Shigeru Tansho

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.

A Rapid Colorimetric Assay for Determination of Leukocyte-Mediated Inhibition of Mycelial Growth of Candida albicans

A staining method with crystal violet (CV) was demonstrated to be useful for a simple, quick and objective assessment of in vitro growth inhibitory activity of leukocytes against Candida albicans cells. Candida cells incubated with murine neutrophils or macrophages for 14 hr in microwells were stained with CV and, after washing with 0.25% sodium dodecyl sulfate (SDS), treated with isopropanol containing HCl (0.04 N) to extract Candida cell‐bound CV. Then the absorbance at 590 nm of the isopropanol extract was photometrically measured. The results showed that the photometrical absorbance was proportional to the amount of3H‐glucose taken up by C. albicans cells, which reflected the number of viable Candida cells.

Inhibition of Candida albicans Growth by Murine Peritoneal Neutrophils and Augmentation of the Inhibitory Activity by Bacterial Lipopolysaccharide and Cytokines

Anti‐Candida activity of murine neutrophils and its regulation by immunomodulators were studied in vitro. Murine neutrophils which were prepared from peritoneal‐exudated cells inhibited the growth of Candida albicans at an effector: target (E/T) ratio of 30/1 or above. This anti‐Candida activity of neutrophils was augmented by lipopolysaccharide from Escherichia coli, murine tumor necrosis factor (TNF), murine interferon‐γ (IFN‐γ) and murine granulocyte macrophage colony‐stimulating factor (GM‐CSF) but not by granulocyte colony‐stimulating factor (G‐CSF) added to the incubation medium. Greater extent of augmentation was obtained when TNF plus GM‐CSF or INF‐γ plus GM‐CSF were used in combination. These results indicate that anti‐Candida activity of murine neutrophils is regulated similarly to that of the human neutrophils reported previously. Therefore murine peritoneal neutrophils can be used as a favorable substitute for human neutrophils in studies on protective machinery against C. albicans infection.

Suppression of Anti‐Candida Activity of Murine Neutrophils by Progesterone In Vitro: A Possible Mechanism in Pregnant Women's Vulnerability to Vaginal Candidiasis

Sex steroid hormones were examined for their effect on mycelial growth of Candida albicans, and the inhibitory activity of casein‐induced murine peritoneal neutrophils against mycelial growth of C. albicans was examined in vitro using a crystal violet staining method or a [3H]glucose incorporation method. Four steroid hormones, danazol, estradiol, estriol and testosterone had no effect on mycelial growth of C. albicans, but progesterone appeared to convert the growth form of C. albicans from hyphal to yeast. Danazol (10–6 m) and progesterone (10–5 m) suppressed anti‐Candida activity of neutrophils of non‐treated mice, while testosterone, estradiol, and estriol did not. The anti‐Candida activity of neutrophils of estradiol‐pretreated mice was clearly suppressed by progesterone even at 10–6 m which corresponded to its plasma concentration in pregnant women in the third trimester. The physiological significance of this suppressive effect of progesterone was discussed in relation to the vulnerability of pregnant women to vaginal candidiasis.

Influences of aflatoxin B1 on reactive oxygen species generation and chemotaxis of human polymorphonuclear leukocytes

The effects of aflatoxin (AF), a hepatotoxic agent and the strongest carcinogen in nature, on polymorphonuclear leukocyte (PMN) chemotaxis and chemiluminescence (CL) were studied. Luminol-dependent CL activity, which reflects the production of reactive oxygen species (ROS) from PMNs, was up-regulated to approximately 150% when PMNs were treated with 0.05 ng/ml of AFB1 upon stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP) or zymosan. On the other hand, PMN CL activity was down-regulated to approximately 25% of the control when PMNs were treated with 50 ng/ml of AFB1 upon stimulation with zymosan. The effect of AFB1 on PMN chemotaxis was also investigated using the Boyden chamber method. The chemotactic ability of PMNs when interleukin (IL)-8 was used as a chemoattractant was inhibited in a dose-dependent manner at AFB1 concentrations of >10 ng/ml. In reverse transcriptase (RT)-PCR analysis, gene expression levels of CXC chemokine receptor (CXCR)1/2, whose ligands are IL-8, granulocyte chemotactic protein (GCP)-2, neutrophil attractant-activation protein (NAP)-2, and epithelial neutrophil-activating protein (ENA)-78, which regulate PMN chemotaxis, were also down-regulated in a dose-dependent manner by 50 ng/ml AFB1. mRNA expression levels of CXCR1/2 were down-regulated to approximately 85% of that in the controls when PMNs were treated with 100 ng/ml AFB1. These results suggest that a high concentration of AFB1 reduces the chemotactic ability of PMNs via the CXCR1/2 cascade indirectly.

