Inhibition of granulocyte phagocytosis of Candida albicans by amphotericin B.

Phagocytic activity of PMN's in five healthy and five burned patients were measured in vitro. Addition of 1 microgram per millilitre of amphotericin B to the assay produced a marked inhibitory effect of the phagocytic activity of PMN against C. albicans.     

Canine mycotic stomatitis due to Candida albicans

Candida albicans, a medically important opportunistic yeast is described as the etiologic agent of stomatitis in dogs. The oral swabs collected from 34 dogs showing symptoms of stomatitis or gingivitis such as anorexia, halitosis, bleeding within the oral cavity, dysphagia, ptyalism (salivation) and submandibular lymphadenopathy were cultured for isolation of the causative agent. C. albicans was isolated from four (11.8%) dogs. The isolates were sensitive to clotrimazole, fluconazole and amphotericin-B but were resistant to nystatin. The routine application of Pal's sunflower seed medium and Narayan stain in microbiological laboratories is highly emphasized. It is recommended that the role of C. albicans, as the etiologic agent of canine stomatitis, should be carefully investigated in various clinical related disorders of dogs as well as in other animals.     

Production of a factor inhibiting macrophage migration to the Candida albicans antigen in mice and their resistance to this microorganism

Resistance to C. albicans, an opportunistic microorganism, has been studied in CBA and C57BL/6 mice, oppositely responsive in the production of the factor inhibiting migration of macrophages to antigen obtained from this fungus. The study has shown that CBA mice, highly responsive in the macrophage migration inhibiting factor, are less resistant to C. albicans, while C57BL/6 mice with low response to this antigen are more resistant to this infective agent. Macrophages play, probably, a certain role in the generalization of the process because not all phagocytized C. albicans cells are digested.     

Interaction of Candida albicans with Human Leukocytes and Serum

A quantitative assay of candidacidal activity based on differential staining of non-viable Candida albicans by methylene blue was developed and applied to studies of leukocytes from normal individuals and patients with fungal and other infections. Serum factors were necessary for optimal phagocytosis of C. albicans but lacked direct candidacidal activity. Normal human neutrophils (38 studies) killed 29.0 +/- 7.4% of ingested C. albicans in 1 hr. Eosinophils and monocytes killed a smaller percentage. Neutrophil candidacidal activity did not require protein or ribonucleic acid synthesis by the leukocyte but was inhibited by anaerobic conditions, potassium cyanide, and colchicine. Leukocytes of a patient with hereditary myeloperoxidase deficiency and of three children with chronic granulomatous disease phagocytized C. albicans normally, yet failed to kill them. Our data suggest that the neutrophil can play an important role in resistance to Candida infection and that the lysosomal enzyme myeloperoxidase and its oxidant substrate hydrogen peroxide are the major participants in neutrophil candidacidal activity.     

Cytokine profile of human septic shock serum inducing cardiomyocyte contractile dysfunction

This study was designed to measure nitrite/nitrate and cytokine levels of serum obtained from septic shock patients and to describe potential depressant effects of human septic serum on rat cardiomyocytes. Serum was prepared from 10 non-septic patients and 10 patients with documented septic shock. Adult rat ventricular myocytes were exposed to 20 % serum in the medium. Cardiomyocyte contractility was assessed by measuring shortening fraction and shortening velocity. Serum levels of nitrite/nitrate, a marker of nitric oxide final metabolites, and cytokines (tumor necrosis factor (TNF)-alpha, interleukin (IL) 1beta, 6, 10, 8 and 12p70) were measured. Compared with serum from non-septic patients, serum of septic shock patients induced rapid reduction of the extent and velocity of shortening in isolated cardiomyocytes. Nitrite/nitrate, TNF-alpha, IL-1beta and IL-12p70 concentrations of tested serum for cardiomyocyte studies were not increased in septic serum compared with controls. In contrast, septic serum that induced a depression of in vitro contractility, had increased levels of IL-6, IL-8 and IL-10. We can conclude that the depression of in vitro contractility induced by septic serum is not directly dependent on elevated levels of nitric oxide metabolites, TNF-alpha or IL-1beta. Our results support the view that other cytokines, including IL-6, IL-8 and IL-10, are potent circulating mediators of myocardial depression in cardiomyocytes.     

