Masuko Suzuki

Protective effect of N-acetyl chitohexaose on Listeria monocytogenes infection in mice

A water‐soluble oligosaccharide, N‐acetyl chitohexaose (NACOS‐6) was able to enhance the protecting effect of BALB/c male mice against Listeria monocytogenes infection, when administered intraperitoneally 24 hr before the challenge with this microbe. Significant decrease in number of microbes within the peritoneal cavity, spleen, and liver from the mice of NACOS‐6‐administered group was not observed 1 day after the infection but 4 days after the infection. Administration of NACOS‐6 enhanced the delayed‐type hypersensitivity response against sheep red blood cells (SRBC) or heat‐killed L. monocytogenes. Splenic T lymphocytes from mice administered NACOS‐6 released macrophage activating factor (MAF). These results suggested that NACOS‐6 was also able to elevate the function of cellular immunity. Macrophages treated with a combination of NACOS‐6 and the culture supernatant of splenic T lymphocytes from mice administered NACOS‐6, “NACOS‐6 sup,” were found to exert a fairly strong growth‐inhibitory effect on L. monocytogenes. Interferon‐γ (IFN‐γ) and interleukin 2 (IL‐2) were able to enhance the growth‐inhibitory effect on L. monocytogenes by the NACOS‐6‐treated macrophages.

Protecting Effect of Chitin and Chitosan on Experimentally Induced Murine Candidiasis

Chitin and chitosan were found to exhibit a protective effect on mice administered these polysaccharides intraperitoneally against infection of the viable cells of Candida albicans NIH A‐207 strain. A significant difference was observed between the protective effects of chitin and chitosan, i.e., chitin was much more effective than chitosan when the C. albicans cells were challenged via the intravenous route. In intraperitoneal inoculations of C. albicans cells, however, chitosan provided stronger resistance for mice than chitin.

Effect of N-Acetylchitohexaose against Candida albicans Infection of Tumor-Bearing Mice

A water‐soluble oligosaccharide, N‐acetylchitohexaose (NACOS‐6), was able to enhance the protective effect against Candida albicans infection in mice during the early phase of tumor‐bearing. A significant decrease in the number of C. albicans cells in the kidneys of NACOS‐6‐treated tumor‐bearing mice was observed 8 days after the fungal infection, or 15 days after the tumor transplantation. The candidacidal activity of polymorphonuclear leukocytes from NACOS‐6‐treated tumor‐bearing mice did not differ from that of NACOS‐6‐untreated tumor‐bearing mice. On the other hand, the candidacidal activities of both macrophages and T lymphocytes increased following administration of NACOS‐6 in the early phase of tumor‐bearing. The culture supernatant of T lymphocytes from NACOS‐6‐treated tumor‐bearing mice also potentiated the candidacidal activity of casein‐induced macrophages. An enhancement of natural killer cell activity of splenic lymphocytes obtained from NACOS‐6‐treated tumor‐bearing mice was also observed.

Effect of N-Acetylchito-Oligosaccharides on Activation of Phagocytes

Four N‐acetylchito‐oligosaccharides, from tetra‐N‐acetylchitotetraose (NACOS‐4) to hepta‐N‐acetylchitoheptaose (NACOS‐7), were found to increase the number of peritoneal exudate cells (PEC) in male BALB/c mice after 3 hr intraperitoneal administration of 50 mg/kg of each oligosaccharide. The number of attracted cells, consisting largely of polymorphonuclear leukocytes (PMN), was proportional to the molecular weights of the administered oligosaccharides, except for NACOS‐7 which displayed the same activity as NACOS‐6. In an in vitro chemotaxis assay using normal mouse leukocytes, it was found that NACOS‐6 displayed stronger effects than muramyl dipeptide. The PEC from NACOS‐6 treated mice showed a higher active oxygen‐generating activity. PMN from normal mouse peripheral blood were also shown to have enhanced active oxygen‐generating activity in vitro. PEC from NACOS‐6 treated mice were shown to possess strong candidacidal activity in vitro.

Gelation of Limulus Amoebocyte Lysate by Simple Polysaccharides

The Limulus lysate gelation activity of several simple polysaccharides including yeast mannans and bacterial dextrans was investigated. The mannans from Saccharomyces cerevisiae wild and mutant strains possessing dense branches showed positive gelation activity at concentrations of 1 μg/ml or more regardless of differences in their chemical structure. However, two synthetic mannans possessing linear structures with α1←2 and α1←6 linkages also gave positive reactions at concentrations of 10 μg/ml or more and 500 μg/ml or more, respectively. The dextran from Leuconostoc mesenteroides IAM 1046 consisting of a dense branching moiety displayed reactivity at concentrations of 100 μg/ml or more, while the dextrans devoid of such branches were negative in this reaction. The optimal concentration for Limulus lysate gelation could not be determined for any of the polysaccharides and lipopolysaccharides (LPS) tested in this study. The gelation activity of the polysaccharides was stable to treatment with 100 mm NaOH at 30 C for 72 hr. The minimum concentration for the gelation activity of LPS treated with 100 mm NaOH under the same conditions was reduced from 10−6~−9 μg/ml to 1–10 μg/ml. The above findings demonstrate that the major part of Limulus lysate gelation activity of LPS depends on the alkali‐degradable lipid A moiety, and that such simple polysaccharides are also able to participate in this activity even though the extent of participation is very low.

