Takafumi Okutomi

Cooperative Anti-Candida Effects of Lactoferrin or Its Peptides in Combination with Azole Antifungal Agents

The effects of lactoferrin (LF), an antimicrobial protein secreted in body fluids, and its peptides in combination with azole antifungal agents were investigated by the micro‐broth‐dilution method in a study of Candida albicans. In the case of LF, its pepsin hydrolysate (LFhyd) or the LF‐derived antimicrobial peptide Lactoferricin® B (LF‐B), the concentrations required to inhibit the growth of Candida decreased in the presence of relatively low concentrations of clotrimazole (CTZ). The minimum inhibitory concentration (MIC) of all azole antifungal agents tested was reduced by 1/41/16 in the presence of a sub‐MIC level of each of these LF‐related substances. Polyene and fluoropyrimidine antifungal agents did not show such a combined effect with these LF‐related substances. The anti‐Candida activity of LF or LF‐B in combination with CTZ was shown to be synergistic by checkerboard analysis. These results indicate that LF‐related substances function cooperatively with azole antifungal agents against C. albicans.

Macrophage-mediated tumor lysis induced by loach egg lectin.

Vertebrate lectin purified from loach egg was tested for induction of tumor lysis mediated by macrophages. Loach egg lectin lysed tumor cells but not normal spleen cells in cooperation with BCG‐ or glucan (TAK)‐elicited peritoneal macrophages of mice. Corynebacterium parvum‐, OK432‐, glycogen‐, lipopolysaccharide‐elicited or resident macrophages were not effective. Neither loach egg lectin nor BCG nor glucan macrophages alone had a cytolytic action on tumor cells. Thus, the vertebrate lectin from loach egg is a mediator in macrophage‐mediated tumor lysis, inducing binding of macrophages to target cells. This lectin‐dependent macrophage‐mediated cytolysis (LDMC) was inhibited by galactose, N‐acetylgalactosamine, fucose, or rhamnose. These results suggest that tumor cells can be recognized via glycoconjugates on cell membrane in addition to tumor‐associated antigen and that some animal lectins participate in macrophage‐mediated tumor lysis.

Anti-Candida activity of calprotectin in combination with neutrophils or lactoferrin.

The effect of an anti‐microbial protein, calprotectin, in combination with neutrophils on the growth of Candida albicans was investigated. The growth inhibition of C. albicans by murine neutrophils was augmented by the addition of a low concentration of calprotectin prepared from rat peritoneal exudate cells. The concentrations of calprotectin causing 50% inhibition of growth of C. albicans in the absence or presence of neutrophils at an effector‐to‐target (E/T) ratio of 30 and 60 were estimated to be 0.45, 0.34 and 0.28 U/ml, respectively. The anti‐Candida activity of calprotectin was completely inhibited by 2 μM of zinc ion, while it only partially lowered the activity of the combination of calprotectin and neutrophils. Lactoferrin, which is an anti‐microbial protein released from neutrophils, strongly inhibited the growth of C. albicans in combination with calprotectin. These results suggest that calprotectin and lactoferrin released from neutrophils may cooperate to inhibit the growth of C. albicans at a local lesion of the infection where there is an accumulation of neutrophils.

Effects of a lipopolysaccharide from Pantoea agglomerans on the cocaine-induced place preference

A lipopolysaccharide from Pantoea agglomerans (LPSp) was purified, and its effect on the cocaine-induced place preference was examined in rats. Cocaine (4 mg/kg, i.p.) produced a significant place preference. Administration of LPSp (5-1000 micrograms/kg, i.p.) alone resulted in neither preference nor aversion for either the drug- or saline-associated place. However, pretreatment with LPSp (500 and 1000 micrograms/kg, i.p.) abolished the place preference that had been induced by cocaine. Furthermore, treatment with LPSp (500 micrograms/kg, i.p.) abolished cocaine (20 mg/kg, i.p.)-induced locomotor enhancement in mice. These results suggest that while LPSp itself may possess neither reinforcing nor locomotor enhancing effects, it blocks both the reinforcing and the locomotor enhancing effects of cocaine. Therefore, LPSp might be useful in pharmacotherapy for prevention of recurrent cocaine abuse.

