the uterus, indicating some other cause for changes in citrate level accompanying the development of adenomyosis.

P300 as indicator of effects of prophylactic cranial radiation.

In order to assess the late adverse effects of cranial radiation on the central nervous system, 33 children with acute lymphocytic leukemia were examined through event-related potential P300, brain response analysis. P300 latency was significantly prolonged in children who received both prophylactic cranial radiation and intrathecal methotrexate. Although a longitudinal study is necessary, we believe that P300 is useful in the assessment of the adverse effects of cranial radiation in children with acute lymphocytic leukemia.

Inhibitory effects of Dai-saiko-to (Da-Chai-Hu-Tang) on the progression of atherosclerotic lesions in Kurosawa and Kusanagi-hypercholesterolemic rabbits.

The inhibitory effects of the traditional herbal medicine Dai-saiko-to (Da-Chai-Hu-Tang) on the progression of the atherosclerotic lesions were studied using the spontaneous familial hypercholesterolemia (FH) model, Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits. Changes in blood chemistry, pathology and low-density lipoprotein (LDL) oxidation were measured in a control group and a Dai-saiko-to-treated group. In the control group, the area of atheromatous plaques of the aorta progressed between week 12 (29.1%) and 26 (51.5%). This progression of atherosclerotic lesions did not happen in the Dai-saiko-to-treated group between week 12 (26%) and 26 (27.4%). Antioxidative effects on LDL were seen in the Dai-saiko-to-treated group in weeks 16 and 18. Dai-saiko-to did not improve the hypercholesterolemia in the KHC rabbits. These results suggest that Dai-saiko-to has inhibitory effects on the development of atheromatous plaque formation in spontaneous FH model rabbits. It is possible that the antioxidative effects of Dai-saiko-to on LDL led to the beneficial effects observed in this study.

Effects of Sho-seiryu-to on experimental allergic rhinitis in guinea pigs.

The effects of Sho-seiryu-to, an antiallergic Kampo medicine, on experimental allergic rhinitis were investigated in actively sensitized guinea pigs. The number of sneezes and scratches by the animals after a topical antigen challenge was significantly inhibited by pretreatment with Sho-seiryu-to (1000 mg/kg per os p.o.). The antigen-induced eosinophil infiltration in the nasal mucosa was significantly inhibited by Sho-seiryu-to (1000 mg/kg p.o.). Sho-seiryu-to (100 mg/kg p.o.) also reduced the increase in dye leakage to the nasal cavity induced by the antigen challenge and the antigen-induced decrease in volume of the nasal cavity was inhibited. Moreover, Sho-seiryu-to (1000 mg/kg p.o.) suppressed the volume change in the nasal cavity induced by leukotriene D4. These results demonstrate that Sho-seiryu-to inhibits experimental allergic rhinitis in guinea pigs, confirming that the agent may be beneficial for the treatment of allergic rhinitis.

Pharmacological characteristics of Sho-seiryu-to, an antiallergic Kampo medicine without effects on histamine H1 receptors and muscarinic cholinergic system in the brain.

The pharmacological characteristics of Sho-seiryu-to, an antiallergic Kampo medicine, were investigated. Forty-eight-hour passive cutaneous anaphylactic (PCA) reaction was significantly inhibited in rats orally administered Sho-seiryu-to (1000 mg/kg). Sho-seiryu-to significantly inhibited increase in vascular permeability induced by histamine. These data confirm previous findings that Sho-seiryu-to has antiallergic activity in animals and suggest that the antagonism of histamine may be an antiallergic mechanism of Sho-seiryu-to. Sho-seiryu-to did not affect locomotor activity or motor coordination in mice. Although ketotifen prolonged sleeping time induced by pentobarbital, Sho-seiryu-to had no such effect. Nor was there any effect on oxotremorine-induced tremor and [3H]-mepyramine binding to histamine H1 receptors in rat brain. Thus, Sho-seiryu-to appears to be useful for treating type I allergy, with relatively few side effects such as sedation and drowsiness due mainly to blockade of histamine H1 and muscarinic receptors in the brain.

Further pharmacological study on Sho-seiryu-to as an antiallergic.

Examination was made of the pharmacological characteristics of Sho-seiryu-to, an antiallergic kampo medicine. Sho-seiryu-to suppressed histamine release from rat peritoneal mast cells, but failed to inhibit the binding of [3H]-mepyramine to histamine H1 receptors in guinea pig cerebral cortex and lung. Sho-seiryu-to had no effect on cutaneous reactions induced by serotonin, platelet-activating factor (PAF), leukotriene (LT) C4 or LTD4. Ketotifen prolonged electrically induced convulsions, while Sho-seiryu-to did not. Sho-seiryu-to did not affect salivation induced by pilocarpine. Sho-seiryu-to thus does not appear to inhibit histamine H1 receptors or inflammation induced by serotonin, PAF, LTC4 and LTD4, but suppresses mast cell activity. Sho-seiryu-to would thus have only a few side effects such as dry mouth and convulsions due mainly to the blockage of the action of muscarinic in salivary glands and histamine in the brain.

Anti-inflammatory effects of mao-bushi-saishin-to in mice and rats.

Effects of Mao-Bushi-Saishin-to (MBS) on anti-inflammatory activities were examined in mice and rats. MBS significantly inhibited the increase in vascular permeability induced by acetic acid, the ear edema induced by arachidonic acid and phorbol ester, and the cutaneous extravasation induced by bradykinin and histamine. MBS, however, was not effective against the serotonin-induced cutaneous permeability increase in mice. MBS significantly inhibited carrageenin-induced hind foot edema and cotton pellet-induced granulation tissue growth in rats. These results show that MBS may exert anti-inflammatory effects through the underlying mechanism(s) of preventing mediator release from mast cells and macrophages.

[Critical role for cell cycle regulators in androgen receptor function].

Androgen plays an important role in the growth of prostate cancer, but the molecular mechanism that underlies development of resistance to antiandrogen therapy remains unknown. Cyclin E has now been shown to increase the transactivation activity of the human androgen receptor (AR) in the presence of its ligand dihydrotestosterone. The enhancement of AR activity by cyclin E was resistant to inhibition by the antiandrogen 5-hydroxyflutamide. Cyclin E was shown to bind directly to the AB domain of the AR, and to enhance its AF-1 transactivation function. These results suggest that cyclin E functions as a coactivator of the AR, and that aberrant expression of cyclin E in tumors may contribute persistent activation of AR function, even during androgen ablation therapy.

[Clinical role of cell cycle regulators in androgen-dependent cancer cell growth].

The functional and quantitative alterations in cell cycle regulators after androgen depletion in an androgen-dependent cancer cell and the interaction between androgen receptor and cell cycle regulators were examined in order to clarify the initial response of cancer cells to anti-androgen therapy. Fluorescence activated cell sorter analysis (FACS) of androgen-dependent cancer cell line (SC-3) cells cultured with or without 1 nM dihydrotestosterone (DHT) revealed that suppression of cell growth after androgen withdrawal was due to G1 arrest. The protein level of cyclin D1 decreased without any apparent change in the amounts of Cdk2, Cdk4, cyclin E or cyclin A. Among various Cdk inhibitors (CKIs) examined, p27 was upregulated at both mRNA and protein levels 24 h after androgen depletion. On the other hand, cyclin E has been shown to increase the transactivation activity of the human androgen receptor (AR) in the presence of DHT. These results suggest that cell cycle regulators are critical targets in the initial response of androgen-dependent cancer cells to androgen depletion and play a key role in the transcriptional activity of AR.