Albicans Antigen Research Articles

Production of granulocyte-macrophage colony-stimulating factor by large granular lymphocytes stimulated with Candida albicans: role in activation of human neutrophil function

In the present study, culture supernatants from larger granular lymphocytes (LGL) that were activated with Candida albicans antigens were shown to stimulate the ability of neutrophils to inhibit fungal growth. Identification of the activation factors showed that granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, was involved. Human peripheral blood mononuclear cells were fractionated by Percoll density centrifugation and each subpopulation of cells was stimulated with C albicans yeast cells. GM-CSF was produced in those fractions enriched for LGL, but not T lymphocytes or adherent monocytes. Additionally, the phenotype of the GM-CSF-producing cell was found to be CD2+, CD16+, HLA-DR+, and negative for CD4, CD8, and CD15. Kinetic studies demonstrated that GM- CSF appeared in the supernatants within 2 days of culture and continued to be produced up to 7 days. Optimal stimulation of LGL was seen at a ratio of 3 heat-killed C albicans yeast cells per LGL. Thus, LGL play an important role in host defense against this opportunistic pathogen by producing cytokines, including GM-CSF, which in turn activates the fungicidal activity of neutrophils.

Production of Granulocyte-Macrophage Colony-Stimulating Factor by Large Granular Lymphocytes Stimulated With Candida Albicans: Role in Activation of Human Neutrophil Function

In the present study, culture supernatants from larger granular lymphocytes (LGL) that were activated with Candida albicans antigens were shown to stimulate the ability of neutrophils to inhibit fungal growth. Identification of the activation factors showed that granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, was involved. Human peripheral blood mononuclear cells were fractionated by Percoll density centrifugation and each subpopulation of cells was stimulated with C albicans yeast cells. GM-CSF was produced in those fractions enriched for LGL, but not T lymphocytes or adherent monocytes. Additionally, the phenotype of the GM-CSF–producing cell was found to be CD2+, CD16+, HLA-DR+, and negative for CD4, CD8, and CD15. Kinetic studies demonstrated that GM-CSF appeared in the supernatants within 2 days of culture and continued to be produced up to 7 days. Optimal stimulation of LGL was seen at a ratio of 3 heat-killed C albicans yeast cells per LGL. Thus, LGL play an important role in host defense against this opportunistic pathogen by producing cytokines, including GM-CSF, which in turn activates the fungicidal activity of neutrophils.

Serum antibody response of gnotobiotic athymic and euthymic mice following alimentary tract colonization and infection with Candida albicans

Colonization and infection evoked specific immunoglobulin responses to Candida albicans antigens in gnotobiotic nu/+ mice which appeared to correlate with clearance of infected mucosal surfaces (tongue and stomach). Conversely, colonized and infected nu/nu mice formed some IgM but no detectable IgG or IgA antibodies against C. albicans antigens. Although chronic mucosal infections of tongue and stomach persisted in nu/nu mice, they were able to resist overwhelming mucosal and systemic infections with C. albicans. Thus, C. albicans specific antibodies may play a role in clearance of mucosal candidiasis (tongue and stomach), but these antibodies do not appear to be necessary for protecting athymic mice against systemic candidiasis of endogenous origin.

Altered monocyte function in uremia

Uremia appears to suppress immune function predisposing patients to infections. When the defect in cellular immunity was studied by exposing mononuclear cells (MNC) from uremic patients and controls to tetanus toxoid, diptheria toxoid, or Candida albicans antigen in vitro, the uremic cells were far less responsive. Monocytes and T cells, which are both involved in the proliferative response to soluble antigens, were isolated from MNC of uremic patients and HLA class II matched controls and incubated with tetanus toxoid. Tetanus toxoid-pulsed uremic monocytes were unable to stimulate the proliferation of HLA identical control T lymphocytes. Lymphocytes from uremic patients, however, were stimulated by tetanus toxoid-pulsed control monocytes. Therefore, the ability of monocytes to function as accessory cells is severely affected by uremia. The uremic monocytes were FcR +, produced IL-1β, and expressed levels of HLA class II antigens comparable to controls. Although the biochemical defect in uremic monocytes remains unknown, the abnormality could explain many of the immunological changes of uremia.

