Occurrence of atypical lymphocytes following intravenous injection of Candida albicans in mice

The occurrence of atypical lymphocytes has been observed in the course of experimental acute infection withCandida albicans in mice. In animals injected intravenously with 2.5 × 10⁶ ofCandida albicans cells, an increased number of monocytes was seen in 24 hours. Monocytes showed toxic vacuolisation in most instances in protoplasm and sometimes in the nuclei. Only a few atypical lymphocytes could be seen at that time. In the following days the number of monocytes diminished and the number of atypical lymphocytes increased. After four days atypical lymphocytes constituted frequently over 20 % of white cells. The autopsy of sacrificed or dead animals with the presence of such elevated percentages of atypical lymphocytes showed enlargement of cervical lymphnodes in all animals. In mice infected with 1.4 × 10³ ofCandida albicans cells, no level higher than 12% of atypical lymphocytes was seen. Pictures were returning to normal with only a few atypical lymphocytes present among the animals which survived for two months after infection withCandida albicans.This work was supported partially byDora Kaplan, Joan Sloan, Cathy Cooper. Memorial Funds and the Roon Foundation.     

Increased carbohydrate content in the thymus and spleen in mice receiving Candida albicans orally (occurence of lymphomas)

Following oral administration ofCancida albicans to non — inbred albino mice in the experimental conditions reported, an increased level of carbohydrate was found in the spleen and thymus. Significant increase of carbohydrate content in these organs was found to occur in the third month of the experiment and remained so, during the ten months of its duration with the exception of the fourth and fifth month. The carbohydrate content in other essential organs was in a similar range in the control and experimental animals. No significant differences in protein level in the control and experimental animals were found. This was regarded as an indication that the incresaed carbohydrate level in the thymus and spleen of experimental animals represents their reaction to the pathological influences of metabolic products ofCandida albicans.Appearance of lymphomas was observed between the seventh and tenth month. Three lymphomas were found in sixty four animals which were autopsied. Previously reported occurrence of lymphomas in mice which received injection ofCandida albicans to the spleen, provides circumstantial support that the observed increase of carbohydrate level as reported here in the spleen and thymus represents the pathological reaction towards metabolic products ofCandida albicans and forms the basis of a collapse of the defense system of the experimental animals.Thymus and spleen appear to act synergically in the reported experimental conditions and as a part of enterodefense system. The possibility of relevance of observed pathology to humans is discussed.This work was supported partially byDora Kaplan, Joan Sloan, Cathy Cooper Memorial Funds and the Roon Foundation.     

Seizure induction by pentylenetetrazol in rodents receivingCandida albicans glycoprotein orally

Oral administration of extracellularCandida albicaus glycoprotein produces increased proneness to seizures in mice and rats when tested with pentylenetetrazol in the experimental conditions reported.Dosages of 0.25 ug/g of body weight in mice and rats applied for a period of five weeks were enough to produce increased proneness to seizures. It appears that the substance is absorbed from the stomach or intestine and also that its detoxication or excretion is at such a slow rate that it accumulates. Introducing to the stomach much larger dosages could cause increased proneness to seizures to be obtained within six hours. Due to the frequent presence ofCandida albicans in humans and the high probability ofCandida albicans glycoprotein production in vivo, it is possible that the level of this substance may accumulate to produce increased proneness to seizures. The states of tenseness, increased sensitivity and hyperactivity could possibly occur in the same pathological process.A renewal of interest in the possible role of intestinal intoxication in human pathology appears to be indicated. Fungi which are harbored in the digestive tract could be the important source of such toxic substances.This work was supported partially byDora Kaplan, Joan Sloan, Cathy Cooper. Memorial Funds and the Roon Foundation.     

Effects of surface active agents on the susceptibility of Swiss mice to Candida albicans

Summary The LD50's ofCandida albicans combined with Plurafac B26, Mulsor 224, Pluronic L62, and Polyethylene Glycol 400 Mono Laurate injected intraperitoneally into female white Swiss mice were calculated by the method ofReed &Muench. Each of the previously mentioned surface-active agent —Candida albicans combinations had a lower LD50 than theCandida albicans control.Plurafac B26 was found to intially decrease the number of leukocytes in the peritoneal cavity and thus enhance the invasiveness ofCandida albicans.     

