Unilateral involvement of kidneys in mice infected with Candida albicans

Mice injected with 10(3) cells of a virulent isolate of Candida albicans via the lateral tail vein developed frequent unilateral abnormalities of the right but not the left kidneys. Initially the number of colony forming units in the right and left kidneys were similar but the number of colonies became consistently higher in the right kidneys as the infection progressed. The frequency of unilateral involvement decreased when the inoculum size was increased to 5 X 10(3) cells. These observations indicate that when growth of C. albicans in vivo is monitored over a period of time starting with a low inoculum, it is critical to be consistent in culturing kidneys from the same side.     

Induction of suppressor cells in vitro by Candida albicans

Normal splenocytes cultured with Formalin-killed Candida albicans were shown to acquire significant suppressor cell activity in a period of 3 days. These cells were found to suppress both the phytohemagglutinin-induced mitogen response as well as the anti-sheep erythrocyte antibody response. Experiments were carried out to determine the nature of the suppressor cell population. Results showed that these cells were not susceptible to treatment with anti-Thy 1 antibody and complement. Panning experiments showed that the suppressor cells were not plastic-adherent or Mac-1 antigen-positive. The suppressor cells were, however, adherent to anti-mouse immunoglobulin (F(ab')2-fragment)-coated dishes. Additional experiments showed that the suppressor cell activity was susceptible to treatment with monoclonal anti-Lyb 2.1 antibody and complement. These results suggest that the suppressor cell induced in vitro by Candida is a member of the B-lymphocyte lineage.     

Immune response in mice infected with Candida albicans in the mycelial form

The effect of the infection with the mycelial form of a Candida albicans strain (Mycology Dept.) upon the immune system in mice was studied.BALB/c mice were infected intraperitoneally in a single dose of a 3×10⁶, 6×10⁶ and 12×10⁶ cell suspension of the strain.Macrophages's activity was studied the days 7,14,21,28, 35, and 42 after inoculation, by the following assays: phagocytosis in vitro, mononucleated phagocytic system by the colloidal carbon clearance technique, the lymphocyte's activity by the direct plaque forming cells technique (PFC) and delayed hypersensitivity (DTH).Infection with the mycelial form did not affect the peritoneal macrophage's phagocytic ability, neither modified the delayed hypersensitivity to sheep red blood cells (SRBC). However, a slight and transient depression of the lymphocyte stimulation was found. Suppression of PFC to SRBC was high when a 12×10⁶ cell suspension was used in contrast to the infection with blastospores.These results suggest that systemic infection by Candida albicans in its mycelial form do not induce a non specific immunosuppression.     

Chlamydospore-like cells of Candida albicans in the gastrointestinal tract of infected, immunocompromised mice

We have demonstrated in a previously described murine model of gastrointestinal (GI) and systemic candidiasis that the antifungal angent cilofungin was efficacious in clearing infection of body organs when administered subcutaneously by infusion, but permitted large numbers of Candida albicans in the GI tract to persist. Yeast and hyphae in these animals were associated primarily with the stratified squamous epithelium of the stomach. Administration of immunocompromising drugs (cyclophosphamide plus cortisone acetate) to animals with persistent GI infection resulted in relapse of systemic candidiasis. Histological examination of the gastric mucosa revealed invasive hyphal elements and yeast as well as multiple chlamydospore-like cells. Comparative histochemical and electron-microscopic examinations of these latter cells produced in host tissue and chlamydospores formed in vitro were conducted. The results suggested that similarities in wall and cytoplasmic composition and ultrastructure exist between these in vivo and in vitro produced C. albicans cells. Exposure of C. albicans to cyclophosphamide during in vitro growth resulted in stimulation of chlamydospore production. No significant effect of cortisone acetate on C. albicans morphogenesis was detected. The murine model used in this study permits investigation of the formation of chlamydospore-like cells of C. albicans during early stages of fungal invasion of cyclophosphamide-treated mice, and of the possible influence of these cells on immunological response of the host to persistent candidiasis of the GI tract.     