Mechanisms of lysosomal enzyme release from human polymorphonuclear leukocytes. Effects of phorbol myristate acetate

PMA enhanced release of the azurophil granule enzyme, beta-glucuronidase, as well as lysozyme, from cytochalasin B-treated PMN's exposed to either zymosan particles or C5a. PMA was active at nanomolar concentrations, was not toxic to the cells, and was most effective when present for brief durations (0-1 min) before exposure of the cells to the stimuli. Beta-glucuronidase was not released in significant amounts from PMN's exposed to PMA alone, in the absence of stimuli such as zymosan or C5a. In contrast, only the specific granule enzyme, lysozyme, was released from unstimulated cells. Electron micrographs of cells exposed to PMA revealed an increase in the number of visible cytoplasmic microtubules as compared to control cells. Enhancement of lysosomal enzyme (beta-glucuronidase) release by PMA appears to be independent of effects on release of specific granule enzymes (lysozyme), but rather is likely due to PMA-induced elevations of cellular cGMP.     

In vivo inhibition of tumor growth of B16 melanoma by recombinant interleukin 1 beta. II. Mechanism of inhibition: the role of polymorphonuclear leukocytes

Recombinant human interleukin 1 beta (IL 1 beta) inhibits growth of B16 melanoma in syngeneic C57BL/6 mice in a dose-dependent manner when given intratumorally, intradermally, or intramuscularly over a period of 5 to 7 days. Inhibition of tumor growth was rapid and measurable within 3 days after the initial injection and occurred regardless of the route of injection. However, only intratumoral (ITU) injections of IL 1 beta resulted in greater than 90% inhibition in tumor growth. This enhanced inhibition of tumor growth was not dependent on T or NK cells since inhibition of tumor growth occurred in nude and Beige mice. Also, a profound lymphopenia occurred in mice receiving IL 1 beta. Inhibition of tumor growth did correlate with an increase in the number of polymorphonuclear leukocytes (PMN's) in the circulation. However, only ITU injections of IL 1 beta increased the number of PMN's within the tumors. IM injections of IL 1 beta, while increasing the number of PMN's in the circulation, did not increase the influx of PMN's into the tumors. Furthermore, the transfer of PMN's directly into B16 tumors caused a 49% reduction in tumor growth without the presence of IL 1 beta. These results suggest that in vivo, PMN's may effectively control the growth of tumors and that IL 1 beta may increase this effectiveness by increasing the number of PMN's in the circulation and by locally stimulating the production of chemotactic factors for PMN's within the tumor.     

Epithelial permeability and the transepithelial migration of human neutrophils

Although polymorphonuclear leukocytes (PMN's) can migrate through every epithelium in the body regardless of its permeability, very little is known about the effect of epithelial permeability on PMN migration and the effect of emigrating PMN's on the permeability of the epithelium. In an in vitro model system of transepithelial migration, human PMN's were stimulated by 0.1 micrometer fMet-Leu-Phe to traverse confluent, polarized canine kidney epithelial monolayers of varying permeabilities. Epithelial permeability was determined by both conductance measurement and horseradish peroxidase (HRP) tracer studies. As epithelial permeability increased, the number of PMN invasion sites as well as the number of PMN's that traversed the monolayer increased. The effect of PMN migration on epithelial permeability was examined using the ultrastructural tracers HRP and lanthanum nitrate. PMN's traversing the monolayer made close cell-to- cell contacts with other invading PMNs and with adjacent epithelial cells. These close contacts appeared to prevent leakage of tracer across invasion sites. Following PMN emigration, epithelial junctional membranes reapproximated and were impermeable to the tracers. These results indicated that, in the absence of serum and connective tissue factors, (a) the number of PMN invasion sites and the number of PMN's that traversed an epithelium were a function of the conductance of the epithelium and (b) PMN's in the process of transepithelial migration maintained close cell-cell contacts and prevented the leakage of particles (greater than 5 nm in diameter) across the invasion site.     