Protective Effect of Oral Administration of: A Traditional Medicine, Juzen-Taiho-To, and Its Components on Lethal Candida Albicans Infection in Immunosuppressed Mice

Protective effects of a kampo medicine, Juzen-taiho-to (TJ-48) and its herbal components against experimental candidiasis in cyclophosphamide-induced immunosuppressive mice were investigated. ICR mice were immunosuppressed by intraperitoneal treatment with cyclophosphamide (day-4) and were orally given TJ-48 or one of its 10 herbal components for 4 consecutive days (day-4--1). They were then challenged intravenously with a lethal dose of Candida albicans (day 0). An oral dose of 1 g/kg/day of TJ-48 prolonged their life span. A similar protective effect was obtained by treatment with its component drugs Ginseng radix, Glycyrrhizae radix, Atractylodis lancea rhizoma or Cnidii rhizoma. These herbal components were suggested to have a main role in the protective effect of Juzen-taiho-to against Candida infection.

Protection of C3H/HE J mice from development of Candida albicans infection by oral administration of Juzen-taiho-to and its component, Ginseng radix: possible roles of macrophages in the host defense mechanisms.

Protective effect of a Japanese traditional herbal medicine, Juzen-taiho-to (TJ-48), which was recently reported to augment host-mediated antifungal actions, in Candida albicans-infected mice was further studied. TJ-48, given orally once daily for 5 consecutive days in a dose of 2 g/kg after intravenous infection of C. albicans, prolonged survival period of infected mice of a C3H/He J strain which is characteristic of functional deficiency of macrophages, but did not that of infected mice of a C3H/He N strain with normal macrophage function. Peritoneal macrophages obtained from C3H/He J mice showed a moderate inhibitory activity against Candida growth in vitro. The anti-Candida activity of the macrophages was augmented by the addition of TJ-48 or some component extracts of TJ-48 to the incubation medium. Among such active component extracts is an extract of Ginseng radix which was demonstrated to enhance the anti-Candida activity of macrophages in vitro and to prolong the survival time of C. albicans-infected C3H/He J mice without effect on C3H/He N mice. On the base of these findings, the mechanisms underlying the protective action of TJ-48 against systemic Candida infection was discussed in relation with its possible activity to activate the macrophage function.

A d-octapeptide drug efflux pump inhibitor acts synergistically with azoles in a murine oral candidiasis infection model

Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis.

Suppression of anti-Candida activity of murine and human neutrophils by glucocorticoids.

Effects of glucocorticoid (GC) compounds on inhibitory activity of neutrophils to mycelial growth of Candida albicans were examined by in vitro crystal violet staining method with 14 hr co‐culture. Both GC hormones (hydrocortisone ≥6 × 10–7 m and corticosterone ≥10–6 m) and anti‐inflammatory GC agents (prednisolone ≥10–7 m and dexamethasone ≥10–8 m) significantly suppressed anti‐Candida activity of murine casein‐induced neutrophils. Anti‐Candida activity of human neutrophils prepared from peripheral blood was also suppressed by hydrocortisone (≥6 × 10–7 m). These GC compounds did not affect the Candida growth in the absence of neutrophils. Steroidal compounds without anti‐inflammatory activity, cholesterol, cholic acid, aldosterone did not suppress neutrophil activity. These results suggest that GCs at their physiological or clinical concentration may suppress anti‐Candida activity of neutrophils in vivo.

Protection of Immunosuppressed Mice from Lethal Candida Infection by Oral Administration of a Kampo Medicine, Hochu-Ekki-To

The protective effect of a Kampo medicine, Hochu-ekki-to (TJ-41) on experimental candidiasis in immunosuppressed mice was investigated. ICR mice were immunosuppressed by injection of prednisolone or cyclophosphamide, given TJ-41 orally and challenged intravenously with Candida albicans (day 0). Treatments with a daily dose of 1 g/kg/day of TJ-41 for 8 days from day-4 or for 4 days from day 0 significantly prolonged the life span of the Candida-infected mice pretreated with prednisolone. The latter treatment appeared to inhibit the colonization of Candida in kidneys of the infected mice. These results suggest that Hochu-ekki-to can be used as a therapeutic agent against candidiasis in patients with glucocorticoid-induced immunosuppression.