Detection of Candida antigenuria in disseminated candidiasis by immunoblotting

Immunoblotting (Western blotting) was used to detect Candida albicans antigens in urine of 10 patients with disseminated candidiasis who had two or more positive blood cultures. Twelve urine samples were examined; and antigenuria was found in five of six patients with C. albicans infections, in one patient with a mixed Candida infection (including C. albicans), and in one of two patients with C. tropicalis infection. All except one specimen was collected from 2 to 12 days after initiation of amphotericin B therapy. Positive samples showed different numbers of bands in Western blots with an antigen that had an apparent molecular weight of 47,000 in common. This antigen was not found in the urine of patients who had more than 5 days of therapy for candidiasis and who were responding to therapy. The results suggest that Western blotting for C. albicans antigens in urine may be a useful method for the diagnosis of disseminated candidiasis and for evaluating antifungal treatment.

Detection of an antibody response in immunocompetent patients with systemic candidiasis orCandida albicans colonisation

Tests to detect circulating antibodies to Candida albicans antigens were performed in sera from 27 immunocompetent patients, 15 of whom had deep-seated candidiasis and 12 of whom were colonised by Candida albicans. For the diagnosis of deep-seated candidiasis in patients with either deep-seated candidiasis or Candida albicans colonisation, counterimmunoelectrophoresis had a sensitivity of 87% and a specificity of 75%. Using immunoblotting it could be shown that antibodies to 35K, 47K, 68K and 88K antigens of Candida albicans occurred more frequently in patients with deep-seated candidiasis than in colonised patients. The presence of dense bands in immunoblots representing antibodies against the 47K and/or 68K antigen served to discriminate significantly between deep-seated and superficial candidiasis (p less than 0.05).

IgE‐, IgA‐ and IgG‐antibody responses to carbohydrate and protein antigens of Candida albicans in asthmatic children

Analysis of IgE, IgA and IgG antibodies directed against Candida albicans antigens in 28 asthmatic children was performed with immunoblotting after SDS-PAGE. Analysis with the purified cytoplasmic protein fraction revealed a major protein allergen with an MW of 46 kD. In addition to the major allergen, 15 other antigenic bands with molecular weights between 16 and 135 kD bound IgE. Ten of 13 anti-C. albicans IgE-positive children had IgE towards the 46 kD major allergen. None of the subjects in the study group or in the non-atopic controls had IgA or IgG antibodies towards this protein. Analysis of the crude surface extract showed that mannan, a carbohydrate, was an intermediate allergen contrary to being the major antigen in IgA and IgG antibody responses.

Transfer of cell-mediated immunity toParacoccidioides brasiliensisin hamsters with dialysable leukocyte extracts

Dialysable leukocyte extracts (DLE) were obtained from lymph nodes and spleen cells of hamsters immunized with Paracoccidioides brasiliensis (immune DLE) or from non-immunized hamsters (non-immune DLE). Sensitivity to P. brasiliensis antigen (PbAg) was transferred by immune DLE to normal recipient hamsters and could be detected by the macrophage migration inhibition (MIF) assay on day three and by a positive skin test to PbAg on day seven after DLE inoculation. This reactivity persisted for 120 days. The specificity of DLE was studied by inoculation of immune or non-immune DLE into normal hamsters and transfer was evaluated 7 days later by the MIF assay and by skin tests to PbAg, Candida albicans antigen (CaAg), sonically treated bacille Calmette-Guérin (BCG) (SBCG) and ovalbumin (OVA). The immune DLE recipients showed a reactivity to PbAg that was significantly stronger than that of the nonimmune DLE recipients although both groups of recipient animals showed reactivity to CaAg and SBCG. The results suggest that both DLE preparations contain a non-specific antigen augmenting factor. This factor may stimulate the minimal background response to the antigens in nonimmunized recipient hamsters.

Production and characterization of a murine monoclonal antibody to Aspergillus fumigatus antigen having IgG- and IgE-binding activity.

By employing hybridoma technology, a monoclonal antibody against Aspergillus fumigatus was produced. This antibody, isotyped as IgM k, reacted with 12 of 16 antigens extracted from 9 different A. fumigatus strains. This antibody also reacted with all 3 Aspergillus flavus antigens studied, but not with Aspergillus niger, Aspergillus terreus, Penicillium notatum or Candida albicans antigens. Western blot analysis indicated that this antibody reacted with two concanavalin A (Con-A) binding bands of the A. fumigatus antigen extract. The specific binding antigens were isolated using monoclonal antibody affinity chromatography. When used in immunoassay this fraction demonstrated strong IgG and IgE antibody-binding activities against patient sera. The antibody levels against the purified fraction were significantly higher in patients' sera than in normal controls. The purified fraction demonstrated comparable reactivity with the crude A. fumigatus extract against patient sera, but the former has the added advantage of being pure and standardizable for dependable and reproducible results.