Structural requirements for hemoglobin to induce fibronectin receptor expression in Candida albicans

Hemoglobin (Hb) is a host factor that induces expression of a promiscuous receptor on Candida albicans for fibronectin (FN) and several other extracellular matrix proteins. FN receptor expression was induced by ferric (Hb(+)Met and Hb(+)CN), ferrous (HbCO and HbO(2)), and cobalt-protoporphyrin derivatives of Hb, whereas globin was inactive. The Hb derivatives all exhibited saturable, dose-dependent kinetics of FN receptor induction, suggesting that Hb may be acting as a receptor ligand. Soluble Hb bound saturably to a low-affinity binding site [K(d) = (1.1+/-0.2) x 10(-6) M] on C. albicans blastospores. However, uptake of (55)FeHb revealed that heme or iron transport into the cell is not required for induction, since internalization of (55)Fe from Hb did not occur until after induction of FN binding. The serum Hb-binding protein, haptoglobin, specifically abrogated this response, indicating that protein structure rather than the heme ligand or iron is necessary for induction of this signaling pathway. C. albicans also adhered to immobilized Hb, which was sufficient to induce FN receptor expression, and to Hb polymers that formed in defined Hb liquid media in the presence of cells. Formation of Hb polymers in solution required metabolic energy, since the aggregation process was halted with azide addition. Collectively, these data demonstrate that C. albicans recognizes polymerized Hb through multivalent low-affinity interactions, and this may be a host environmental cue that triggers extracellular matrix receptor expression at a septic site.     

Bacterial colonization of the ovarian bursa in dogs with clinically suspected pyometra and in controls

Septic peritonitis occurs relatively commonly in dogs. Secondary septic peritonitis is usually associated with perforation of intestines or infected viscera, such as the uterus in pyometra cases. The aim of this study was to evaluate the bacterial flora in the ovarian bursae of intact bitches as a potential source of contamination. One hundred forty dogs, clinically suspected of pyometra, were prospectively enrolled. The control group consisted of 26 dogs that underwent elective ovariohysterectomies and 18 dogs with mammary gland tumors that were neutered at the time of mastectomy. Bacteriology samples were taken aseptically at the time of surgery from the bursae and the uterus in all dogs. Twenty-two dogs that were clinically suspected of pyometra had sterile uterine content (“mucometra” cases); the remaining 118 had positive uterine cultures (“pyometra” cases) and septic peritoneal fluid was present in 10% of these cases. Of the 118 pyometra cases, 9 had unilateral and 15 had bilateral bacterial colonization of their ovarian bursae. However, the bacteria from the ovarian bursa were similar to those recovered from the uterine pus in only half of the cases. Furthermore, positive bursae were also seen in one mucometra dog (unilateral) and in four control dogs (two unilateral and two bilateral). The data illustrate that the canine ovarian bursa can harbor bacteria. The biological importance of these isolations remains unclear.     

Effects of histones on phagocytosis and phagocytic metabolism]

Enhanced phagocytosis and no intracellular glycogen breakdown was found in guinea-pig peritoneal polymorphonuclear leukocytes (PMN) incubated together with heat-killed staphylococci (Twort) treated with a calf thymus histone fraction (P II), for 1 hr at 37 degrees C. Glcose consumption and lactate production of these PMN's were as high as in phagocytosis of the same germ untreated with P II, and their oxygen consumption was depressed significantly.     

EVALUATION OF CANDIDA ISOLATION MEDIUM IN FOOD HYGIENE IN HUNGARY

Aniline Blue containing Yeast and Mold agar, or Candida Isolation Agar (CIA, DIFCO) was evaluated and found successful in presumptive identification of 8 strains of laboratory cultures of Candida albicans based on the ability of C. albicans to grow and give characteristic fluorescence at 365 nm UV light. C. tropicalis (3 strains) was able to grow but did not fluoresce. The CIA medium was also useful for the recovery of C. albicans from inoculated milk and orange juice. The recovery was complete when peptone water was used as the diluent, however, when milk or orange juice were used as the diluent of the respective food, recovery was reduced to about 40%. No C. albicans was recovered in this study from hands of factory workers (dairy, brewery and canning) or from 12 types of foods tested as well as from food processing environment. More samplings are needed to further ascertain the role of C. albicans in Food Microbiology.     

