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Aim: To investigate the fungicidal activity of rod-shaped gold nanoparticles (AuR NPs) against Candida strains isolated from hematooncological patients and representative strains of filamentous fungi. Methods: Colony-counting assays, colorimetric and fluorometric methods and atomic force microscopy were employed. Results: AuR NPs were characterized by their potent fungicidal activity against all tested isolates, regardless of the species or drug susceptibility, at concentrations that are nontoxic to the host cells. The membrane-permeabilizing properties of AuR NPs and induction of reactive oxygen species were recognized as crucial for fungicidal activity. Conclusions: The results provide a rationale for the development of nonspherical Au NPs as effective antifungals or drug-delivery carriers to improve therapy for fungal infections.

Human low-molecular weight salivary mucin (MUC7) is a small, secreted glycoprotein coded by MUC7. In the oral cavity, they inhibit the colonization of oral bacteria, including cariogenic ones, by masking their surface adhesions, thus helping saliva to avoid dental caries. The N-terminal domain is important for low-molecular weight (MG2) mucins to contact with oral microorganisms. In this study, we aimed to identify the N-terminal coding region of the MUC7 gene between individuals with and without caries. Forty-four healthy dental students were enrolled in this study; 24 of them were classified to have caries [decayed, missing, filled-teeth (DMFT) = 5.6] according to the World Health Organization (WHO) criteria, and 20 of them were caries-free (DMFT = 0). Simplified oral hygiene index (OHI-S) and gingival index (GI) were used to determine the oral hygiene and gingival conditions. Total protein levels and salivary total protein levels and salivary buffer capacity (SBC) were determined by Lowry and Ericsson methods. DNA was extracted from peripheral blood cells of all the participants and genotyping was carried out by a polymerase chain reaction (PCR)-sequencing method. No statistical differences were found between two groups in the terms of salivary parameters, oral hygiene and gingival conditions. We detected one common single nucleotide polymorphism (SNP) that leads to a change of asparagine to lysine at codon 80. This substitution was found in 29.0 and 40.0%, respectively, of the groups with and without caries. No other sequence variations were detected. The SNP found in this study may be a specific polymorphism affecting the Turkish population. Further studies with extended numbers are necessary in order to clarify this finding.

The immunoregulatory effect of Artin M and jacalin from extract of Artocarpus integrifolia seeds (jack extract) against infection with Candida albicans was investigated. Swiss mice received jack extract containing 500 μg protein/ml PBS intraperitoneally (i.p.) or PBS alone and after 72 h were infected i.p. with C. albicans CR15 (10 7 ) and sacrificed after 30 min, 2, 6, 24, and 72 h. ELISA analysis revealed that in jack extract-treated mice IFN-γ was predominantly produced versus IL-10 in control mice. These results suggest that jack extract induced a protective immune response, since C. albicans clearance was complete at 72 h postinfection. Jack extract presents two lectins (Artin M and jacalin) with distinct biological properties. Artin M was able to induce IL-12 production by macrophages. Also, Artin M in different concentrations, associated with jacalin or in jack extract induced both IFN-γ and IL-17 production. As a consequence, phagocytic and candidacidal activity increased significantly. Alanine aminotransferase activity (ALT) was used as parameter for damage of the liver. The activity of ALT correlated with inoculum size that increased significantly in control group, however, mice pretreated with jack extract 3 days before infection presented normal ALT. Mice pretreated with jack extract that received a lethal inoculum of Candida presented 90% survival versus 20% among controls or mice pretreated with jacalin. Thus, the results suggest that Artin M by itself, associated with jacalin or present in jack extract is able to induce protective Th1 and Th17 immune responses against Candida albicans infection.

