Kinin-converting aminopeptidase from human serum

A kinin-converting aminopeptidase from human serum (HuPA) which converts kallidin (lysylbradykinin, LBK) to bradykinin (BK) was purified about 900-fold by a three-step procedure involving ammonium sulfate fractionation, continuous loading Sephadex gel electrophoresis, and gel filtration on Sephadex G-200; the final yield was about 7 per cent. A bioassay was developed in which BK formed in the presence of excess LBK or methionyllysylbradykinin (MLBK), pH 7.8, 37°, was separated on carboxymethylcellulose columns and assayed on the isolated guinea pig ileum. L-Aminoacyl-β-naphthylamidase activities of HuPA measured on L-lysine, L-arginine- and L-leucylnaphthylamides (Lys-NA, Arg-NA and Leu-NA) were parallel to the kinin-converting activity in all purification steps. The activity of the purest preparation on LBK and MLBK, measured at the approximate enzyme:substrate molar ratio 1:1500, was respectively 267 and 174 nmoles BK/min/mg of protein. Considering the activity on Met-NA as 100, the activities on other naphthylamides were: Leu-NA, 65; Arg-NA, 46; Lys-NA, 29; Asp-NA, 0; Glu-NA, 0. A few tri- and dipeptides, angiotensin II and its amide, polylysine and polyarginine were poorer substrates. The following substances were inhibitors: 1,10-phenanthroline, puromycin, EDTA and 2,3-dimercapto-l-propanol. 1,10-Phenanthroline or EDTA inhibition could be partially reversed by Co²⁺, Mn²⁺ and Zn²⁺. The molecular weight was estimated as 95,000 on Sephadex gel filtration. On agarose gel microelectrophoresis, the enzyme migrated as α₁-globulin before the electrophoresis step and had a migration intermediate between α₂- and β-globulin following this step. In this microelectrophoresis, only one band of arylamidase activity was detected on Lys-, Arg- and Leu-NA. The purest preparation was still contaminated with albumin but was free of kininase activity.     

Enhancement of interleukin 1 and interleukin 2 releases by ubenimex

The effect of ubenimex on the release of interleukin 1 (IL-1) and interleukin 2 (IL-2) from immuno-competent cells was studied. Ubenimex enhanced release of IL-1 from mouse peritoneal macrophages at 1.0 and 100 micrograms/ml in vitro and the release at 1.0 microgram/ml was larger. When ubenimex was administered to mice IL-1-releasing activity of the peritoneal macrophages was enhanced 3 and 5 days after the administration but not enhanced 1 day after the administration. Ubenimex also enhanced IL-2 release from rat spleen cells at 0.1 and 10 micrograms/ml, when concanavalin A (Con A) was added in the IL-2-releasing system. The enhancement was still observed with mouse spleen cells, when serum was further added. Moreover, thymocyte-proliferating activity was attained in the broths which rat spleen cells were incubated with ubenimex from 0.1 to 10 micrograms/ml in the absence and presence of Con A.     

Antitumor cells found in tumor-bearing mice given ubenimex

Antitumor effector cells in spleens of tumor-bearing mice given ubenimex were investigated. The administration of ubenimex, starting 7 days after the tumor inoculation, was effective in inhibiting growth of IMC carcinoma. Spleen cells taken from these mice showed a marked suppression of the tumor growth by the WINN assay. The antitumor activity of spleen cells was reduced by treatment to remove T cells or NK cells, whereas spleen cell preparations enriched for T cells showed the strongest antitumor activity. Moreover, NK activity against YAC-1 cells was found in the spleen. These results indicate that the administration of ubenimex to IMC carcinoma-bearing mice generates cytotoxic T cells and NK cells in the spleen. The antitumor effect of ubenimex was not observed in X-ray-irradiated and in anti-asialo GM1 serum-treated mice.     