Differential antigen presentation by heat-treated peripheral blood mononuclear cells and Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL): Heated EBV-LCL present alloantigen and soluble antigen but are deficient in the stimulation of autologous EBV-LCL primed T cells

Heat-treated PBM (1 hr at 45°C) cannot present soluble Candida albicans antigens (CAN) or stimulate in the mixed lymphocyte culture (MLC) reaction. This is despite their continued expression of serologically defined class II MHC antigens. In contrast, heat-treated EBV-LCL present soluble CAN and stimulate allogeneic T cells in the MLC. Heated EBV-LCL stimulate strong secondary responses from allogeneic alloprimed T-cell lines in the primed lymphocyte test (PLT), while heated PBM stimulate only weak secondary allogeneic responses. To test whether this difference was due to a subtle difference in the thermal stability of the functional expression of class II MHC antigens on PBM and EBV-LCL cells, the EBV-LCL cells were heated for 1 hr at temperatures from 45°C to 60°C. Even after treatment at 60°C, the heated EBV-LCL strongly stimulated alloreactive T cells in MLC and PLT reactions. Heated EBV-LCL are not nonspecifically mitogenic, as they do not stimulate autologous T-cell lines primed to alloantigens. However, the weak response of alloprimed T-cell lines to heated allogeneic PBM can be augmented by coculturing with autologous heated EBV-LCL, suggesting heated EBV-LCL maintain a metabolic activity necessary for allogeneic stimulation that is deficient in heated PBM. While heated EBV-LCL stimulate allogeneic alloprimed T-cell lines, they no longer stimulate autologous EBV-LCL primed T-cell lines; irradiated EBV-LCL stimulate both strongly. This suggests the involvement of a heat labile antigenic or metabolic factor in the T-cell recognition of autologous but not allogeneic EBV-LCL.

Cell surface and intracellular expression of two Candida albicans antigens during in vitro and in vivo growth

Intracellular and cell surface expression of two antigenic determinants of Candida albicans was studied by reacting ultrathin sections of the fungus with monoclonal antibodies and locating the sites of antibody-antigen reactions by use of colloidal gold-coupled secondary antibody and electron microscopic techniques. Comparisons were made between fungal cells allowed to develop in vitro and cells ( in vivo) recovered from infected mice. Differences between in vitro antigen expression and expression in vivo were particularly noted during early germination events. Antigen expression in vivo appears to be concentrated in deeper cell wall layers and beneath the plasmalemma especially on germ tubes. Cells which develop in vivo and in vitro have antigen associated with an outer flocculent layer and on the innermost cell wall layer of mother cells and hyphae. A middle electron transparent cell wall layer is usually devoid of antigen. Comparison of antigen expression during in vivo growth in early (intraperitoneal) infection versus late disseminated (kidney) disease suggests that both of the antigens are expressed in greater quantity on the surface of germ tubes than on mother cells in kidney tissue as compared to intraperitoneally-grown cells. Results also suggest that at least one of the antigens, which is a substituted mannan, can be found on a lamello-tubular intracellular structure.

Possible functional impairment of Langerhans' cells in vitiliginous skin. Reduced ability to elicit dinitrochlorobenzene contact sensitivity reaction and decreased stimulatory effect in the allogeneic mixed skin cell lymphocyte culture reaction

We used patch testing to compare the ability to elicit contact sensitivity to dinitrochlorobenzene (DNCB) of uninvolved with vitiliginous skin of 31 patients with vitiligo. The induction of DNCB contact sensitivity was possible in the vitiliginous skin in the same way as in normal skin. The DNCB contact sensitivity reactions, however, were generally diminished in vitiliginous skin, although the number of cases showing similar DNCB contact reactivity between normal and vitiliginous skin increased when the sensitization procedure was performed in vitiliginous skin instead of normal skin. On the other hand, delayed skin reactions to intradermally injected Candida albicans antigen were not suppressed in vitiliginous skin. The number of Langerhans' cells was not decreased in vitiliginous skin as compared with that of normal skin. The epidermal cells derived from vitiliginous skin, however, tended to show a lower stimulatory effect in the allogeneic mixed skin cell lymphocyte culture reaction than those from normal skin. These results suggest a possibility of functional impairment of Langerhans' cells in vitiliginous skin.