Oral Diseases (2011) 17, 577–583 Objective: To investigate the fungistatic and fungicidal activity of hyaluronic acid (HA) and the influences of HA on the anticandidal activities of lysozyme and the peroxidase system. Materials and Methods: HA, hen egg-white lysozyme, and the bovine lactoperoxidase system were used. Candida albicans ATCC 10231, 18804, and 11006 strains were used in the experiments. The fungistatic activity of HA was determined by measuring the optical densities of the cultures. The candidacidal activity of HA and the influences of HA on the candidacidal activities of lysozyme and the peroxidase system were determined by comparing the numbers of colony-forming units. Results: Hyaluronic acid displayed inhibitory effects on the growth of C. albicans, and the inhibitory effects were proportional to HA concentration. HA did not have any measurable candidacidal activity. HA showed inhibitory effects on the candidacidal activities of lysozyme, and the peroxidase system that was proportional to HA concentration. HA at 1.0–2.0 mg ml−1 almost completely inhibited the candidacidal activities of lysozyme and the peroxidase system. Conclusions: Hyaluronic acid possesses fungistatic activity but no candidacidal activity. HA showed inhibitory effects on the candidacidal activities of lysozyme and the peroxidase system.

Resveratrol is a natural stilbene synthesised by plants. This compound has been shown to inhibit the growth of Candida albicans TIMM 1768 efficiently. Till date, no information is available for other Candida species. The evaluation of the antimicrobial activity of resveratrol was analysed by the inhibition of the growth and metabolism assays. Our data indicate that resveratrol is not effective against Candida albicans and non-C. albicans species (C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei) in vitro. The potential candidacidal activity could not be confirmed.

Candida albicans is an opportunistic pathogen prevalent in AIDS patients and oral candidiasis. Azole-based drugs are currently used in the treatment of candidiasis. Histidine-rich peptides (histatins), are the natural inhibitors of candida species present in human salivary secretions. Sequence comparison of histatins revealed the common motif –KRKFHE– in active peptide fragments. Molecular modeling analysis showed structural similarity between this segment of histatins and azole-based drugs. The helical conformation adopted by histatin-5 may be stabilized by two side chain–side chain interactions (Phe . . . His and Arg . . . Glu). Based on sequence comparison of histatin peptides and molecular modeling, a synthetic 10-residue peptide derived from histatin-5 was helical and possessed significant anti candida activity. This peptide may be used as a template to develop histatin-based drugs for treating oral candidiasis.

Previous investigations indicate that alpha-melanocyte-stimulating hormone (alpha-MSH) and certain synthetic analogues of it exert antimicrobial effects against bacteria and yeasts. However, these molecules have weak activity in standard microbiology conditions and this hampers a realistic clinical use. The aim in the present study was to identify novel peptides with broad-spectrum antimicrobial activity in growth medium. To this purpose, the Gly10 residue in the [DNal(2')-7,Phe-12]-MSH(6-13) sequence was replaced with conventional and unconventional amino acids with different degrees of conformational rigidity. Two derivatives in which Gly10 was replaced by the residues Aic and Cha, respectively, had substantial activity against Candida strains, including C. albicans, C. glabrata, and C. krusei and against gram-positive and gram-negative bacteria. Conformational analysis indicated that the helical structure along residues 8-13 is a key factor in antimicrobial activity. Synthetic analogues of alpha-MSH can be valuable agents to treat infections in humans. The structural preferences associated with antimicrobial activity identified in this research can help further development of synthetic melanocortins with enhanced biological activity.

Objective The purpose of this study was to investigate the candidacidal activity of the glucose oxidase-mediated lactoperoxidase system at various levels of glucose and glucose oxidase. Design Candida albicans ATCC strains 18804, 10231, 11006, bovine lactoperoxidase (25 and 50 μg/mL), and KSCN (1 mM) were used. Different levels of glucose oxidase (1, 5, 10, and 20 units/mL) and glucose (0.03, 0.3, and 3.0 mg/mL) were added to complete the system. The candidacidal activity of the system was examined by preincubating its components for 0–60 min, and then with C. albicans . Candidacidal activity was determined by comparing the numbers of CFU and calculating the percent loss of cell viability. Results The system displayed 13.9–27.4% (without preincubation) to 28.6–34.3% (preincubation for 60 min) loss of viability at 25 μg/mL of bovine lactoperoxidase, 10 units/mL of glucose oxidase, and 0.03 mg/mL of glucose; similar results were obtained with 20 units/mL of glucose oxidase or 0.06 mg/mL of glucose. The candidacidal activity of the system increased markedly as the glucose concentration increased. The candidacidal activity displayed 87.2% (without preincubation) to 100.0% (preincubation for 60 min) at 3.0 mg/mL of glucose. At 3.0 mg/mL of glucose, the system containing 1 or 5 units of glucose oxidase also showed significant levels of candidacidal activity. Conclusions The candidacidal activity of the glucose oxidase-mediated lactoperoxidase system at physiological concentrations of salivary glucose was moderate, but was greatly elevated with increases of glucose level.