Biological activity of the main metabolites of ubenimex in humans

The biological activity of the two main metabolites of ubenimex in humans, (-)-N-[(2S,3R)-3-amino-2-hydroxy-4-(4'-hydroxy)phenylbutyryl]-L-leucine (OH-ubenimex) and (2S,3R)-3-amino-2-hydroxy-4-phenylbutyric acid [2S,3R)-AHPA) was examined. OH-Ubenimex was almost identical in inhibitory activity against mouse peritoneal resident macrophage aminopeptidases (APases) and the growth of IMC carcinoma in mice to ubenimex. In contrast, the inhibition of; mouse spleen cell APase activities in vitro, blastogenesis of mouse T and B cells in vitro, delayed cutaneous hypersensitivity in mice, and the growth of C1498 leukemia and HeLa S3 cells in vitro was weaker than ubenimex. Macrophage APase activity was only slightly inhibited by (2S,3R)-AHPA which also had practically no activity in the other biological assays.     

Synthesis of p-hydroxyubenimex

p-Hydroxyubenimex, (2S,3R)-3-amino-2-hydroxy-4-p-hydroxyphenylbutyryl-L-leucine, was synthesized starting from D-tyrosine. The structure and stereochemistry of the synthesized product were confirmed by comparison with p-hydroxyubenimex that was chemically transformed from ubenimex, an aminopeptidase inhibitor of microbial origin. Compared to ubenimex, p-hydroxyubenimex is more active against aminopeptidase B but less active against leucine aminopeptidase. By using the synthetic p-hydroxyubenimex as a reference sample, one of the metabolites of ubenimex was identified as p-hydroxyubenimex. The (2R,3R)-stereoisomer of p-hydroxyubenimex was also prepared. However, its activity against aminopeptidases was much weaker.     

Physical exercise may improve macrophage phagocytic activity of tumor bearing mice.

The influence of an endurance training on the phagocytic activity of macrophages from tumor bearing mice was studied. Female NMRI mice were trained on a treadmill. The training period differed from 3 to 6 weeks for different experimental groups and was performed either before or after tumor inoculation, or both. Either L-1210 or S-180 tumor cells were inoculated into the peritoneal cavity or the interscapular region, respectively. The phagocytic activity of peritoneal and spleen macrophages was estimated at different intervals. The trained animals' macrophages showed increased phagocytic activity especially evident in the group that had been trained before and after tumor inoculation. It was concluded that physical exercise might enhance phagocytosis of macrophages in tumor bearing animals and that this effect strongly depends on the duration and onset of the training program.     

Mechanism of action of BCG vaccine on neoplastic proliferation and host immune responses.

BCG (Bacillus Calmette-Guerin) vaccine, Tice strain, caused a threefold increase in spleen weight of normal animals and a fourfold increase in spleen weight of sarcoma-bearing mice. In the latter group, the BCG vaccine caused infiltration of the sarcoma cells into the peritoneum and tumor metastasis in the spleen. Spleen lymphocytes from mice immunized with neuraminidase-treated sarcoma or from mice that had overcome an inoculum (100 cells) and a challenge (10(4) cells) of sarcoma P-1798 were cytotoxic against 51 Cr- or 14C-2-thymidine-labeled sarcoma cells. The serum of these mice enhanced the cytotoxic activity and inhibited the migration of the syngeneic lymphocytes. These serums also inhibited the migration of peritoneal macrophages from guinea pigs immunized with the sarcoma cells. BCG vaccine enhanced the development and growth of sarcoma P-1798; i.e., 50-100 viable sarcoma cells produced solid tumors in 8% of the untreated animals but in 100% of the BCG-treated animals. The serum of BCG-treated sarcoma-bearing animals inhibited the spleen lymphocyte-mediated cytotoxic action. The spleen lymphocytes from the BCG-treated sarcoma-bearing animals had no effect against 51Cr- or 14C-2-thymidine-labelled sarcoma cells. The data indicate that the serum from BCG-treated sarcoma-bearing animals blocks the spleen lymphocyte-mediated cytotoxic activities directed against proliferation and growth of the sarcoma.     

Enhancement of mouse immune system by pyrrolomycin B

Pyrrolomycin B enhanced both humoral immune response and delayed-hypersensitivity against sheep red blood cells in mice. In spleen cell culture it was a weak inhibitor of mitogenesis. However, in combination with concanavalin A, there was stimulation of mitogenesis in spleen cell culture. Pyrrolomycin B also enhanced phagocytosis of yeasts by peritoneal macrophages after in vivo administration to mice. Thus, pyrrolomycin B, formerly isolated as an antibiotic agent, is an immunopotentiator possibly acting on the membrane of lymphocytes or macrophages.     