Cellular and humoral immune responses to Candida albicans in subcutaneously infected mice

Pure mycelial and yeast cultures of Candida albicans were produced in a low sulphate medium. Groups of mice were injected subcutaneously with increasing doses of viable or heat-killed mycelial or yeast cells and the kinetics of delayed-type hypersensitivity (DTH), anti-mycelial and anti-yeast antibodies were studied. Both the dose and the morphological phase of C. albicans showed an influence on the development of the DTH, but the viability is the factor which showed the highest influence on this reaction, since on the one hand mice infected with viable yeast or mycelial cells developed higher DTH levels than mice injected with heat killed cells, and on the other hand this factor seems to play an important role in the kinetics of DTH response. The enzyme-linked immunosorbent assay has been adapted to detect antibodies to yeast and mycelial phase cytoplasmic antigens of C. albicans. In contrast with the DTH reactions, neither dose, morphological phase nor viability played an important role on the antibody titer developed. However, the use of mycelial cytoplasmic antigens seems to be better than the yeasts to detect anti -Candida antibodies over the last days studied.     

Immune responses to yeast and mycelial forms of Candida albicans in intraperitoneally infected mice

Candida albicans E-139 produced pure mycelial and yeast cultures in a low sulphate medium at different temperatures. The influence of the morphological phase, dose and viability of the fungi on the kinetic of delayed-type hypersensitivity (DTH) and anti-mycelial and anti-yeast antibodies have been studied in mice injected intraperitoneally. The mycelial form elicited higher DTH levels than the yeast phase. This effect seems to be related to its antigenic properties. The effect of dose on the immune response depends on the viability of the fungus. The mycelial cytoplasmic antigens were more effective than the yeast ones in detecting antibodies induced during the experiments, particularly during the later stages of the observation periods, suggesting that such antigens may be useful in the serodiagnosis of Candida infections.     

Interactions of Candida albicans with epithelial cells

The fungus, Candida albicans, interacts with epithelial cells in the human host both as a normal commensal and as an invasive pathogen. It has evolved multiple complementary mechanisms to adhere to epithelial cells. Adherent C. albicans cells can invade epithelial surfaces both by penetrating into individual epithelial cells, and by degrading interepithelial cell junctions and passing between epithelial cells. Invasion into epithelial cells is mediated by both induced endocytosis and active penetration, whereas degradation of epithelial cell junction proteins, such as E‐cadherin, occurs mainly via proteolysis by secreted aspartyl proteinases. C. albicans invasion of epithelial cells results in significant epithelial cell damage, which is probably induced by lytic enzymes, such as proteases and phospholipase secreted by the organism. Future challenges include identifying the epithelial cell targets of adhesins and invasins, and determining the mechanisms by which C. albicans actively penetrates epithelial cells and induces epithelial cell damage.     

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.     

Systemic candidiasis in mice immunized with Candida albicans ribosomes

In view of our previous findings that vaccination of mice with Candida albicans ribosomes protects them against experimental systemic candidiasis, the aim of this study was to investigate the effect of this vaccination on the course of infection in immunized animals. Since the kidney is the maj or target in systemic candidal infection, we concentrated in this research on studying the histopathology and determining quantitatively the candidal colonization of this organ. The experiments were carried out at various time intervals after intravenous inoculation with live C. albicans. The colonization of kidneys in immunized mice was markedly lower than that in controls. The maximal difference in renal colonization between immunized and non immunized animals was observed when relatively low challenge doses were used. The inhibition of candidal multiplication in immunized mice seemed to be correlated to their increased resistance against lethal challenge, as expressed by a significantly higher survival rate. Histopathological changes and fungal elements were found in kidneys of control mice as early as 20 h post infection, while the kidneys of immunized mice did not seem affected by the disease. Moreover, even 3 days post infection, the kidneys of vaccinated animals still seemed normal. In conclusion, apparently the ribosomal vaccination leads to diminished colonization of the major site of infection in candidiasis, thus affording protection to the immunized animals against these infections.     

Low dose of Concanavalin-A enhances innate immune response and prevents liver injury in mice infected with Candida albicans

The mechanisms through which Candida albicans is recognized by immune cells and how it triggers host defence are not completely understood. In this study, we evaluated the effect of Concanavalin-A on the clearance of C. albicans by infected mice and their production of proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Subgroups of 5 animals were pretreated with Con-A (250 mug mL(-1) PBS) and after 96 h were infected intraperitoneally with 10(7) cells of C. albicans CR15 (an isolate from a HIV+ person); 30 min, 2, 6, 24 or 72 h after infection the mice were sacrificed. Phagocytosis of C. albicans by peritoneal macrophages increased 30 min after infection in mice pretreated with Con-A. The liver presented the greatest number of CFUs, and this number was reduced by pretreatment with Con-A. Control animals infected with C. albicans presented a significant increase in plasmatic alanine aminotransferase, which was not observed in mice treated with Con-A. Two hours after infection the production of TNF-alpha in the liver of mice pretreated with Con-A was significantly increased. These results suggest that a single dose of Con-A caused a beneficial modulating action of the inflammatory response during infection with C. albicans.     