Production of glycoprotein by Candida albicans in a synthetic medium and its influence on the growth of newborn mice

Summary Growth ofCandida albicans on a synthetic medium for a period of 6 weeks produces glycoprotein substance in the approximate amount of 150 mg per liter. The polysaccharide component is formed by glucose and mannose in the approximate ratio 3:1. The protein component is composed of at least 15 different amino acids. Half percent of glucosamine was also found. The glycoprotein substance is water soluble and toxic to Swiss mice. LD50 is 0.75 mg/g of body weight when injected intravenously. Subcutaneous injection to newborn Swiss mice produced inhibition of growth. The degree of inhibition varied with the dosage. On one occasion when using a small amount of compound, stimulation of growth was also seen.This work was supported by Damon Runyon Memorial Fund Grant 720 and Joan Sloan Memorial Fund.     

Rapid and extensive vacuolation of the budding yeastsSaccharomyces cerevisiae andCandida albicans by amphotericin B

The effect of amphotericin B (AMPH) on vacuolation in the budding yeastsSaccharomyces cerevisiae andCandida albicans was studied. The minimum inhibitory concentration of AMPH for growth ofS. cerevisiae andC. albicans was 1 µg/ml. In untreated control cultures, mature cells had large central vacuoles in the exponential phase, which hampered the detection of vacuolation effect. Small buds in untreated exponential phase cells, however, only rarely showed vacuoles under the light microscope. Treatment with 0.2 µg/ml of AMPH for 20–30 min induced extensive vacuolation not only in mothers but also buds ofS. cerevisiae. Extensive vacuolation lasted 4 h or more, and growth rate of the cells was much reduced for 8 h or more. Vacuolation itself was not fatal: on removal of the drug most cells gradually recovered from vacuolation and eventually multiplied. A similar effect of AMPH was also observed inC. albicans but at a higher concentration (0.5 µg/ml).     

Seizure induction by pentylenetetrazol in animals experimentally infected with Candida albicans

Summary The prolonged presence ofCandida albicans in the spleen of experimental rats and mice predisposes them to pentylenetetrazol induced seizures. The seizure threshold of the experimental animals is lowered. This becomes statistically significant at ten months after the operation. In the experimental animals the latent period was sometimes reduced. The convulsions in experimental rats and mice were prolonged, sometimes repeated, and on three occasions (2 rats and 1 mouse) ended in death. Electroencephalographic studies confirmed the increased proneness to seizures in the animals to whichCandida albicans was introduced to the spleen.This work was supported by the Damon Runyon Grant no 720, and Joan Sloan Memorial Fund.     

Occurrence of lymphomas and lupus erythematosus-like syndrome in the course of experimental Candida albicans infection in mice

Occurrence of four lymphomas was seen following injection of 1.4×10³ Candida albicans cells to the spleen of eighty non - inbred albine mice. These neoplastic changes occurred in three to seven months of the duration of the experiment. Extensive invasion of the subcutaneous tissue by the tumor was found in one animal. Two of the animals with lymphoma also had changes in the kidneys compatible with lupus erythematosus and presence of LE cells in the blood. In other animals most frequent and extensive pathological changes were found in the kidneys and in order of frequency were as follows: thickening of the basement membrane, fibrinoid degeneration, presence of hematoxylin bodies, wireloop formation. In total, the presence of positive LE cells in blood was found in seven instances and suggestive LE cells was observed in eight animals. Finding of perisplenitis, exudative pleuritis and pericarditis gave additional support to the resemblance of observed pathological changes with human lupus erythematosus. The fact that occurrence of lymphomas and lupus erythematosus together has been reported in humans provides additional interest to the results reported.Results presented here and previously suggest that there may be more extensive interrelationship among variety of pathological changes observed than is accepted until now. Better understanding of host — parasite relationship ofCandida albicans, and possible other fungi, could advance out knowledge of pathogenesis of these diseases.This work was supported partially byDora Kaplan, Joan Sloan, Memorial Funds and the Roon Foundation.     