Enhancement by ubenimex (bestatin) of host resistance to Candida albicans infection.

Ubenimex is a low molecular weight microbial metabolite which has been demonstrated to have antitumor and immunomodulatory activities. In this study, the protective effect of ubenimex on Candida albicans infection was investigated in normal and immunosuppressed mice. In normal mice, treatment with ubenimex at 0.5, 5 and 25 mg/kg for 5 days prior to infection prolonged survival time in a dose-dependent manner. In immunosuppressed mice treated with a single dose of cyclophosphamide 4 days prior to infection, ubenimex treatment at 5 mg/kg for 5 days significantly increased the number of survivors. Ubenimex-treated mice had a significant increase in number of peritoneal exudate cells with neutrophils as well as enhanced functions, including phagocytosis and active oxygen production. These results suggest the potential usefulness of ubenimex as a prophylactic agent for the management of patients with opportunistic fungal infections.     

Manganese chloride enhances natural cell-mediated immune effector cell function: effects on macrophages

A single intramuscular injection of MnC12 in mice caused an increase in macrophage functional activity. Spleen cell antibody-dependent cell-mediated cytotoxicity against both chicken erythrocytes and P815 tumor cell targets was enhanced 24 h following a single injection of MnC12. Enhanced antibody-dependent cell-mediated cytotoxicity activity following MnC12 treatment was not associated with a change in spleen cellularities compared with saline-injected mice. Resident peritoneal macrophages from mice injected intramuscularly with MnC12 displayed enhanced phagocytic activity for chicken erythrocytes in the presence or absence of opsonizing antibody. Enhanced cytolytic activity against P815 mastocytoma target cells and enhanced cytostatic activity against MBL-2 lymphoma target cells was also observed for nonelicited resident peritoneal macrophages from mice injected intramuscularly with MnC12. There were no differences in the cellularity or relative number of adherent cells obtained from the peritoneal cavity of saline or MnC12-injected mice. These enhanced macrophage functions were associated with the induction of increased interferon levels in mice injected with MnC12.     

The antioxidative and immunostimulating properties of D-glucosamine

The objective of the present study was to investigate the antioxidant activity and immunostimulating property of glucosamine (GlcN) using various in vitro and in vivo tests. Results showed that GlcN possessed excellent antioxidant activities as manifested by strong chelating effect on ferrous ions and protection of macromolecules such as protein, lipid, and deoxyribose from oxidative damage induced by hydroxyl radicals. The immunostimulating effects of GlcN were further evaluated through various immunological tests. GlcN showed excellent activity of enhancing splenocyte proliferation. Neutral red pinocytosis and NO production in mouse peritoneal macrophages were significantly augmented. Oral administration of GlcN to mice for 20 days significantly enhanced the serum antibody level in mice in response to sheep red blood cells (SRBC), increased the relative organ weight of spleen and thymus tissue, and promoted the delayed-type hypersensitivity (DTH) against SRBC as compared with control group. In conclusion, the present investigation reveals GlcN is biologically functional in antioxidative activities and immunostimulating properties.     

Effect of hot water extract from Agaricus blazei Murill on antibody-producing cells in mice

We investigated the immunopotentiating activities of boiled water-soluble extracts from desiccated Agaricus blazei Murill (ABM). Effect of ABM extract on antibody production was investigated by method of hemolytic plaque-forming cells (PFC) against sheep red blood cells (SRBC) antigen. ABM extracts significantly (p<0.01) increased the number of PFC in spleen with intraperitoneal administration at doses of 25 mg/kg as compared with control group. The populations of Mac-1- or CD25-positive cells significantly (p<0.01, p<0.001) increased, but in CD19-positive cells, there were no differences in ABM-treated mice as compared with control mice. The expressions of IL-6 and IL-1beta mRNA were augmented by ABM extract in both peritoneal macrophages and spleen cells. These results suggested that ABM extract might be an effective stimulator for T cell and macrophage to IL-1beta and IL-6 release, resulting in augmentation of antibody production against SRBC antigen.     