Membrane attack complex formation on yeast as trigger of selective release of terminal complement proteins from human polymorphonuclear leukocytes

It has recently been shown that measurable amounts of complement proteins, C6 and in particular C7, are released from human polymorphonuclear leukocytes (PMNs). The aim of the present study was to investigate the impact of opsonized Candida albicans on this release. Stimulation with opsonized C. albicans led to a rapid and sustained increase of C6 and C7 in the cell culture supernatant beginning within 5 min of placing in co-culture, whereas co-culture with unopsonized C. albicans or C. albicans mock-opsonized with inactivated human serum did not affect the release. In contrast, even after stimulation employing opsonized C. albicans, no release of the complement component C8 and only trace amounts of C9 were detected. The presence of the membrane attack complex (MAC) on C. albicans after opsonization was demonstrated by indirect immunofluorescence. Opsonization of C. albicans with human serum deficient in or depleted of a terminal complement component resulted in only minor stimulation of C6 and C7 release, although C3 deposition on the surface of C. albicans was not affected as determined by direct immunofluorescence. Detailed analyses with inactivated or deficient sera showed that detection of C6 and C7 was not due to insufficient washing of the opsonized yeast prior to co-culture and suggest that only a small proportion of these proteins was derived from the membrane bound and then cleaved off MAC. Thus, these findings imply that MAC on the fungal surface may represent an additional trigger for the release of C6 and C7 from PMNs, suggesting a new role for the terminal complement complex (TCC) on target membranes as modulator of PMN functions locally at the site of inflammation.     

Multilocus sequence typing confirms synonymy but highlights differences between Candida albicans and Candida stellatoidea

We used multi-locus sequence typing (MLST) to investigate 35 yeast isolates representing the two genome-sequenced strains plus the type strain of Candida albicans, four isolates originally identified as Candida stellatoidea type I and 28 representing type strains of other species now regarded as synonymous with C. albicans. DNA from all 32 C. albicans synonyms readily formed PCR products with the C. albicans MLST primer sets. Their sequences placed all of them within the existing C. albicans clade structure, represented by 1516 isolates. One isolate, originally received as Mycotorula sinensis, was resistant to flucytosine, but no other unusual susceptibilities were found to polyene, azole or echinocandin antifungal agents. The four isolates of C. stellatoidea type I coclustered with two other sucrose-negative isolates, originally identified as examples of Candida africana, in a group of strains highly distinct from the majority of C. albicans. Our results not only confirm the synonymity of all the isolates with C. albicans but also confirm an obvious genotypic difference in the case of C. stellatoidea type I.     

Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicans enolase, an abundant, immunodominant glycolytic enzyme.

We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the highly expressed EF-1 alpha proteins of C. albicans. Northern (RNA) blot analysis showed that the enolase cDNA hybridized to an abundant C. albicans mRNA of 1.5 kb present in both yeast and hyphal growth forms. The polypeptide product of the cloned cDNA, which was purified as a recombinant protein fused to glutathione S-transferase, had enolase enzymatic activity and inhibited radioimmunoprecipitation of a single C. albicans protein of molecular weight 47,000. Analysis of the predicted C. albicans enolase showed strong conservation in regions of alpha helices, beta sheets, and beta turns, as determined by comparison with the crystal structure of apo-enolase A of S. cerevisiae. The lack of cysteine residues and a two-amino-acid insertion in the main domain differentiated C. albicans enolase from S. cerevisiae enolase. Immunofluorescence of whole C. albicans cells by using a mouse antiserum generated against the purified fusion protein showed that enolase is not located on the surface of C. albicans. Recombinant C. albicans enolase will be useful in understanding the pathogenesis and host immune response in disseminated candidiasis, since enolase is an immunodominant antigen which circulates during disseminated infections.     