Generation of suppressor cells in the response of human lymphocytes to a polysaccharide from Candida albicans

MPPS stimulation of human lymphocytes is characterized by a 2-way cooperation phenomenon, i.e., T help of B cells and B help of T cells. In this system suppressor cells are generated after 6 days of culture. They were characterized as Ia- T cells. Their suppressor activity affected both T and B cell stimulation, and it may involve 2 different mechanisms: direct cell suppression, and interaction with soluble helper factors.     

Genetic control of susceptibility to infection with Candida albicans in mice

Candida albicans is an opportunistic pathogen that causes acute disseminated infections in immunocompromised hosts, representing an important cause of morbidity and mortality in these patients. To study the genetic control of susceptibility to disseminated C. albicans in mice, we phenotyped a group of 23 phylogenetically distant inbred strains for susceptibility to infection as measured by extent of fungal replication in the kidney 48 hours following infection. Susceptibility was strongly associated with the loss-of-function mutant complement component 5 (C5/Hc) allele, which is known to be inherited by approximately 40% of inbred strains. Our survey identified 2 discordant strains, AKR/J (C5-deficient, resistant) and SM/J (C5-sufficient, susceptible), suggesting that additional genetic effects may control response to systemic candidiasis in these strains. Haplotype association mapping in the 23 strains using high density SNP maps revealed several putative loci regulating the extent of C. albicans replication, amongst which the most significant were C5 (P value = 2.43×10⁻¹¹) and a novel effect on distal chromosome 11 (P value = 7.63×10⁻⁹). Compared to other C5-deficient strains, infected AKR/J strain displays a reduced fungal burden in the brain, heart and kidney, and increased survival, concomitant with uniquely high levels of serum IFNγ. C5-independent genetic effects were further investigated by linkage analysis in an [A/JxAKR/J]F2 cross (n = 158) where the mutant Hc allele is fixed. These studies identified a chromosome 11 locus (Carg4, Candida albicans resistance gene 4; LOD = 4.59), and a chromosome 8 locus (Carg3; LOD = 3.95), both initially detected by haplotype association mapping. Alleles at both loci were inherited in a co-dominant manner. Our results verify the important effect of C5-deficiency in inbred mouse strains, and further identify two novel loci, Carg3 and Carg4, which regulate resistance to C. albicans infection in a C5-independent manner.     

A novel suppressor tRNA from the dimorphic fungus Candida albicans

Unfractionated tRNAs from a number of prokaryotes and eukaryotes were examined for their ability to promote termination codon readthrough in a cell-free system isolated from Saccharomyces cerevisiae. tRNA from the dimorphic fungus Candida albicans was found to have significant UGA and UAG readthrough activity and this activity was present in tRNA extracted from both the yeast and the hyphal phase of the fungus. Unusually the efficiency of readthrough activity in vitro was not affected by the [psi] determinant. C. albicans tRNA was fractionated by one-dimensional and two-dimensional gel electrophoresis and both readthrough activities appeared to be associated with a single species of tRNA.     

Characteristics of biofilm formation by Candida albicans

A variety of manifestations of Candida albicans infections are associated with the formation of biofilms on the surface of biomaterials. Cells in biofilms display phenotypic traits that are dramatically different from their free-floating planktonic counterparts, such as increased resistance to anti-microbial agents and protection form host defenses. Here, we describe the characteristics of C. albicans biofilm development using a 96 well microtitre plate model, microscopic observations and a colorimetric method based on the use of a modified tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide, XTT) to monitor metabolic activities of cells within the biofilm. C. albicans biofilm formation was characterized by initial adherence of yeast cells (0-2 h), followed by germination and micro-colony formation (2-4 h), filamentation (4-6 h), monolayer development (6-8 h), proliferation (8-24 h) and maturation (24-48 h). The XTT-reduction assay showed a linear relationship between cellular density of the biofilm and metabolic activity. Serum and saliva pre-conditioning films increased the initial attachment of C. albicans, but had minimal effect on subsequent biofilm formation. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize C. albicans biofilms. Mature C. albicans biofilms consisted of a dense network of yeasts cells and hyphal elements embedded within exopolymeric material. C. albicans biofilms displayed a complex three dimensional structure which demonstrated spatial heterogeneity and a typical architecture showing microcolonies with ramifying water channels. Antifungal susceptibility testing demonstrated the increased resistance of sessile C. albicans cells against clinically used fluconazole and amphotericin B as compared to their planktonic counterparts.     