Involvement of host macrophages in the immunoadjuvant activity of amphotericin B in a mouse fungal infection model

We have recently reported the in vivo augmentation of resistance to experimental Candida albicans injection by amphotericin B in mice and have shown that this event is concurrent with the appearance in the spleen of a highly candidacidal cell population reactive in vitro against 51Cr-labeled yeast cells. In the present study we characterize these in vitro fungicidal effectors as macrophages and describe the conditions of amphotericin B treatment most suitable for inducing candidacidal activity. We also report that macrophages from intact mice can be activated in vitro to become cytotoxic against Candida. The possible mechanisms through which the amphotericin B activated macrophages exert their increased anti-Candida activity are also investigated.     

Immunomodulating activity of CVT-E002, a proprietary extract from North American ginseng (Panax quinquefolium).

The activity of CVT-E002, an aqueous extract containing mainly oligosaccharides and polysaccharides from North American ginseng (Panax quinquefolium), as an immunobooster on murine spleen cells and peritoneal macrophages, was studied in-vitro. CVT-E002 stimulated the proliferation of normal mouse spleen cells, of which the major responding subpopulation was identified as B lymphocytes. CVT-E002 also activated peritoneal exudate macrophages leading to enhanced interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production. In addition, CVT-E002 stimulated in-vivo immunoglobulin G (IgG) production in treated mice. These results identify some of the immunomodulating activities of CVT-E002 and suggest its use clinically for the modulation of immune responses.     

Immunological and anti-inflammatory effects of clarithromycin: inhibition of interleukin 1 production of murine peritoneal macrophages

The immunological and anti-inflammatory effects of clarithromycin (CAM), a new oral macrolide antibiotic, were examined in in vitro models such as lymphocyte transformation (LTF) of murine spleen cells, interleukin 1 (IL-1) production of murine peritoneal macrophages and IL-1-induced proliferation of C3H/HeJ mice thymocytes; the results were compared with those achieved by erythromycin (EM). CAM suppressed these responses much more than EM. Murine peritoneal macrophages precultured with CAM showed diminished IL-1 production, but macrophages precultured with EM did not, indicating that CAM has suppressive effects on the early phase of IL-1 production of murine peritoneal macrophages. Suppressive effects of CAM on IL-1 production by macrophages and proliferation of lymphocytes were independent of prostaglandin biosynthesis, since this drug had no effect on cyclooxygenase activity. Additional immunosuppressive and anti-inflammatory activities of CAM may explain its superior clinical effect.     

Different cellular responses of dexmedetomidine at infected site and peripheral blood of emdotoxemic BALB/c mice

Various sedative agents, including dexmedetomidine (dex), induce immunosuppression, and enhance infection progression. However, there was no information on how anesthetic affects local and systemic cellular immune function. We conducted this study to examine the impact of dex on the differentiation and function of immune cells at site of inflammation and in peripheral blood during endotoxemia of mice. In BALB/c mice with and without endotoxemia, we evaluated the influence of two dosages of 5 and 50 mcg/kg/h intravenous dex on immune cells: including number of T cells (CD3), B cells (CD19), natural killer cells (CD8a), monocytes (CD11b), and macrophages (Mac‐3) in peripheral blood, the activities of macrophages in peripheral blood and in peritoneal lavage, and proliferation of B and T cells and of natural killer cells activity in the spleen. Endotoxemia increased the number of CD3 T cells, CD 19 B cells and macrophages in the peripheral blood, augmented macrophage activity in the peritoneum, and increased T cell proliferation and natural killer cell activity in the spleen. Further administration of 5 mcg/kg/h dex attenuated systemic increase in number of T cells, B cells, and macrophages during endotoxemia and 50 mcg/kg/h dex significantly attenuated the increase in activity of macrophages in the peripheral blood during endotoxemia. In the peritoneum, however, 5 mcg/kg/h dex preserved and 50 mcg/kg/h dexmedetomidine enhanced the activity of macrophages during endotoxemia. Increased in proliferation of T cells in spleen during endotoxemia was attenuated by both doses of dex. Last, 50 mcg/kg/h dex enhanced natural killer cells activity during endotoxemia. While preserving the effects of endotoxemia on macrophage's activity in the infection site and natural killer cell's activity in the spleen, dex decreased systemic fulminant immune reaction in endotoxemia, by attenuating the augmented response in the number of T cells, B cells and macrophages, activity of macrophages in the peripheral blood, and proliferation of T cells in spleen during endotoxemia. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1416–1422, 2015.     