Role of antibodies and effect of BCG vaccination in experimental candidiasis in mice

The role of humoral antibodies and the effect of BCG vaccination were studied in the experimental candidiasis in mice. The antibody suppressed, B-cell deficient animals were prepared by repeated administration of rabbit anti-mouse--antiserum to the new born mice from birth onwards. Such immunodeficient animals along with controls were infected intravenously with Candida albicans, to study the course of candidal infection. It was observed that B-cell-deficient animals were found to be more susceptible to candidal infection than the controls, as indicated by their steady loss of body weight, longer mean time to death and higher viable counts of candidal cells in different organs. The anti-candidal antibodies were absent in all B-cell-deficient animals but present in the controls. These results suggest that antibodies make a contribution in protection against candidal infection in mice. The BCG vaccinated animals were prepared by repeated intravenous administration of BCG to mice and these vaccinated animals along with unvaccinated controls were challenged intravenously with C. albicans, to study the course of candidal infection. It was observed that BCG vaccination prolonged meantime to death and reduced the number of candidal cells in their kidneys.     

Antibody formation in experimental immunizations with Candida albicans ribosomal fractions

Sera of mice immunized with ribosomal fractions of Candida albicans showed the presence of anti-C. albicans antibodies, detected by the gel-immunodiffusion, agglutination and immune adherence tests.Candida infections are among the most prevalent opportunistic yeast infections, attacking debilitated individuals, and against which there is no effective prophylactic treatment currently available (1, 2, 3, 4, 5). In view of the succes reported in experimental immunizations with ribosomal fractions from various bacteria and some fungi, as summarized by Youmans and Tewari (7, 8), a similar approach for immunization in experimental candidiasis appears reasonable. The present work describes preliminary results on circulating antibodies elicited in the course of immunizations with ribosomal fractions of Candida albicans.Ribosomal preparations were obtained from mechanically disrupted cell-pellets of C. albicans by differential centrifugation and purification in a 15% sucrose and 5% ammonium sulfate solution (in sodium-magnesium-Tris buffer), using a modification of the procedure described by Rubin (6). Concentration of ribosomal-RNA was determined by the absorbance at 260 nm; ribosomal-protein concentration by the Lowry reaction; and purity of the ribosomal preparation checked by the ratio of absorbance at 260 nm to 280, and at 260 to 235 nm. Mice (ICR strain) were immunized with these ribosomal preparations in amounts of 50–100 g ribosomalprotein/mouse, by 2–3 subcutaneous inoculations with Frend's adjuvant, with a 10–21 day interval between the inoculations.This work constitutes part of Ruth Levy's research study towards the Ph.D. degree.     

Prophylactic Effect of Enterococcus faecalis FK-23 Preparation on Experimental Candidiasis in Mice

The prophylactic effects of heat-killed cells of Enterococcus faecalis FK-23 (FK-23 preparation) on experimental candidiasis were investigated in normal and leukopenic mice. In cyclophosphamide-induced leukopenic mice, oral or intraperitoneal administration of the FK-23 preparation at a daily dose of 1.25 or 5 mg/mouse for 3 consecutive days prior to Candida albicans infection significantly prolonged survival periods of the infected mice, and decreased viable counts of C. albicans recovered from their kidneys. In normal mice, the FK-23 preparation administered at dosages ranging from 0.63 to 10 mg/mouse/day for 3 consecutive days was ineffective, while in leukopenic mice, the FK-23 administered orally caused a facilitated recovery in the number of white blood cells including neutrophils. Furthermore, intraperitoneal administration of the FK-23 preparation into mice augmented the anti-Candida activity of immunocompromised peritoneal exudate cells obtained from the animals. These results suggested the potential usefulness of the FK-23 preparation as a prophylactic agent for the management of patients with opportunistic fungal infections.     

Amylolytic activity of yeasts

The total amylolytic activity of 14 selected fermentation type I members of the generaSaccharomyces, Zygosaccharomyces, Candida andTorulopsis was studied. Fractions with α-glucosidase activity, the specificty of which was tested for maltose and sucrose, were isolated on carboxymethyl-cellulose from the intracellular contents of two strains ofCandida albicans and one ofCandida stellatoidea. The fraction from the strainCandida albicans 29-3-109, which is more virulent for mice, displayed the greatest α-glucosidase activity, moderate activity was present in the strainCandida stellatoidea 29-64-1, while the lowest activity was found in the less virulent strain ofCandida albicans, 29-3-19.     