Augmentation of production of TNF-alpha and anti-tumour activity by an amphotericin B preparation for clinical use in mice

Effects of amphotericin B on production of endogenous tumour necrosis factor alpha (TNF-alpha) and anti-tumour activity in mice was examined. Intravenous administration of Fungizone, an amphotericin B preparation complexed with deoxycholate, augmented the induction of endogenous TNF in response to a second stimulus with intravenous doses of FK23 (heat-killed Enterococcus faecalis). This augmentation was observed when more than 1.8 microg of Fungizone was injected intravenously before intravenous dosing of FK23. The time interval between priming injection of Fungizone and secondary injection of FK23 for the maximal effect was 3 h. Similar augmentation of TNF production was also observed in amphotericin B-primed and FK23-injected mice. Correspondingly, anti-tumour activity of the combined, intravenous injection of Fungizone and FK23 with a 3-h interval was examined. Growth of Meth A fibrosarcoma was clearly inhibited by this combination but not by administration of either one alone. These results suggest that amphotericin B is able to elicit anti-tumour activity, perhaps through activation of the immune system, and in particular augmentation of the induction of endogenous TNF.

Antinociceptive effect of lipopolysaccharide from Pantoea agglomerans on streptozotocin-induced diabetic mice.

The antinociceptive effect of lipopolysaccharide from Pantoea agglomerans (LPSp) in streptozotocin-induced diabetic mice was examined. Although subcutaneous (s.c.) administration of LPSp produced a dose-dependent inhibition of the tail-flick response in both non-diabetic and diabetic mice, the antinociceptive response was greater in diabetic mice than in non-diabetic mice. The antinociceptive effects of LPSp in both diabetic and non-diabetic mice were significantly antagonized by s.c. administration of naltrindole, a selective delta-opioid receptor antagonist or nor-binaltorphimine, a selective kappa-opioid receptor antagonist, but not by beta-funaltrexamine, a selective mu-opioid receptor antagonist. These results suggest that LPSp produces a marked antinociceptive effect in diabetic mice through the activation of delta- and kappa-opioid receptors.

In vitro and in vivo antifungal activities of FX0685, a novel triazole antifungal agent with potent activity against fluconazole-resistant Candida albicans.

To evaluate the therapeutic potential of FX0685, a new triazole antifungal agent, for the treatment of opportunistic fungal infections, particularly systemic candidiasis and aspergillosis, in vitro and in vivo studies were performed using fluconazole (FLC), itraconazole (ITC) and/or amphotericin B (AMB) as reference drugs. A preliminary in vitro study showed that the antifungal activity of FX0685 against FLC-susceptible Candida albicans, several non-C. albicans Candida species and Cryptococcus neoformans was superior to that of FLC and comparable or superior to those of ITC and AMB, while the anti-Aspergillus fumigatus activity of FX0685 was to varying degrees lower than that of ITC. FX0685 appeared to be comparable to FLC and ITC in the treatment of murine systemic C. albicans and pulmonary A. fumigatus infection, respectively. The biological property of FX0685 was characterized by its potent in vitro and in vivo activity against FLC-resistant C. albicans. Part of this unique property was explained by the finding that it retained potent inhibitory activity against those CYP51 molecules in which amino acid substitutions confer a phenotype of resistance to FLC and some other azole derivatives. All of these results lead to the possibility that FX0685 may be a potential antifungal drug candidate for the treatment of various clinical forms of systemic candidiasis, including those caused by FLC-resistant C. albicans, as well as for the treatment of pulmonary aspergillosis.

Antigens Associated with Various Cytotoxic Activities of Murine Peripheral Macrophages

BCG‐ or glucan‐elicited murine peripheral macrophages released a cytotoxin in the presence of loach egg lectin, whereas proteose peptone‐, glycogen‐, or thioglycollate‐elicited or resident macrophages did not. The macrophages that released cytotoxin coincided with those that showed lectin‐dependent macrophage‐mediated cytolysis (LDMC) in response to loach egg lectin. The cytotoxin released by BCG‐elicited macrophages in response to loach egg lectin had a molecular weight of 55 K daltons. The macrophages that released cytotoxin and other cytotoxic macrophages such as those that showed LDMC‐ and antibody‐dependent macrophage‐mediated cytolysis (ADMC) were examined by using several antibodies to surface antigens of macrophages. The results showed that murine peripheral macrophages could be divided into three types. Resident macrophages (Type I) which had common macrophage antigens (Mac‐1 and B12) showed only LDMC in response to wheat germ agglutinin. Some elicited macrophages (Type II) were asialo GM1‐positive and showed both ADMC and LDMC in response to wheat germ agglutinin. Activated macrophages (Type III) showed LDMC in response to loach egg lectin and cytotoxin‐release, but had no antigen detectable with monoclonal anti‐macrophage antibody (C14). These three types of macrophages were clearly distinguished diagrammatically by their roof‐shaped, rocket‐shaped and irregular‐shaped profiles of activities and antigens. These data suggest that several selected surface antigens of macrophages are associated with distinct cytotoxic stages of peripheral macrophages.