米糠多糖抗肿瘤作用及其作用的部分机制

目的研究稻糠中提取物米糠多糖（ｒｉｃｅｂｒａｎｐｏｌｙｓａｃｃｈａｒｏｓｅ,ＲＢＳ）对Ｂａｌｂ／ｃ小鼠Ｍｅｔｈ－Ａ纤维肉瘤有无抑瘤作用以及有关细胞免疫学机制。方法设立米糠多糖处理组,环磷酰胺处理组及空白对照组。对各组小鼠的抑瘤情况,腹腔巨噬细胞吞噬能力,巨噬细胞表面Ｆｃ受体数量以及外周血中Ｔ细胞所占比例进行检测对比。结果米糠多糖组Ｂａｌｂ／ｃ小鼠同空白对照组和环磷酰胺处理组比较,有明显抑瘤作用;小鼠腹腔巨噬细胞吞噬能力,巨噬细胞表面Ｆｃ受体数量以及外周血中Ｔ细胞所占比例均有明显改善。结论米糠多糖对Ｂａｌｂ／ｃ小鼠Ｍｅｔｈ－Ａ纤维肉瘤有明显抑瘤作用,该作用与提高细胞免疫功能有关。     

Protective effects of hochu-ekki-to, a Chinese traditional herbal medicine against murine cytomegalovirus infection.

The innate immunity against murine cytomegalovirus (MCMV) at the early phase of infection is mediated by NK cells and macrophages. We studied the effects of hochu-ekki-to (HET), a traditional Chinese herbal medicine, on the regulation of innate immunity mediated by NK cells and macrophages. We found the oral administration of HET to increase both the number of leukocytes in the spleen and liver and the splenic NK cell cytotoxicity associated with the increased induction of serum IFN-alpha/beta after an MCMV infection but it had no effect on liver NK cells. However, no differences were found in the serum IL-12, IFN-gamma, TNF-alpha and nitric oxide (NO) production in the culture of macrophages between the HET- and PBS-treated mice on day 2 after MCMV infection. In addition, HET-treated splenic and peritoneal macrophages were found to show a higher intrinsic resistance against in vitro MCMV infection than that of PBS-treated mice. Therefore, the HET-induced effects on NK cells and macrophages selectively reduced the viral load in the spleen but not in the liver at an early phase of MCMV infection. HET may thus be useful in the treatment of human cytomegalovirus infection which commonly occurs in HIV-infected AIDS patients.     

Enhancement of colony formation of mouse bone marrow cells by ubenimex

Ubenimex enhanced colony formation of bone marrow cells from CDF1 mice induced by L920 cell supernatant, which shows a macrophage-colony-stimulating activity (M-CSA), and also enhanced the colony formation induced CDF1 mouse spleen cell conditioned medium, which shows a granulocyte and macrophage-colony-stimulating activity. The maximal effect was obtained at 0.01 microgram/ml. But, ubenimex showed no effect on the nature of the colonies induced by each CSA. By preincubation of the bone marrow cells with ubenimex, M-CSA-induced colony-forming and the M-CSA-binding activities of the cells were increased. These results suggest that ubenimex enhances the CSA-induced colony formation of bone marrow progenitor cells of CDF1 mouse by increasing the amount of the CSA-binding to the cells.     

Immunomodulatory activity of polysaccharide from Acanthopanax obovatus roots.

The effects of the Acanthopanax obovatus polysaccharide (AOPS) as well as its combination with cyclophosphamide (CY) or prednisolone on immune responses were investigated in mice. AOPS (250 mg/kg i.p. x 5) increased the spleen weight and the number of spleen cells, and augmented the phagocytosis of peritoneal macrophages both in normal mice and in immunosuppressed mice. In a haemagglutinin assay AOPS increased the production of specific antibodies and antagonized the suppressive effect of CY. AOPS not only enhanced the degree of in vitro spleen cell-mediated red blood cells (SRBC) hemolysis (quantitative hemolysis of SRBC) but also restored the suppressive effect of CY completely. From these results, AOPS was shown to have an enhancing and a modulating activity on immune responses.