Effect of epinephrine on neutrophil kinetics in rabbit lungs

The effect of epinephrine on neutrophil (PMN) kinetics in rabbit lungs was evaluated by measuring the retention of radiolabeled PMN's in the lung, the exchange rate between the marginated and circulating pools of PMN's, and the erythrocyte (RBC) transit time. Epinephrine treatment decreased RBC transit times and increased exchange rates in the regions with the shortest transit times but did not change the pulmonary recovery of radiolabeled PMN's. When regions of similar RBC transit time were compared, epinephrine did not affect PMN retention at short transit times but did produce greater retention at long transit times. These data suggest that the major effect of epinephrine was to increase the proportion of the lung having short RBC transit times and fast exchange rates between the marginated and circulating pools. However, this effect did not decrease the overall retention of PMN's most likely because it was balanced by recruitment of additional capillary segments, which increased PMN retention in regions with longer transit times.     

Prevalence of Candida albican serotypes in blood isolates in Chile, and first report of Candida dubliniensis candidemia

Our main goal was to determine the prevalence of C. albicans serotypes isolates from blood cultures and identify the presence of C. dubliniensis. We studied 47 strains identified as C. albicans by conventional methods, 28 were isolated from children and 19 from adult patients. The strains were re-identified by standard methods and phenotypic screening as xylose assimilation and growth at 42 degrees C. API ID 32C (bioMérieux) was employed with the C. dubliniensis suspected strains and confirmation was made by molecular fingerprinting using random amplified polymorphic DNA (RAPD). The C. albicans serotype was determined by agglutination with antiserum anti-antigen 6 from cell wall (Candida Check, Iatron Inc., Japan) and the in vitro susceptibilities were evaluated by a microdilution method. From 47 strains, 46 were confirmed as C. albicans, 31 of them (67%) were serotype A. Adult patients presented a high prevalence of serotype A (95%) and children presented a frequency of 52% of the serotype B (p<0.05). We confirmed the identification of C. dubliniensis in one strain isolated from an infant. All serotype B strains were susceptible to fluconazole, itraconazole and amphotericin B. On the other hand, 3% and 6% of serotype A strains were "susceptible dose dependent" to fluconazole and itraconazole, respectively. C. albicans serotype A was predominant in adult candidemia and its distribution was homogenous in children patients. All strains were highly susceptible to antifungals. We report here the first case of C. dubliniensis candidemia in South America.     

Characterization of Candida antigens by crossed-immunoaffinoelectrophoresis

The antigens of three Candida albicans strains (3153 A, 3156 B and CBS 1905) and one C. tropicalis strain were studied by means of crossed-immunoaffinoelectrophoresis with the corresponding polyvalent antisera. Most antigens (from 63.8% to 77.7% depending on the strain) were bound to concanavalin A-sepharose and about 20% to blue cibacron-sepharose for all the strains tested. Free concanavalin A, wheat germ lectin-sepharose and Helix pomatia lectin-sepharose revealed differences between C. albicans 3153 A and C. albicans CBS 1905 on the one hand and C. albicans 3156 B and C. tropicalis on the other, since affinity percentages were from 4.2 to 10.2 and from 14.2 to 20.0 respectively. Among 10 previously described species-specific antigens of C. albicans, 4 were never bound and 5 were bound to concanavalin A-sepharose which was considered an unsuitable agent for antigen purification since it retained 77% of C. albicans antigens. One important species-specific antigen was bound to blue cibacron sepharose and the corresponding purification could be undertaken. Similar results were found for 12 species-specific antigens of C. tropicalis. Blue cibacron-sepharose as well as wheat germ lectin or Helix pomatia lectin-sepharose were found suitable agents for purification of some of them.     

Observations on some unusual cell types in the enigmatic worm Xenoturbella (phylum uncertain)

The inner epithelially organized gastrodermis of the enigmatic simple worms of the genus Xenoturbella contains numerous partly phagocytized cells of two kinds, ciliated cells (PCCs) and muscle cells (PMCs). PCCs and PMCs have features of undifferentiated cells and do not derive from differentiated adult cells. Homology of phagocytized cells to pulsatile bodies in acoel and nemertodermatid flatworms is therefore rejected. The phagocytized cells might represent an hitherto unknown process of regeneration in Xenoturbella. The phagocytized material contains as much DNA as in all mitochondria and nuclei of the living cells. This is probably caused by lack of digestion of nucleic acids. The genome size of Xenoturbella bocki was determined. It has a C-value of about 0.55 pg.