和厚朴酚通过ROS的积累和破坏细胞膜杀死白色念珠菌

[目的]研究在体外情况下和厚朴酚对白色念珠菌的抑制作用及其可能机制。[方法]采用微量稀释法测定和厚朴酚对白色念珠菌的最低抑菌浓度（MIC₈₀）和最低杀菌浓度（MFC）;用透射电镜观察不同浓度和厚朴酚对白色念珠菌超微结构的影响;采用Annexin V-FITC/PI染色法分析不同浓度和厚朴酚对白色念珠菌细胞凋亡的影响;用DCFH-DA染色法测定不同浓度和厚朴酚对白色念珠菌细胞内活性氧积累的影响;用JC-1染色法分析不同浓度和厚朴酚对白色念珠菌线粒体膜电位的影响;用碘化丙啶染色、考马斯亮蓝G-250染色检测和厚朴酚对白色念珠菌细胞膜通透性的影响;通过测定加入麦角甾醇后,和厚朴酚对白色念珠菌的抑制作用的变化,检测和厚朴酚对白色念珠菌细胞膜的影响。[结果]和厚朴酚对白色念珠菌具有很强的抑制作用,MIC和MFC分别为16 μg/mL和32 μg/mL。对白色念珠菌细胞壁、细胞膜和胞浆均有明显的影响。和厚朴酚是通过增加活性氧的产生和破坏线粒体功能来诱导白念珠菌的细胞凋亡和坏死。它也影响细胞膜的通透性,这可能和细胞壁的破坏和与麦角固醇的结合有关。[结论]和厚朴酚通过产生活性氧并伴随着一系列的细胞损伤这种复杂的机制从而对白色念珠菌产生抑制作用,使和厚朴酚成为一种潜在的抗真菌药物。     

Effect of Candida albicans dsDNA in Gastrointestinal Candida Infection

Neonates are highly sensitive to infections because they are biased to develop Th2 immune responses. When exposed to certain agents, such as DNA vaccines or CpG DNA motifs, neonates are capable to mount adult-like Th1 protective responses. This study investigates the capacity of Candida albicans (C. albicans) dsDNA to induce host resistance in newborn mice against gastrointestinal C. albicans infection. The protective properties of dsDNA are related to an increased number of spleen CD4+ T cells secreting IFN-γ. In infected DNA-treated mice, an enhanced production of IFN-γ by Peyer’s patch cells was observed together with reduced colonization and histopathological changes in the stomach. Our results indicated that C. albicans dsDNA administration in neonates elicited the protective immune response against gastrointestinal Candida infection.     

Synergy of fluconazole with macrophages for antifungal activity againstCandida albicans

The possible synergy between macrophages and fluconazole for antifungal activity against different isolates ofC. albicans was studied. The susceptibility ofC. albicans isolates to fluconazole (FCZ), when incubated in RPMI-1640 with 10% fetal bovine serum (FBS) and 10% fresh mouse serum (test medium, TM) was determined by using a quantative culture methodology. Multiplication of isolate Sh27 was strongly inhibited by FCZ, even at 1.0 µg/ml. However, FCZ even at 100 µg/ml was not fungicidal. Resident murine peritoneal macrophages (MP) incubated for 48 h in RPMI-1640+10% FBS (tissue culture medium, TCM), then challenged with Sh27 in TM for 24 h, were fungistatic (20±9%,n=4). Cultured macrophages synergized with FCZ (10 µg/ml) for fungicidal activity when co-cultured with Sh27 in TM for 24 h (46±8%) and for 48 h (74±5%),n=3. Macrophages and FCZ (10 µg/ml) could not synergize for significant killing of a less FCZ-sensitiveC. albicans isolate 94-164. Multiplication of a FCZ-resistant isolate (94–20) was not inhibited by FCZ at 10 µg/ml TM; however, macrophages and FCZ (10 µg/ml) could synergize for fungistatic (64%), but not fungicidal, activity.     

The pathological activity of metabolic products of candida albicans on newborn mice

Summary Concentrated filtrates ofCandida albicans have an inhibiting effect on the growth of newborn mice. Occasionally, this inhibition of growth is very distinct and the clinical picture corresponds to human progeria.Increased dosages of subcutaneous injections of filtrates did not increase the frequency of appearance of distinctly retarded growth in mice, but the mice showed a great area of baldness.In the number of mice, which did not show progeria, there were degenerative changes in liver and kidneys morphologically suggesting glycogenosis. The same type of changes were also observed when contents of disruptedC. albicans cells were injected into newborn mice. In the later case there was positive histochemical staining for the deposits of glycogen.The induction of pathological changes through fungus filtrates is a newly described biological phenomenon, which may play a greater role in pathology.     

