Judith E. Domer

Candida Cell Wall Mannan: A Polysaccharide with Diverse Immunologic Properties

It is clear that mannan has the potential to influence multiple biologic functions in vivo and in vitro, including both mannan-specific and mannan-nonspecific activities. Based on in vitro studies, various mechanisms have been proposed for the regulatory activities observed, ranging from interference with normal PMNL and monocyte function to the induction of T suppressor cells. It may well be, in fact, that different mechanisms function at different levels depending upon the specific phenomenon being influenced. Approaches to determining the mechanisms involved in these regulatory phenomena, however, have been complicated by the fact that many studies were performed with mannan extracted in the laboratory by traditional methods and used as such without further purification. Most laboratory-acquired mannans appear to be heterogeneous mixtures containing polymers of differing size and charge. When such mixtures have been separated on the basis of size or charge, it has been shown that biologic function can be correlated with individual fractions, and that a single bulk preparation of mannan can contain components with opposing biologic activities. Resolution of the specific mechanisms involved in the regulatory phenomena described, therefore, will not be complete until homogeneous preparations of mannan are employed to investigate the mechanisms.

Separation of immunomodulatory effects of mannan from Candida albicans into stimulatory and suppressive components

Mannan extracted from Candida albicans was studied for its immunomodulatory activity on in vivo antibody responses to type III pneumococcal polysaccharide (SSS-III), a helper-T-cell-independent antigen, and to sheep erythrocytes (SRBC), a helper-T-cell-dependent antigen. In some studies, the antibody response to SSS-III was converted to a helper-T-cell-dependent response by attaching it to a carrier (horse erythrocytes, HRBC); this complex then was used to immunize mice primed with a subimmunogenic dose of HRBC. Mannan enhanced the antibody response to both SSS-III and SRBC when administered at the same time or 1 or 2 days after immunogen. However, when both mannan and SSS-III were coated onto HRBC for immunization, either enhancement or suppression was noted; the effect depended upon the amount of mannan used. Larger amounts stimulated, whereas smaller amounts suppressed, the antibody response to SSS-III. The enhancing and suppressive components of mannan could be separated by molecular size or charge by chromatography on Sepharose 4B or on DEAE-Sephadex A-50 columns, indicating that mannan extracts contain individual components having opposing immunomodulatory properties. These components can be separated on the basis of molecular size and charge.

Innate and acquired immune responses against Candida albicans in congenic B10.D2 mice with deficiency of the C5 complement component

Congenic mice, sufficient or deficient with respect to the C5 component of complement, were evaluated for their innate and acquired immune responses to Candida albicans. When unimmunized mice were challenged intravenously and sacrificed at intervals for cultural analyses of kidneys, it was clear that C5-sufficient mice were able to deal more effectively with C. albicans during the first week after challenge than C5-deficient mice. When immunized mice were challenged intravenously to assess the development of protective responses, an intact complement cascade appeared to contribute to the more rapid clearance of fungi during the first few weeks following challenge, but by the fourth week after challenge, the numbers of fungi had decreased significantly in both types of mice and were at levels which were not significantly different. No significant differences were detected in the development of delayed hypersensitivity or Candida-specific antibody between C5-sufficient and C5-deficient mice either. C5-deficient mice did have slightly elevated levels over the C5+ mice, but this may simply reflect the prolonged antigenic load during the first 3 weeks following intravenous challenge in both immune and nonimmune animals. The later-acting complement components, while appearing to contribute to the early inhibition of the growth of C. albicans in the nonimmune animal, had no adverse effect on the development of specific immune responses, in that delayed hypersensitive responses were equivalent between the two groups, antibody response was not significantly altered and the ultimate outcome of challenge in immunized animals was not affected.