Prospective study on humoral immune response induced by fungal infection in patients with hematologic malignancies.

To evaluate the immune response in an immunosuppressed population, antibodies against commercially available Candida albicans antigens were prospectively studied during 37 episodes of acute stomatitis caused by C. albicans and 36 episodes complicated by deep-seated mycoses in 62 adult patients with hematologic malignancies. During uncomplicated stomatitis in patients with acute leukemia, the mean peak IgM, IgG and IgA class enzyme-linked immunosorbent assay (ELISA) units differed significantly from the basic level preceding fungal infection. Mean time until peak values was 2.7-3.8 weeks after diagnosis of stomatitis. During systemic mycoses the antibody response was similar. Among patients with other hematologic malignancies, predominantly lymphomas, several were terminally ill and responded infrequently by antibody production. Similar results were given by Ouchterlony immunodiffusion and counterimmunoelectrophoresis. Thus, patients with acute leukemia showed an antibody response to fungal infection; the peak values, however, were somewhat delayed.

Immunoblot analyses of Candida albicans-associated antigens and antibodies in human sera

We tested 10 patient sera for the presence of immunoglobulin G (IgG) antibodies to Candida albicans and for C. albicans antigens by immunoblot analysis (i.e., electrotransfer blot radioimmunoassay) (G. E. Smith and M. D. Summers, J. Virol. 39:125-137, 1981). We evaluated sera from two patients at risk for candidiasis, five patients with systemic candidiasis documented by culture, and two patients who had experienced transient candidemia. Both the specificity and the relative amount of IgG antibodies to C. albicans in each serum sample were readily visualized by this technique, as was the absence of antibody from serum of neonatal and immunocompromised patients. No antibody species appeared to be uniquely associated with candidiasis patients (i.e., each antibody species present in the candidiasis patient was also present in sera of normal individuals or "at-risk" patients). IgG from rabbits immunized with whole cells or with a cytoplasmic fraction of C. albicans was used to detect C. albicans antigens in patient sera. Although several antigens were detected in the sera from patients with candidiasis, the same antigens were also detected in sera from patients at risk and in normal human serum. No antigens were detected in human serum when preimmune rabbit sera were used. These results suggest that the antigens detected by the rabbit antisera were human serum proteins that cross-reacted with C. albicans antigens. These findings may have important implications in studies of both the pathobiology of C. albicans and the serodiagnosis of candidiasis.

Failurel of Immunotherapy for Metastatic Renal Cell Carcinoma

Prompted by reports of tumor regression among patients with metastatic renal cell carcinoma treated by injection of an aggregated autologous or allogeneic tumor antigen with a nonspecific adjuvant, we studied the efficacy of this therapy in 23 patients. Following nephrectomy or excision of a metastatic lesion 16 patients were treated with an aggregated autologous tumor antigen combined with Candida albicans antigen. There were only 2 minimal responses. Seven patients judged unsuitable for an operation were treated with an aggregated allogeneic antigen derived from 12 autologous antigen preparations and combined with Candida albicans antigen. There were no objective responses. We conclude that these treatment regimens have minimal activity against metastatic renal cell carcinoma.

Functional heterogeneity of human antigen-presenting cells: presentation of soluble antigen but not self-Ia by monocytes.

