Candida-specific antibodies during experimental vaginal candidiasis in mice

Protective host defense mechanisms against vaginal Candida albicans infections are poorly understood. Although cell-mediated immunity (CMI) is the predominant host defense mechanism against most mucosal Candida infections, the role of CMI against vaginal candidiasis is uncertain, both in humans and in an experimental mouse model. The role of humoral immunity is equally unclear. While clinical observations suggest a minimal role for antibodies against vaginal candidiasis, an experimental rat model has provided evidence for a protective role for Candida-specific immunoglobulin A (IgA) antibodies. Additionally, Candida vaccination-induced IgM and IgG3 antibodies are protective in a mouse model of vaginitis. In the present study, the role of infection-induced humoral immunity in protection against experimental vaginal candidiasis was evaluated through the quantification of Candida-specific IgA, IgG, and IgM antibodies in serum and vaginal lavage fluids of mice with primary and secondary (partially protected) infection. In na?ve mice, total, but not Candida-specific, antibodies were detected in serum and lavage fluids, consistent with lack of yeast colonization in mice. In infected mice, Candida-specific IgA and IgG antibodies were induced in serum with anamnestic responses to secondary infection. In lavage fluid, while Candida-specific antibodies were detectable, concentrations were extremely low with no anamnestic responses in mice with secondary infection. The incorporation of alternative protocols-including infections in a different strain of mice, prolongation of primary infection prior to secondary challenge, use of different enzyme-linked immunosorbent assay capture antigens, and concentration of lavage fluid-did not enhance local Candida-specific antibody production or detection. Additionally, antibodies were not removed from lavage fluids by being bound to Candida during infection. Together, these data suggest that antibodies are not readily present in vaginal secretions of infected mice and thus have a limited natural protective role against infection.     

Candida-specific cell-mediated immunity is demonstrable in mice with experimental vaginal candidiasis.

Women with recurrent vulvovaginal candidiasis often demonstrate a down-regulation of cell-mediated immunity (CMI) to Candida albicans detected by a lack of cutaneous delayed-type hypersensitivity (DTH) to Candida antigens. However, the role of systemic CMI as a host defense mechanism against recurrent vulvovaginal candidiasis is not well understood, in part because of the lack of a well-defined murine model of vaginal candidiasis. The present study was undertaken to determine: (i) whether soluble Candida culture filtrate antigens (CaCF) could be used to induce and detect Candida-specific CMI in mice and (ii) whether these antigens would be useful in detecting systemic CMI in mice given an experimental Candida vaginal infection. To this end, mice were immunized subcutaneously with CaCF in complete Freund's adjuvant, and within 7 days they developed Candida-specific DTH reactivity detected by footpad swelling (increase in footpad thickness, 0.36 mm) 24 h after footpad challenge with CaCF. Adoptive transfer studies showed that the DTH responsiveness was elicited by CD4+ DTH T cells. In mice given a vaginal inoculum of C. albicans blastoconidia (5 x 10(5)), footpad challenge with CaCF resulted in positive DTH responses (0.24 mm) as early as 1 week, responses similar to immunization in 2 to 3 weeks (0.33 mm), and sustained low levels of DTH reactivity (0.15 mm) through 10 weeks of vaginal infection. Vaginal lavage cultures revealed that peak vaginal Candida burden occurred 1 week post-vaginal inoculation (10(5) CFU) and declined 16-fold by week 10. These results provide evidence that Candida-specific systemic CMI is generated and can be detected longitudinally in mice with Candida vaginitis by a multiantigen preparation of Candida organisms which both initiates and detects Candida-specific CMI.     

Effects of preinduced Candida-specific systemic cell-mediated immunity on experimental vaginal candidiasis.

It has been postulated that systemic cell-mediated immunity (CMI) is an important host defense factor against recurrent vaginal infections caused by Candida albicans. Using an estrogen-dependent murine model of vaginal candidiasis, we have previously shown that mice inoculated vaginally with C. albicans acquire a persistent vaginal infection and develop Candida-specific Th1-type systemic CMI. In the present study, experimental vaginitis was monitored in the presence of preinduced systemic Candida-specific CMI. Mice immunized systemically with C. albicans culture filtrate antigens (CaCF) in complete Freund's adjuvant (CFA) had Th1-type reactivity similar to that of vaginally infected mice. CaCF given to mice intravenously induced Candida-specific suppressor T (Ts) cells. Mice preimmunized with CaCF-CFA and given a vaginal inoculum of C. albicans had positive delayed-type hypersensitivity (DTH) reactivity from the time of vaginal inoculation through 4 weeks. Conversely, mice infected in the presence of Ts cells had significantly reduced DTH responses throughout the 4-week period in comparison with naive infected mice. However, the presence of Th1-type Candida-specific DTH cells or Ts cells, either induced in mice prior to vaginal inoculation or adoptively transferred at the time of inoculation, had no effect on the vaginal Candida burden through 4 weeks of infection. A similar lack of effects was obtained in animals with lower Candida population levels resulting from a reduction in or absence of exogenous estrogen. These results suggest that systemic Th1-type CMI demonstrable with CaCF is unrelated to protective events at the level of the vaginal mucosa.     

