A randomized double blind placebo-controlled clinical trial of Hochuekkito, a traditional herbal medicine, in the treatment of elderly patients with weakness N of one and responder restricted design.

OBJECTIVE: To evaluate the effects of Hochuekkito, a traditional Japanese and Chinese medicine, in the treatment of elderly patients with general weakness. To devise a suitable study design for assessing the clinical effectiveness of traditional herbal medicines. METHODS: Fifteen elderly patients (mean +/- SD: age 78.4 +/- 7.8; m/f 3/12) participated in this study. A multicenter, prospective, randomized, double-blind, placebo-controlled study with N of one and responder restricted design was performed. After the run-in period, the patients were divided into responders and non-responders. Only responders were entered in the study, and were randomized into three groups: an active-placebo group, a placebo-active group and an active-active group. The study consisted of two 6-week terms with a 2-week washout period in between. We assessed the Short Form 36 Health Survey (SF-36) and Profile of Mood States (POMS) as an endpoint of quality of life (QOL). In addition, we assessed the biodefense status by measuring the natural killer cytolytic activity (NK activity), IL-2 producing activity of peripheral lymphocytes, lymphocyte proliferating activity and lymphocyte cell-surface antigens. RESULTS: The physical component summary of the SF-36 analysis significantly improved in the Hochuekkito-treated group. Four components (A-H: anger-hostility, F: fatigue, T-A: tension-anxiety, C: confusion) out of six improved in the Hochuekkito-treated group in the POMS analysis. Lymphocyte proliferating activity improved in the Hochuekkito-treated group but not significantly. Concerning the surface antigens of peripheral lymphocytes, the population of CD3 positive cells and CD3CD4 double positive cells increased in the Hochuekkito-treated group. CONCLUSION: We revealed that Hochuekkito improved the QOL and immunological status of elderly patients with weakness by randomized controlled trial. Our study design might be useful for assessing the efficacy of traditional herbal medicine in the future.

Protective effect of keishi-bukuryo-gan and its constituent medicinal plants against nitric oxide donor-induced neuronal death in cultured cerebellar granule cells.

Keishi-bukuryo-gan (Gui-Zhi-Fu-Ling-Wan) (KBG) is a traditional Chinese/Japanese medical (Kampo) formulation that has been administered to patients with "Oketsu" (blood stagnation) syndrome. In the process of neuronal cell death induced by brain ischemia, excessive generation of nitric oxide (NO) free radicals is implicated in the neurotoxicity. In the present study, we examined the protective effects of KBG and its constituent medicinal plants against NO donors, sodium nitroprusside (SNP) and 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC18)-induced neuronal death in cultured rat cerebellar granule cells (CGCs). MTT assay showed cell viability to be significantly increased by the addition of KBG extract (KBGE) (100 microg/ml), Cinnamomi Cortex extract (CCE) (3, 10 and 30 microg/ml), Paeoniae Radix extract (PRE) (100 microg/ml) and Moutan Cortex extract (MCE) (10 and 30 microg/ml) compared with exposure to SNP (30 microM, 24 h) only. Also, cell viability was significantly increased by the addition of KBGE (100 and 300 microg/ml), CCE (30 and 100 microg/ml), PRE (100 and 300 microg/ml) and MCE (30 and 100 microg/ml) compared with exposure to NOC 18 (100 microM, 48 h) only. Persicae Semen extract and Hoelen extract did not protect against NO donor-induced neuronal death. These results suggest that KBG has protective effect against NO-mediated neuronal death in cultured CGCs and that it is derived from Cinnamomi Cortex, Paeoniae Radix and Moutan Cortex.

Effect of Shimotsu-to (a Kampo medicine, Si-Wu-Tang) and its constituents on triphasic skin reaction in passively sensitized mice.

Previous studies have reported that mice passively sensitized with anti-DNP (dinitrophenol) IgE antibody exhibited IgE-mediated skin reaction with an immediate phase response (IPR) at 1 h and a late phase response (LPR) at 24 h after the challenge of DNFB (dinitrofluorobenzene). We recently found that a third phase inflammatory reaction with intense and persisting infiltration of eosinophils, named very late phase response (vLPR), was induced by DNFB challenge peaking at 8 days. In this study, we examined the effects of a Kampo medicine, Shimotsu-to (Si-Wu-Tang), and its constituent crude drugs on triphasic skin reaction in passively sensitized mice. Shimotsu-to inhibited ear swelling in LPR and vLPR after DNFB challenge in a dose-dependent manner, and slightly diminished the scratching behavior considered to be associated with pruritus in IPR. The inhibitory effect on LPR and vLPR was partly due to Cnidii Rhizoma (Senkyu) in Shimotsu-to formulation, especially its fraction 5 containing cnidilide. On the other hand, Angelicae Radix (Toki) rather than Cnidii Rhizoma (Senkyu) in Shimotsu-to, inhibited the scratching behavior, although it did not inhibit the ear swelling in IPR. These findings indicate that the Shimotsu-to formulation is useful for the inhibition of cutaneous inflammatory diseases.

Multilineage hematopoietic recovery by a single injection of pegylated recombinant human megakaryocyte growth and development factor in myelosuppressed mice.