Induction of LAK-like cells in the peritoneal cavity of mice by inactivated Candida albicans

We have investigated the effect of multiple administrations of inactivated Candida albicans (CA) cells on induction of non-MHC-restricted antitumor cytotoxic responses both in normal and congenitally athymic (nude) mice. Intraperitoneal inoculation of CD2F1 mice with five doses of 2 x 10(7) CA cells over a 2-week interval was associated with the induction of peritoneal exudate cells (PEC) that mediated natural killer cell activity. These cells, in contrast to those elicited by a single dose of CA, killed both NK-sensitive and NK-resistant tumor target cells in vitro. This broad-spectrum, antitumor cytotoxicity peaked 1 day after the last injection of CA, and decreased to control values within 6 (NK-resistant) or 14 (NK-sensitive target cells) days. Cytotoxicity could be recalled to a high level by a boosting injection of CA or a major mannoprotein-soluble antigen (MP) from the Candida cell wall, given 30 days after multiple CA treatment. Upon a 24-hr in vitro incubation, CA-induced peritoneal immunoeffectors lost their killing activity unless human recombinant interleukin-2 (rIL-2) was added to cultures. The non-MHC-restricted cytotoxic PEC activity induced by CA was mainly associated with nonadherent, nonphagocytic large granular lymphocytes (LGL) which exhibited the following phenotypes: (i) asialo GM1+, Lyt 2.2-, and partially Thy 1.2+ (effectors active against NK-sensitive targets) and (ii) asialo GM1+, Lyt 2.2-, and Thy 1.2+ (effectors active against NK-resistant targets). Nude mice also responded to multiple CA inoculations by displaying high cytotoxic activity against NK-sensitive targets and significant cytotoxicity against NK-resistant targets. This cytotoxicity could be recalled on Day +30, and the cytotoxic effectors involved were highly sensitive to anti-asialo GM1 plus complement treatment. Overall, the results add further experimental evidence to the wide range of immunomodulatory properties possessed by C. albicans, and demonstrate that the majority of antitumor cytotoxic activity induced by fungal cells was due to lymphokine-activated killer (LAK)-like effectors.     

Delayed-type hypersensitivity (DTH) in BCG-sensitized mice I. Lack of suppressor T cell activity on DTH to sheep red blood cells.

Mice pretreated with an intravenous (i.v.) injection of BCG (BCG-sensitized mice) and then immunized intravenously with a high dose (10(8)--10(9)) of sheep red blood cells (SRBC) 2 weeks later developed strong delayed-type hypersensitivity (DTH) to SRBC, as in mice pretreated with cyclophosphamide (CY) (CY-treated mice) and then immunized with SRBC 2 days later; normal mice given the same dose of SRBC did not show such DTH. The mechanism of this strong DTH to SRBC which developed in BCG-sensitized mice was studied, by comparing it with that in CY-treated mice. The transfer of either whole spleen cells or thymus cells, but not serum, obtained from mice immunized with i.v. injections of 10(9) SRBC 4 days previously (hyperimmune mice) did not suppress either the induction or the expression of DTH to SRBC in BCG-sensitized mice, but suppressed those in CY-treated mice. The suppressor cells were SRBC-specific T cells. Adoptive transfer of DTH to SRBC by spleen cells from either BCG-sensitized mice of CY-treated mice to hyperimmune recipients failed. The adoptive transfer of DTH from BCG-sensitized mice to normal recipients also failed if the spleen cells from hyperimmune mice were cotransferred. Whole body irradiation (600 rad) of mice 2 hr before or after the time of immunization with SRBC reduced significantly DTH to SRBC in both BCG-sensitized and CY-treated mice. It was noticed that the total number of spleen cells in BCG-sensitized mice was 3--4 times larger than that in CY-treated mice. From these results, we conclude that the entity of effector T cells of DTH to SRBC induced in BCG-sensitized mice and in CY-treated mice was not different in terms of susceptibility to suppressor T cells and irradiation, but that the total numbers of effector T cells generated in these mice differed remarkably, resulting in the above-described different responsiveness to suppressor T cells transferred passively.