In their report, Rauch et al. did not find candidacidal activity of α-melanocyte-stimulating hormone (α-MSH) in Sabouraud dextrose broth. The lack of killing activity by the natural α-MSH in broth medium may occur because of accelerated Candida replication or peptide degradation by fungal enzymes. It should be considered that in physiological conditions, there is sustained peptide release by host cells in an autocrine/paracrine manner. However, when the procedure described in the paper published in the Journal Leukocyte Biology was used, the investigation by Rauch et al. found that concentrations of α-MSH in the high micromolar range have candidacidal activity.

The aim of this study was to assess the in vitro anticandidal activity of Indole-3-carbinol (I3C) and its mode of action against Candida albicans. The results indicated that I3C exhibited candidacidal activity in an energy-independent manner without hemolytic effect on human erythrocytes. To understand the anticandidal mode of action of I3C, gel retardation and fluorescence quenching experiments were conducted against Candida albicans. The results suggest that I3C may exert anticandidal activity by binding to DNA. The present study indicates that I3C has considerable anticandidal activity, deserving further investigation for clinical applications.

Tumor-bearing mice showed a significant resistance against Candida albicans intravenous infection. Longer survival was observed in groups of mice inoculated with fungal cells 2-3 weeks after tumor transplantation with allogeneic sarcoma 180, syngeneic methylcholanthrene-induced Meth A fibrosarcoma, and MM 46 mammary carcinoma than in non-tumor-bearing mice inoculated only with fungal cells. This effect was not observed when the mice were infected only 1 week after tumor transplantation. A significant decrease in the number of C. albicans cells in the kidneys was observed in mice inoculated with fungal cells 2-3 weeks after tumor transplantation. In the tumor-bearing mice treated with cyclophosphamide (CY), a remarkable decrease in both the number of peripheral blood polymorphonuclear leukocytes (PMNs) and the defense against challenge with C. albicans cells was observed, as compared with the CY-untreated groups (normal and tumor-bearing mice). A marked increase in the calcium concentration in serum and number, candidacidal activity, active oxygen level, and myeloperoxidase activity of PMNs was observed in the 2-3-week tumor-bearing mice. From these results, it is suggested that PMNs, which accumulated in the 2-3-week tumor-bearing mice, play an important role in the protection from C. albicans infection by increasing the number and the types of killing factors.

Objective: To delineate neutrophil function of preterm and full-term neonates of Chinese origin. Methods: Chinese neonates admitted to two major Hong Kong neonatal units were randomly selected for the study. Candidacidal activity, phagocytosis, chemotaxis and nitroblue tetrazolium reduction scores were evaluated from blood. Results: Except for chemotactic activity, the neutrophil functions of Chinese preterm neonates were similar to Chinese full-term neonates. The candidacidal activity of males was significantly lower when compared with female neonates. Compared with adult controls, both term and preterm infants had similar phagocytic activities although their candidacidal activities and chemotactic index were significantly decreased. Neutrophil function of Chinese infants was nearer to that of adult controls than historically reported for western infants. Conclusion: Preterm Chinese neonates had similar neutrophil function to full-term infants. Neutrophil function of Chinese infants was more mature than that of western infants.

Disruption of neutrophils causes the release of a cytoplasmic protein which can inhibit the growth of Candida albicans without killing the organisms. The present study was undertaken with a combined system in which candidacidal and candidastatic activities of human neutrophils could be compared. In this system it was found that disruption of either half or all of the neutrophils in the samples markedly improved the ability of the cells to handle an inoculum of Candida yeast cells over a 48-h period, even though the disrupted cells had primarily candidastatic activity, with very little candidacidal activity.