Local Th1/Th2 cytokine production during experimental vaginal candidiasis: potential importance of transforming growth factor-beta.

Host defense mechanisms against vaginal Candida albicans infections are poorly understood. Despite the protective role of T helper (Th)1-type cell-mediated immunity (CMI) against mucosal C. albicans infections, studies using an estrogen-dependent murine model of vaginal candidiasis have shown a lack of effect of systemic Th1-type CMI against a vaginal C. albicans infection, and a lack of changes in local T cells during infection. In the present study, the local Thl- (interleukin [IL]-2, interferon [IFN]-gamma and IL-12) and Th2- (IL-4, IL-10 and transforming growth factor [TGF]-beta1) type cytokines were evaluated in vaginal tissue during an experimental C. albicans infection. Results showed constitutive expression of TGF-beta1 in vaginal tissue of naive mice that was two-fold higher than the levels of the other cytokines examined. These high levels of TGF-beta1 were further increased as a result of pseudoestrus and/or infection, and were corroborated at the messenger RNA level. Furthermore, the levels of TGF-beta in naive or infected mice were significantly higher in the vagina compared to other areas of the genital tract. Finally, TGF-beta1 predominated as well in the draining, but not non-draining, lymph nodes during infection. These results suggest that TGF-beta1, a potent immunoregulatory cytokine, may be important in the lack of demonstrable CMI at the vaginal mucosa against C. albicans.     

Increased susceptibility of secretor factor gene Fut2-null mice to experimental vaginal candidiasis

Fut2-LacZ-null mice, which are a model of the human ABO and Lewis nonsecretor group, display increased susceptibility to experimental yeast vaginitis, indicating a role for alpha(1,2)fucosylated cervical glycans in mucosal defense. However, the lack of significant effect of competitive inhibition by exogenous neoglycoproteins in this study emphasizes the complexity of Candida-epithelial cell adhesion events.     

Effects of reproductive hormones on experimental vaginal candidiasis

Vulvovaginal candidiasis (VVC) is an opportunistic mucosal infection caused by Candida albicans that affects large numbers of otherwise healthy women of childbearing age. Acute episodes of VVC often occur during pregnancy and during the luteal phase of the menstrual cycle, when levels of progesterone and estrogen are elevated. Although estrogen-dependent experimental rodent models of C. albicans vaginal infection are used for many applications, the role of reproductive hormones and/or their limits in the acquisition of vaginal candidiasis remain unclear. This study examined the effects of estrogen and progesterone on several aspects of an experimental infection together with relative cell-mediated immune responses. Results showed that while decreasing estrogen concentrations eventually influenced infection-induced vaginal titers of C. albicans and rates of infection in inoculated animals, the experimental infection could not be achieved in mice treated with various concentrations of progesterone alone. Furthermore, progesterone had no effect on (i) the induction and persistence of the infection in the presence of estrogen, (ii) delayed-type hypersensitivity in primary-infected mice, or (iii) the partial protection from a secondary vaginal infection under pseudoestrus conditions. Other results with estrogen showed that a persistent infection could be established with a wide range of C. albicans inocula under supraphysiologic and near-physiologic (at estrus) concentrations of estrogen and that vaginal fungus titers or rates of infection were similar if pseudoestrus was initiated several days before or after inoculation. However, the pseudoestrus state had to be maintained for the infection to persist. Finally, estrogen was found to reduce the ability of vaginal epithelial cells to inhibit the growth of C. albicans. These results suggest that estrogen, but not progesterone, is an important factor in hormone-associated susceptibility to C. albicans vaginitis.     

Th1 and Th2 cytokine secretion patterns in murine candidiasis: association of Th1 responses with acquired resistance.