The readily extracted lipids of Histoplasma capsulatum and Blastomyces dermatitidis

1. 1. Cell walls and cell sap (lyophilized preparation of whole cell minus cell wall) of both yeast and mycelial phases of Histoplasma capsulatum and Blastomyces dermatitidis were extracted with chloroform-methanol and the lipids thus derived were compared. 2. 2. There was little difference between the total readily extracted lipid of the cell sap preparations of yeast and mycelial phases of H. capsulatum. Cell saps of B. dermatitidis generally contained less lipid than H. capsulatum; the mycelial phase contained about half that of the corresponding yeast phase preparations. Most cell walls had less than 3% lipid, with less m mycelial phase than corresponding yeast phase walls. 3. 3. Cell wall and cell sap extracts regardless of source contained phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, triglycerides, diglycerides, sterols, sterol esters and free fatty acids. Triglycerides were the greatest single component in the extracts; phosphatidyl choline and phosphatidyl ethanolamine were the most concentrated phospholipids. Quantitative densitometry of these three classes of compounds revealed little difference between cell wall and cell sap preparations, or yeast and mycelial phase preparations, that could not be explained on the basis of incubation time or different rates of metabolism in different media. 4. 4. The major differences between cell walls and homologous cell sap, and yeast phase and mycelial phase, involved primarily the quantities of oleic and linoleic acids in the extracts. These two fatty acids together, however, accounted for 75–80% of the total fatty acids of all extracts; the relative proportions of the two distinguished the preparations. In general, oleic acid predominated in yeast phase extracts; linoleic acid was either equal or greater in concentration in the mycelial phase extracts. Cell walls usually contained more oleic acid and less linoleic acid than homologous cell saps. This was particularly obvious when the fatty acids of the lipid classes were examined. Even though differences were demonstrated between cell wall and cell sap preparations, and yeast and mycelial phase preparations, little difference could be demonstrated between analogous lipid extracts of H. capsulatum and B. dennatitidis.

Immunodeficient CBA/N mice respond effectively to Candida albicans.

An immune defect in CBA/N mice diminishes their ability to respond adequately to certain well-defined antigens. Since the contribution of T and B cells to immunity in candidiasis has not been clearly defined, it was hoped that CBA/N mice might prove a useful model for the study of specific responses to Candida albicans. Therefore, immunodeficient CBA/N and immunocompetent CBA/J mice were immunized by two cutaneous inoculations of viable C. albicans B311 given 2 weeks apart and challenged iv 14 days after the second inoculation. Delayed-type hypersensitivity (DTH) was tested with a membrane-derived antigen (B-HEX) 7 days following the second inoculation, and lymphocyte stimulation with B-HEX, a cytoplasmic antigen (SCS), and mitogens was done at 12 days. Antibody to SCS was determined by ELISA 2 days after DTH testing and 28 days after iv challenge, at which time the animals were sacrificed for quantitative culture of kidneys and brains. Naive CBA/N mice were no more susceptible to challenge than CBA/J mice in that the mean log colony-forming units (CFU) were 3.79 and 5.48, respectively. Both strains responded to immunization by a similar reduction in CFU, a marked DTH response (e.g., reactions at 24 hr were 1.12 mm for CBA/N and 1.34 mm for CBA/J), and significant and similar quantities of antibody. The immune defect in CBA/N mice had no demonstrable effect on the development of immune responses to infection with C. albicans.

Glycohydrolase contamination of commercial enzymes frequently used in the preparation of fungal cell walls

Commercial enzyme preparations frequently used in the preparation of fungal cell walls, viz., proteases, a lipase, and a phosphatase, were examined for the presence of contaminating glycohydrolase activity, since such activity could result not only in the removal of cytoplasmic constituents but also in the removal of portions of the wall itself. Glucosidase activities were detected in a protease of fungal origin, in a lipase from wheat germ, and in a phosphatase from potatoes. Additionally, two commercial protease preparations from Streptomyces griseus contained β-1,3-glucanase activity in significant amounts, a third contained trace amounts of the glucanase, but a fourth was totally free of glycohydrolase activity. The protease preparations from S. griseus released laminaribiose from yeast-phase cell walls of Histoplasma capsulatum chemotypes I and II, but only trace amounts of glucose were released. One protease was examined more closely and was found to be optimally active on laminarin at pH 5.5 and 50°C. It was also highly active on the same substrate at pH 8.0 and 37°C, however. A protease preparation from Aspergillus oryzae released glucose from the yeast-phase cell walls of H. capsulatum chemotypes I and II as well as from cell walls of Blastomyces dermatitidis, suggesting that the preparation contained both α- and β-glucanases.