Histatins constitute a group of small, cationic multifunctional proteins present in the saliva of human and some nonhuman primates. The most significant function of histatins may be their anti-fungal activity against Candida albicans and Cryptococcus neoformans. Histatins have been extensively studied at both the protein and gene levels. The structure-function relationship of histatins with respect to their candidacidal activity has also been studied by means of recombinant histatin variants, as well as by chemically synthesized histatin fragments. The mechanism of histatins' action on Candida albicans is not clear, but it appears to be different from that of azole-based anti-fungal drugs which interrupt ergosterol synthesis. During the past 20 years, fungal infections have become more prevalent as a result of the emergence of AIDS, as well as, paradoxically, modern medical advances. The toxicity of current anti-fungal medicine, the emergence of drug-resistant strains, and the availability of only a few types of anti-fungal agents are the major disadvantages of current anti-fungal therapy. Therefore, the importance of the search for new, broad-spectrum anti-fungals with little or no toxicity cannot be overemphasized. The following properties make histatins promising anti-fungal therapeutic agents: (1) They have little or no toxicity; (2) they possess high cidal activities against azole-resistant fungal species and most of the fungal species tested; and (3) their candidacidal activity is similar to that of azole-based antifungals. Current research efforts focus on the development of improved histatins with enhanced cidal activity and stability, and of suitable and effective histatin delivery systems. These and other approaches may help to outpace the growing list of drug-resistant and opportunistic fungi causing life-threatening, disseminating diseases. The histatins with improved protective properties may also be used as components of artificial saliva for patients with salivary dysfunction.

The effects of fluconazole on the candidacidal activity of human neutrophils were investigated. Three clinical isolates of Candida albicans were studied in a colony count assay and the presence of fluconazole during incubation of yeasts with neutrophils had no effect on neutrophil candidacidal activity. These results, using a different assay and three additional isolates, confirm previous studies showing that fluconazole does not interfere with the ability of neutrophils to kill C. albicans .

Mouse phagocytic cells in in vivo diffusion chambers had either candidacidal or candidastatic activity depending on the kind of phagocyte studied, the type of mouse whence the phagocytes came, and whether the chamber inoculum of Candida albicans consisted primarily of yeasts or hyphae. Killing of C. albicans occurred when yeasts were placed into chambers with membranes with 3.0-micrometer pores and implanted intraperitoneally into normal mice or thymus-deficient (nude) mice. Although C. albicans remains in chambers with 3.0-micrometer pores, host phagocytic cells can migrate into the chambers. Killing also occurred when yeasts were combined with normal or nude mouse neutrophils in chambers made with membranes with 0.45-micrometer pores, which restrict migration of host cells, but not diffusion of soluble factors. Populations of cells rich in macrophages were candidastatic for yeasts when the phagocytes came from normal mice but candidacidal when obtained from nude mice. Results of gradient fractionations of peritoneal exudates indicated that more than one cell type may be responsible for candidacidal activity by nude mouse macrophage-rich cells. Hyphal-phase cells of C. albicans appear to be more resistant than yeast-phase cells to killing by normal and nude mouse phagocytic cells.

Simultaneous measurement of neutrophil migration, phagocytic activity, candidacidal and bactericidal activity were made during quadruple chemotherapy of advanced Hodgkin's disease (HD). Measurements were also made in normal individuals, hospital patients not on chemotherapy, untreated patients with advanced HD and patients off chemotherapy for over a year. Neutrophil migratory activity was usually normal in untreated HD patients and those on chemotherapy, but less than 20% of all tests showed depressed values, some of which were corrected by plasma. Similar results were found with neutrophil phagocytosis. Abnormalities in these functions were found in both early and late cycles, but there was a tendency for migration to deteriorate during later chemotherapy cycles. Neutrophil candidacidal and bactericidal activity were frequently depressed in patients on treatment and there was deterioration in candidacidal activity during the chemotherapy cycle. These abnormalities of killing activity were frequently corrected in control plasma. Neutrophil function is normal in most patients with advanced HD and in patients in remission. In a minority of patients on treatment there are marked functional defects, especially in killing activity. These defects are partly cell-associated and partly plasma-related. Susceptibility to infection during chemotherapy of HD may be partly due to defective neutrophil function.