These studies were undertaken to examine the phenotype of the antigen-presenting cells (APC) circulating in human peripheral blood. Cells adherent to glass were found to be efficient APC, restoring antigen-induced 3H-thymidine incorporation to T4-positive T cells that had been rigorously depleted of contaminating APC. In order to identify the APC within the glass-adherent cells (AC), these cells were stained with a number of monocyte-specific monoclonal antibodies (Mo-Mab) including 3C10, 63D3, and 61D3, and the Mo-Mab-positive and -negative cells were separated with the fluorescence-activated cell sorter. This method of preparation yielded Mo-Mab(+) AC populations that were more than 98% positive for the relevant Mab when reanalyzed with the fluorescence-activated cell sorter. Less than 1% of the Mo-Mab(-) AC populations were positive when reanalyzed with the Mab used for the separation. However, each Mo-Mab(-) AC population was contaminated with variable numbers (4-60%) of Mo as detected by morphologic criteria, histochemical analysis for esterase activity, or staining with a different Mo-Mab. Both Mo-Mab(+) and (-) AC populations were found to be similarly effective APC, with as few as 500 cells/well supporting responses to streptokinase-streptodornase, tetanus toxoid, and Candida albicans antigen. In the absence of antigen, only 3C10(-), 63D3(-), or 61D3(-) AC consistently stimulated 3H-thymidine incorporation of autologous T4 cells; large numbers (greater than 5 X 10(3)/well) of APC were necessary to induce this response. These results support the conclusion that cells identified by Mo-specific Mab are capable of functioning as APC, inducing 3H-thymidine incorporation in response to exogenous antigens. However, Mo-Mab(+) AC are not unique in this activity since Mo-Mab(-) AC also appeared to be able to present antigen. These Mo-Mab(-) AC appear to contain the majority of cells inducing autologous T4-cell reactivity.

Pathological observations in experimental candida infection of sensitized guinea pigs.

Guinea pigs immunized intramuscularly with heat-killed or viable Candida albicans were infected intracutaneously with C. albicans. Animals with negative delayed hypersensitivity against C. albicans antigen showed similar lesions with non-immunized controls. Delayed hypersensitivity-positive guinea pigs, which were detected in the animals immunized with heat-killed C. albicans in CFA and IFA, demonstrated a delay of the resolution of the inflammatory tissue reaction and, in the animals immunized with C. albicans in CFA, developed a granuloma. These results suggest that both humoral and cell-mediated immunities do not play a significant role for protection against candidiasis and at a late stage of infection, cell-mediated immunity may play a secondary role of the enhancement of resistance to candida infection associated with granuloma formation.

Antigen-dependent heterogeneity of human migration inhibitory factor

Human migration inhibitory factor (MIF) produced by peripheral blood mononuclear cells stimulated with purified protein derivative, tetanus toxoid, streptokinase-streptodornase, or Candida albicans antigen was analyzed by gel filtration and isoelectrofocusing. In all cases, supernatants harvested after a 24-hr exposure of the mononuclear cells to the antigen yielded only one MIF species with an isoelectric point of 5. In contrast, isoelectrofocusing of supernatants obtained from cells exposed to the antigen for an additional 24 hr demonstrated that different antigens induce the elaboration of different MIF species. Streptokinase-streptodornase and tetanus toxoid induced the production of one MIF species with an isoelectric point of 5 (pH 5-MIF). Stimulation of cells with Candida antigen elaborated a MIF species with an isoelectric point of 3 (pH 3-MIF). In contrast, stimulation of cells with purified protein derivative induced the production of both pH 3-MIF and pH 5-MIF.

Nature of the cells regulating migration inhibition factor production in C57BL/6 mice with low response toCandida albicans antigen

Nature of the cells regulating migration inhibition factor production in C57BL/6 mice with low response toCandida albicans antigen

Antigenic Relationships Among Some Candida Species Studied by Crossed-Line Immunoelectrophoresis: Taxonomic Significance

In crossed immunoelectrophoresis with the corresponding pooled rabbit antisera, Candida tropicalis and Candida albicans antigen preparations produced 65 and 63 immunoprecipitates, respectively. The cross-reactions between these antigens and antigens from 15 other yeast strains representing nine species were studied by crossed-line immunoelectrophoresis, using C. tropicalis and C. albicans antigen-antibody systems as references. We found no qualitative differences between strains of the same species or among the strains of C. albicans, Candida stellatoidea, and Candida claussenii. In both the C. albicans and C. tropicalis reference systems the degrees of cross-reaction between C. albicans or C. tropicalis and the nine other species revealed the following three groups: C. albicans, C. tropicalis, C. stellatoidea, and C. claussenii, which shared 85 to 100% of their antigens with C. albicans and C. tropicalis; Candida parapsilosis, Candida guilliermondii, and Candida sake, which shared 50 to 70% of their antigens with C. albicans and C. tropicalis; and Candida utilis, Candida pseudotropicalis, Candida krusei, and Candida zeylanoides, which shared about 30% of their antigens with C. albicans and C. tropicalis. Some antigens seemed to be species specific, whereas others seemed to be specific for a group of species within the genus Candida and should be useful for rapid identification of medically important yeasts.