Chemotactic Response of Human Neutrophils to N -Acetyl Chitohexaose in vitro

N‐Acetyl chitohexaose (NACOS‐6) was able to display chemotactic response of human neutrophils in vitro. In order to analyze the mechanism, a series of chemotaxis studies by means of neutrophils treated with inhibitors of phospholipase A2, cyclooxygenase, or lipoxygenase to NACOS‐6 was conducted. The treatment of neutrophils with inhibitors of phospholipase A2 or cyclooxygenase resulted in decrease of number of migrated cells. However, the lipoxygenase inhibitors did not exhibit the same effect. On the other hand, the treatment of neutrophils with inhibitors of phospholipase A2 or lipoxygenase resulted in decrease of chemotactic response to Formyl‐Met‐Leu‐Phe (FMLP), although the cyclooxygenase inhibitors did not inhibit chemotaxis of neutrophils. Neutrophils added to exogenous prostaglandin E2 (PGE2) caused an enhanced chemotactic response to NACOS‐6. These results indicate that the mechanism of chemotactic response to NACOS‐6 was different from that of FMLP, and that the response was enhanced by PGE2 released from the neutrophils with stimulation of NACOS‐6.

Antitumor Activity of Chitosan and Chitin Immobilized 5-Fluorouracils through Hexamethylene Spacers via Carbamoyl Bonds:

To provide a macromolecular prodrug of 5-fluorouracil (5FU) with reduced side-effects, an affinity for tumor cells and exhibiting the high antitu mor activity, the covalent attachment of 5FU to chitosan and chitin through hexamethylene spacers via carbamoyl bonds was carried out. In vivo testing against p-388 lyphocytic leukemia in female CDF1 mice by intraperitoneal in jection (i.p.) and the in vivo growth-inhibitory effect on Meth-A fibrosarcoma in SPF-C3H/He mice by subcutaneous injection (s.c.)/intravenous injection (i.v.) were evaluated. The effects of the degree of polymerization of chitosan and the degree of 5FU substitution per glucosamine unit on the prolongation of life were investigated. The chitosan-5FU and chitin-5FU conjugates exhibited high survival effects and chitosan-5FU conjugate showed significant growth- inhibitory effect on Meth-A fibrosarcoma. These chitosan-5FU and chitin-5FU conjugates did not display any acute toxicity in the 800 mg/kg dose range.

Preparation and antitumor activity of o-palmitoyldextran phosphates, o-palmitoyldextrans, and dextran phosphate.

Dextran was modified by introduction of palmitoly and phosphate groups. The derivatives were analyzed by sequential periodate oxidation-sodium borohydride reduction. Only one of these derivatives showed a significant growth-inhibitory effect when administered alone, while a second derivative showed, in combination therapy with an ineffective dose of Mitomycin C, a marked synergistic effect.

Candidacidal Effect of Peritoneal Exudate Cells in Mice Administered with Chitin or Chitosan

Recently, several papers have been published on the mechanism of the protective effect of microbial infection and on the role of reactive oxygen intermediates (ROI) and myeloperoxidase (MPO) (2-4, 6-8), demonstrating that polymorphonuclear leukocytes (PMN) play an important role in the killing of Candida albicans by generating large amounts of ROI (2), while macrophages (MO) were able to kill the yeast form cells of C. albicans by releasing a cationic protein (4). However, the role of these phagocytes in the killing process of C. albicans cells has not been fully elucidated . In the preceding paper of this series, we reported that both chitin (poly-Nacetyl-D-glucosamine) and chitosan (de-N-acetylated product of chitin) showed

Gelation of Limulus Lysate by Synthetic Dextran Derivatives

The Limulus test has been considered specific for the presence of bacterial endotoxins. To synthesize a simple model of endotoxin, palmitoyldextran phosphate was prepared by modification of dextran by palmitoylation and phosphorylation. The present studies indicated that a variety of polysaccharide derivatives, such as palmitoyldextran phosphate, palmitoyldextran, and dextran phosphate, give a positive Limulus test and show pyrogenic activity, except for low molecular dextran derivatives. On the other hand, polysaccharides, such as dextran, starch (soluble), chitosan, xylan, and lentinan, were negative in these assays. The gelation reaction of Limulus lysate by modified dextran derivatives may depend on the molecular weight or modification of polysaccharides by palmitoylation and/or phosphorylation to a great extent.