Mouse peritoneal macrophages were infected in vitro with Candida albicans, and the phagocytic and candidacidal activities were estimated by microscopic examination of Giemsa-stained cells. Activated macrophages obtained from either BCG-vaccinated animals or by in vitro exposure of normal macrophages to phytohemagglutinin-induced lymphokines exhibited higher phagocytic and candidacidal activities than did normal macrophages. However, activated macrophages obtained by in vitro exposure of macrophages to candida-induced lymphokines exhibited the highest phagocytic and candidacidal activities. The incorporation of immune mouse serum into the culture medium also enhanced the phagocytic and candidacidal activities of the normal macrophages but failed to improve the function of the activated macrophages. These results suggest that both activated macrophages and antibodies may be required for controlling candida infections in mice.

It has been shown that isoprinosine, an antiviral drug, possesses immunoregulatory properties. To complement such studies, the effects of increasing concentrations of isoprinosine on several functions of polymorphonuclear leukocytes (PMN) [chemotaxis, adhesiveness, particle ingestion, quantitative reduction of nitroblue tetrazolium dye (NBT), candidacidal activity] were investigated. At higher concentrations (greater than 10/gmg/ml) this drug produced a dose-related depressive effect, more pronounced on chemotaxis than on NBT reduction. A significant decrease of adhesiveness was observed only with the highest concentration (1000 μg/ml) of isoprinosine; the other tests were not affected. Thus, these in vitro experiments could not demonstrate any stimulatory effect of isoprinosine on normal human PMN. However, these results do not exclude the possibility of a beneficial effect of the drug on functionally altered PMN.

Studies of host-parasite relationships at the cellular level, using Candida albicans and rabbit alveolar macrophages or guinea pig neutrophils are presented. Guinea pig neutrophils killed the intracellular candida cells presumed by the myeloperoxidase-halide-hydrogen peroxide system. In contrast, rabbit alveolar macrophages did not kill the intracellular candida cells although their phagocytic rate was almost comparable to that of neutrophils. Phagocytizing macrophages were eventually destroyed by the intracellular proliferation of candida cells and formation of germ tubes and pseudomycelia. No significant improvement of candidacidal activity was observed with macrophages from normal and immunized rabbits in immune serum. The mode of phagocytosis by macrophages and neutrophils were also studied under the scanning electron microscope.

The candidacidal activity of mouse neutrophils was determined by a colorimetric method, using a tetrazolium dye that detects living, but not dead, yeast cells. The assay is carried out in microtitre trays, read using a multiwell scanning spectrophotometer (ELISA reader), and can be analysed by computer.

Single and repeated intravenous administration of cyclophosphamide significantly decreased the candidacidal activity of rabbit peritoneal macrophages. Using higher doses of the drug, a more pronounced decrease, persisting up to 10 d, was observed. The phagocytic index has not changed significantly 10 d after cyclophosphamide injection as compared with controls. No changes in the phagocytic activity were recorded. The decreased candidacidal activity may be one of the causes of serious microbial infections in cyclophosphamide-treated patients.

Phagocytosis and killing of ingested Candida albicans by rabbit polymorphonuclear neutrophils were studied in vitro using cells and sera from normal and immunized animals. Optimal phagocytosis was observed when neutrophils and sera obtained from immunized animals were used. The phagocytic index reached its peak at the end of 1 h. The fate of ingested Candida was studied by supravital staining. The highest killing activity was shown by the combination of neutrophils and serum from immunized animals at the end of 1 h. There was no enhancement of this activity on further incubation. Neutrophils derived from non-immunized animals in the presence of normal serum were least effective in candidacidal activity. The candidacidal activity and the inhibitory effect on the germtube formation by the ingested Candida were most marked in the neutrophils obtained from immunized animals.