Candida albicans mannan-specific, delayed hypersensitivity down-regulatory CD8+ cells are genetically restricted effectors and their production requires CD4 and I-A expression.

There is considerable controversy over the induction and activity of down-regulatory cells active in various antigen-specific and antigen-nonspecific systems. We have been studying the nature of such cells in a Candida albicans mannan (MAN)-specific system for some time and report here the requirements for CD4+ and I-A+ cells during the inductive phase for the development of CD8+ effector cells, as well as the requirement for genetic compatibility for effector activity of CD8+ cells. Since we have shown previously that CD8+ down-regulatory cells were present in spleens of MAN-treated mice 4 days following the administration of MAN to naive mice, as determined by their ability to suppress delayed hypersensitivity (DH) when transferred to immunized recipients, we treated mice with monoclonal antibodies specific for CD4 and I-A at various times before, with or after the administration of MAN to assess the role of CD4+ and I-A+ cells in the development of the CD8+ effector cell. Both anti-CD4 and anti-I-A given before or up to 30 h after the administration of MAN abrogated the ability of splenocytes from MAN-treated mice to down-regulate MAN-specific DH in immunized recipients. Moreover, transfers of down-regulatory cells between H-2-incompatible strains of mice, specifically CBA/J and BALB/cByJ, provided evidence that the effector cell for the down-regulatory activity was also restricted genetically in its activity. Taken together, the data presented indicate that genetically compatible cells are required for both the inductive and effector stages of down-regulation of MAN-specific DH, suggesting that cell-cell cooperation is required for both stages and that CD4+ cells are required in a pathway leading to the development of the CD8+ effector cell.

Immunity to Fungal Infections

Medically-important fungi potentially capable of initiating life-threatening disease can be categorized roughly into two groups, viz., primary pathogens and opportunists. The primary pathogens are those fungi which regularly cause disease in individuals with no known underlying clinical conditions, while the opportunists seldom create problems for the healthy person, but instead attack the patient whose normal defenses are compromised by iatrogenic factors or disease.

Introduction to Candida

Candidiasis is the prototypic opportunistic fungal disease. Candida spp. reside most commonly in the gastrointestinal tract1,2 and vagina, where they are normally held in check by as-yet incompletely defined local factors that include competition with the resident bacterial flora and innate effectors of nonspecific mucosal resistance.3,4 Infrequently, but under a wide variety of clinical circumstances, these normally inconspicuous commensals can multiply on or invade through the mucosal surface to initiate local or systemic disease. Various immunologic and nonimmunologic conditions can predispose to such opportunistic infections.5 Among the former are certain malignancies6–8 and the use of cytotoxic or immunosuppressive therapy.9,10 Nonimmunologic predisposing factors include diabetes,11 trauma,12 pregnancy,13 antibiotic therapy,7 and hyperalimentation.14 Not infrequently, such predisposing factors occur in combination.

Lipid, monosaccharide and chitin content of cell walls of Sepedonium

Cell walls and cell sap of 2 strains of Sepedonium were lipid-extracted and the readily-extracted lipids examined. The lipid-extracted cell walls were treated with ethylenediamine, and unfractionated, as well as fractionated, cell walls were analyzed for their sugar and chitin content. The lipids of all preparations consisted of phospholipids, triglycerides, diglycerides, sterols, sterol esters and free fatty acids. Linoleic acid was, with 1 exception, the major fatty acid in all extracts; palmitic, stearic and oleic acids were also present. Mannose and glucose were the only sugars found in the cell walls; much of the glucose was resistant to mild acid hydrolysis. These sugars together accounted for approximately 20% of the unfractionated cell walls of both strains. Glucose and acetylglucosamine were released from the cell walls of both strains by a crude chitinase preparation, and the acetylglucosamine represented 19% and 28% of the cell walls of the 2 strains.