Previous studies have shown that daily multiple administration of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) markedly stimulates thrombopoiesis and effectively ameliorates thrombocytopenia, and in most cases anemia and neutropenia, in myelosuppressed animals. In this study, we evaluated the effects of a single intravenous injection of PEG-rHuMGDF on hematopoietic recovery after sublethal total-body irradiation in mice. A single injection of PEG-rHuMGDF (1 to 640 microg/kg) 1 hour after irradiation accelerated platelet, red blood cell (RBC), and white blood cell (WBC) recovery in a dose-dependent fashion. In the bone marrow of vehicle-treated mice, megakaryocytic, erythroid, and myeloid progenitors, as well as day 12 colony-forming unit-spleen (CFU-S), were dramatically decreased much earlier than the nadirs of peripheral blood cells, whereas megakaryocytes were modestly decreased. Treatment with PEG-rHuMGDF (80 microg/kg, an optimal dose) 1 hour after irradiation resulted in more rapid recovery of these four hematopoietic progenitors and also significantly facilitated megakaryocyte recovery. In addition, the same PEG-rHuMGDF administration schedule expanded bone marrow cells capable of rescuing lethally irradiated recipient mice. As the interval between irradiation and PEG-rHuMGDF treatment was longer, its effects on hematopoietic recovery were attenuated. In contrast to the effects of PEG-rHuMGDF, a single injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) 1 hour after irradiation exclusively accelerated WBC recovery, but only to a similar extent as PEG-rHuMGDF (80 microg/kg) treatment even when rhG-CSF doses were escalated to 1,000 microg/kg. This appeared related to different pharmacokinetics of these two factors after a single injection in irradiated mice. The concentrations of PEG-rHuMGDF after injection persisted in the plasma for a longer time compared with rhG-CSF. These results indicate that a single injection of PEG-rHuMGDF at an early time after irradiation is able to effectively improve thrombocytopenia, anemia, and leukopenia with concomitant accelerated recovery of both primitive and committed hematopoietic progenitors in irradiated mice. Our data also show that compared with the rhG-CSF shown to exert multilineage effects on hematopoiesis, PEG-rHuMGDF has more wide-ranging effects on peripheral blood cell recovery.

Induction and characterization of secretory differentiation in human fetal bronchial epithelial cell line (HFBE) cultured on collagen gel in growth hormone and vitamin A-supplemented medium.

A type of secretory differentiation was induced and characterized in a human fetal bronchial epithelial cell line (HFBE), which was grown on a collagen substratum in a basal differentiative medium (BDM) containing growth hormones and with supplementation of various concentrations of vitamin A (VA). HFBE cells grown on a collagen gel in BDM with or without VA assumed a spindle shape with thick cytoplasm arranged in strands running parallel to each other. Under a phase-contrast microscope, cells cultured in the absence of VA possessed a small number of bright inclusion bodies, which proved to be positive to PAS and almost negative to alcian-blue (AB) staining. Electron microscopy revealed well-developed rough endoplasmic reticulum (rER), enlarged Golgi apparatus and a small number of high-density granules resembling serous or Clara cell granules. HFBE cells treated with VA at levels higher than 6 mu/ml showed a remarkable increase of the secretory granules and contained amorphous material in the rER. Addition of a low concentration of VA (6 ng/ml) only stimulated the growth of HFBE cells. In contrast, higher concentrations of VA significantly inhibited the growth and 3H-thymidine incorporation into DNA in a dose-dependent manner. HFBE cells cultured on collagen gel with VA secreted products with 2 different molecular weights into the medium. A high molecular weight-product, consisting of void volume fractions from a Bio-gel A 15-m column, was identified as hyaluronic acid based on the results obtained from the DEAE-ion exchange chromatography and specific enzymatic digestion. A low molecular weight-product fractionated on the A 15-m was tentatively identified as mainly neutral glycoproteins containing N-linked glycans. While the secretion of hyaluronic acid was inhibited by VA in a dose-dependent manner, the secretion of the neural glycoproteins was most enhanced by VA in the range from the physiological concentration of 600 ng/ml to 6 micrograms/ml. These biochemical data on the secretory products, together with the morphological findings, demonstrate that the HFBE cell line serves as a new model for investigating the cellular differentiation of human lung epithelium.

Secretory differentiation and cell type identification of a human fetal bronchial epithelial cell line (HFBE).

A human fetal bronchial epithelial cell line (HFBE) grew in an undifferentiated pattern under conventional culture conditions. Despite a somewhat fibroblastic shape the cells maintained immunoreactivity to cytokeratin, carcinoembryonic antigen and epithelial membrane antigen. When grown on a collagen gel in a growth-hormone-supplemented medium, their spindle shape became more conspicuous. With an additional supplement of vitamin A (6 micrograms/ml), most of the cells underwent differentiation by producing many bright inclusion bodies which proved to be strongly positive with periodic acid-Schiff and weakly positive with alcian blue staining. Electron microscopy revealed a well-developed rough endoplasmic reticulum, an enlarged Golgi apparatus and many highly electron-dense secretory granules resembling those of Clara cells. Biochemical analysis demonstrated that HFBE cells cultured on collagen gel with vitamin A secreted hyaluronic acid and neutral glycoproteins containing mainly N-linked glycoproteins whose glycans were of a complex type. A monoclonal antibody (SEC-41) generated against the neutral glycoproteins detected a glycoprotein of approximately 52 kDa in the spent culture medium of differentiated HFBE cells. This antibody also reacted with the intracytoplasmic secretory granules in these cells. When tested on frozen sections of lung tissue, the immunohistochemical reactivity of the SEC-41 antibody was confined to Clara cells, some type II pneumocytes in the adult lung, and respiratory epithelial cells in the fetal lung. Moreover, this antibody could detect secretory glycoprotein in broncho-alveolar lavages from two patients. This paper clearly demonstrates that cells derived from human fetal bronchial epithelium can be cultivated in an undifferentiated precursor state and, under appropriate culture conditions, can be stimulated to undergo differentiation into a Clara cell type.