This study shows that malnourished children have impaired candidacidal activity of leucocytes, a finding which runs parallel to the higher rates of isolation of Candida sp. from throat secretions. In well nournished subjects the mean candidacidal ability was 44.5% in the moderately undernourished group it was 17.6%, and in the severely malnourished group it was 13.7%. This impaired candidacidal capacity may be important in the establishment of large numbers of Candida sp. which are commonly found in the upper intestine and in the pathogenesis of diarrhoea in children with malnutrition.

Storage limits for granulocytes have not been defined. The purpose of this study was to determine these limits for neutrophils collected by discontinuous-flow centrifugation and stored at 4 degrees-6 degrees C. The parameters studied were total leukocyte and absolute cell counts, viability measured by dye exclusion, morphology, percentage phagocytic neutrophils, number of Candida organisms ingested per phagocytic neutrophil, candidacidal activity by differential staining, and chemotaxis under agarose. There was a progressive loss of neutrophils on storage that was statistically significant by 48 hr. Phagocytosis was the best preserved function. Microbial killing measured by candidacidal activity was less well preserved. Chemotaxis was the most poorly maintained parameter. There was mild impairment at 24 hr and a severe functional loss at 48 hr. The data suggest the following: (1) the first functions lost on storage are the most highly integrated, i.e., chemotaxis, followed by microbial killing and then phagocytosis; and (2) assuming that these functional losses are irreversible, storage of normal neutrophils used for transfusion should be limited to approximately 24 hr because a severe defect in migration occurs between the first and second days.

Optimal yields of pulmonary macrophages (PuM) from lavage of normal murine lungs have been obtained by the study of several different lung lavage fluids. Approximately 1 X 10(6) PuM can be consistently recovered from mice weighing 20 to 30 g, using 15 ml of phosphate buffered saline with 0.1% EDTA, with or without 10% fetal calf serum at 37 degrees C. Use of Terasaki tray wells for monolayer formation has allowed a relatively small number of cells (75,000) to be used to prepare multiple replicate wells from each mouse. Simultaneous functional comparisons with other cell types, e.g., peritoneal macrophages (PeM) were performed using this practical and economical system. Data on the phagocytosis and killing of Candida albicans by PuM and PeM are presented as evidence of the utility of this miniaturized system.

In vitro phagocytosis of thirteen Candida berkhout species by rat leukocytes was studied to assess a possible correlation between pathogenicity and phagocytosis Yeast-leukocyte suspensions were mixed up for 3 h and phagocytic index, germ-tube formation and leukocyte candidacidal activity were evaluated. Highest values for phagocytosis were reached in all cases at the end of the first hour. Leukocyte candidacidal activity was absent. Only C. albicans produced germ-tubes. The various phagocytosis indices were determined depending on the Candida species assayed. Under these conditions, the more pathogenic species presented the lower indices of phagocytosis. It is determined that the in vitro phagocytic index may bear a close relationship with the pathogenicity of the Candida berkhout.

Neutrophil cell function in psoriasis was studied through polymorphonuclear cell adherence to nylon wool, chemotaxis under agarose, and phagocytosis of Candida albicans. Differences in PMN adherence were not found between psoriatics and controls. The chemotactic response to zymosan activated serum (ZAS) did not differ between psoriatic and normal PMNs. The psoriatic serum exhibited chemoattracting properties equal to ZAS. Psoriatic serum was shown to be chemoattracting to psoriatic and normal PMNs whereas normal serum was only to psoriatic cells. Differences in recognition mechanisms between psoriatic and normal cells are proposed. Phagocytosis of Candida by psoriatic PMNS was earlier and more prolonged and candidacidal activity was earlier and definetely increased at given times compared to normal PMNs. A protective role of psoriatic PMNs in Munro's microabcesses against bacterial invasion is postulated.

Normal human eosinophils possess appreciable intrinsic candidacidal activity. The leukocyte microbicidal deficiency of chronic granulomatous disease is manifested by eosinophils as well as by neutrophils and monocytes.