Antibiotic tetaine — a selective inhibitor of chitin and mannoprotein biosynthesis in Candida albicans

The antibiotic tetaine inhibits in Candida albicans the biosynthesis of two important cell wall constituents, chitin and mannoprotein. This effect is a consequence of inactivation of the enzyme glucosamine-6-phosphate synthetase. Due to the lack of glucosamine-6-phosphate the effective secretion of mannoprotein enzymes, acid phosphatase and invertase, by Candida albicans spheroplasts is inhibited. In the presence of tetaine, probably a modified mannoprotein, lacking a branched polymannan, is synthesized. The antibiotic action decreases the viability of Candida albicans cells, especially that of mycelial forms of this fungus.Abbreviations GlcNAc N-acetyl-d-glucosamine - GlcN-6-P d-glucosamine-6-phosphate - ManNAc N-acetyl-d-mannosamine - -MM -methylmannoside - EGTA 1,2 di/2-aminoethoxy/ethane - N,N,N,N tetra-acetic acid     

Purification and characterization of novel antifungal peptide,mouse beta defensin-1, in Escherichia coli

Mouse beta defensin-1 (mBD-1) is a cationic peptide with broad antimicrobial activity. The mBD-1 gene was cloned and fused with TrxA to construct pET32-mBD1, which was transformed into E. coli BL21 (DE3). The optimal expression conditions of fusion protein TrxA–mBD1 were: cultivation at 32°C in 2 × YT medium, induction with 0.2 mM isopropylthio-d-galactoside (IPTG), and post-induction expression for 8 h. The fusion protein was highly soluble (90.0%) and accounted for 65% of the total soluble protein; and its volumetric productivity reached 0.67 g/l, i.e., 0.14 g/l of recombinant mBD-1. At 5 μM, purified recombinant mBD-1 killed 50% of Candida albicans. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In vitro susceptibility ofPityrosporum ovale (Malassezia furfur) to human androgenic steroids

Cells ofPityrosporum ovale that colonize human pilosebaceous units are constantly exposed to cutaneous androgenic steroids. The aim of our study was to find out whetherP. ovale is susceptible to these hormones. Three strains ofP. ovale were grown in vitro in the presence of various concentrations oftestosterone, dehydroepiandrosterone, androstenedione, androstanedione, 5--dihydrotestosterone andprogesterone (10, 100, and 1000 µg/ml; agar dilution assays). In addition, three strains ofCandida albicans were also exposed to equal concentrations of the same androgens. As a result, allP. ovale strains were suppressed by 1000 µg/mlandrostenedione, which was the strongest inhibitor. The other androgenic steroids also significantly reducedP. ovale growth at different concentrations, depending on the hormone used and the strain tested.Progesterone was inhibitory at the highest concentration for oneP. ovale strain only.Candida albicans was not affected by any of the androgens. These findings demonstrate an in vitro susceptibility ofP. ovale to high concentrations of human androgenic steroids. A relevance of this interaction for the in vivo fungus-host relation is not apparent.     

Candida albicans antigens studied in complement fixation test and agar gel diffusion test

Summary The antigens prepared simultaneously from seven type ACandida albicans strains by cell disintegration by means of sonic vibrations, grinding with the sea-sand, autolysis and the antigens precipitated with acetone from fluid cultures were studied in complement fixation test and agar gel diffusion test against selected rabbit immune sera. The complement fixation test was performed in plexiglass plates as chessboard titration. The agar gel diffusion test was performed in petri dishes according toOuchterlony and according toBjörklund's specific inhibition of precipitation. The activity of studied antigens determined in complement fixation test was high. The antigen titers were ranging from 1/512 to 1/8192. The reaction observed in agar gel diffusion test were also strong and precipitation spectra consisted of 1–3 lines. The reaction pattern obtained in both tests depended on the character of the analysed reagents. TheBjörklund's modification was successfully applied to serological grouping of the strains under study. Sixteen human sera collected from patients with symptoms of Asthma bronchiale and Bronchitis chronica, from whose sputumCandida albicans was isolated, were tested in both tests. The researches made on human sera were intended as preliminary tests.Head of the department: Prof. Dr.Z. Przybylkiewicz.