Two chemically mutagenized agerminative variants of Candida albicans were used to immunize mice against challenge with highly virulent cells of the parent strain. Although both mutants (Vir- 3 and Vir- 13) resulted in nonlethal infection and could be recovered from mouse organs for many days after the intravenous inoculation of 10(7) to 10(6) cells, significant protection to systemic challenge with virulent C. albicans was induced by only one (Vir- 3) of the two variants. Anticandidal resistance in Vir- 3-infected mice was associated with the occurrence in vivo of strong delayed-type hypersensitivity to Candida antigen, detection in vitro of highly fungicidal effector macrophages, and presence in the serum of a large proportion of Candida-reactive antibodies of the immunoglobulin G2a isotype. Bulk cultures of purified CD4+ lymphocytes from mice infected with either mutant were compared for their ability to produce gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-6 in vitro. After stimulation with specific antigen, CD4+ cells from Vir- 3-immunized mice released large amounts of the Th1-specific cytokines, IFN-gamma and IL-2, at a time when CD4+ cells from Vir- 13-infected mice predominantly secreted the characteristic Th2 cytokines, IL-4 and IL-6. These results were confirmed by quantitative analysis of cytokine-producing Th1 and Th2 cells. In addition, only mice infected with Vir- 3 displayed a high frequency of CD8+ cells with the potential for in vitro lysis of yeast-primed bone marrow macrophages. Purified CD4+ cells from Vir- 3-infected mice, but not a mixture of these cells with CD4+ lymphocytes from mice infected with Vir- 13, could adoptively transfer delayed-type hypersensitivity reactivity onto naive mice. Taken together, these data suggest that both Th1 and Th2 CD4+ lymphocytes may be activated during experimental C. albicans infection in mice.     

Humoral immunity in vaginal candidiasis.

Serum antibody titers to Candida albicans were estimated in 37 women with recurrent vaginal candidiasis and in 148 normal American and Finnish subjects, using the passive-hemagglutination technique. The antibody titers ranged from 0 to 16 in normal individuals and 4 to 256 in vaginal candidiasis patients. Antibodies to C. albicans in the sera of vaginal candidiasis patients were found to be the secretory immunoglobulin A type, as determined by gel filtration and double-diffusion tests. The results were confirmed by the indir-ct fluorescent-antibody technique. Our findings suggest that, in vaginal candidiasis, the antibody response is mainly local, consisting of secretory immunoglobulin A, some of which finds its way into systemic circulation.     

Th 1-type immunity is incompatible with successful pregnancy

Several years ago, the rather provocative question was raised: is successful pregnancy a T helper 2 (Th2) phenomenon? Implicit in this argument is the corollary that unsuccessful pregnancy is a Th1-type situation. Here, evidence from murine and human pregnancy is presented to show that, since Th1-type cytokines mediate pregnancy loss, a shift towards Th1-type immunity may help resolve ‘unexplained’ pregnancy failure.     

Role for dendritic cells in immunoregulation during experimental vaginal candidiasis

Vulvovaginal candidiasis (VVC) caused by the commensal organism Candida albicans remains a significant problem among women of childbearing age, with protection against and susceptibility to infection still poorly understood. While cell-mediated immunity by CD4+ Th1-type cells is protective against most forms of mucosal candidiasis, no protective role for adaptive immunity has been identified against VVC. This is postulated to be due to immunoregulation that prohibits a more profound Candida-specific CD4+ T-cell response against infection. The purpose of this study was to examine the role of dendritic cells (DCs) in the induction phase of the immune response as a means to understand the initiation of the immunoregulatory events. Immunostaining of DCs in sectioned murine lymph nodes draining the vagina revealed a profound cellular reorganization with DCs becoming concentrated in the T-cell zone throughout the course of experimental vaginal Candida infection consistent with cell-mediated immune responsiveness. However, analysis of draining lymph node DC subsets revealed a predominance of immunoregulation-associated CD11c+ B220+ plasmacytoid DCs (pDCs) under both uninfected and infected conditions. Staining of vaginal DCs showed the presence of both DEC-205+ and pDCs, with extension of dendrites into the vaginal lumen of infected mice in close contact with Candida. Flow cytometric analysis of draining lymph node DC costimulatory molecules and activation markers from infected mice indicated a lack of upregulation of major histocompatibility complex class II, CD80, CD86, and CD40 during infection, consistent with a tolerizing condition. Together, the results suggest that DCs are involved in the immunoregulatory events manifested during a vaginal Candida infection and potentially through the action of pDCs.     

Genetic susceptibility to vaginal candidiasis.

To enable future studies on host resistance factors and therapy, inbred and outbred mouse strains were tested for susceptibility to vaginal candidiasis. Groups of mice were given 0.5 mg estradiol 3 days before and 4 days after intravaginal challenge with a suspension of Candida albicans. On day 1 after challenge, a swab was used to quantitate infection in all groups and to assure equivalent infection levels. On day 6, this was repeated and the experiment was terminated. BALB/c, the reference strain in repeated experiments, was susceptible, showing persistent infection with levels of cfu at day 6 falling within a range between a twofold decrease and a fourfold increase in relation to day 1 levels. CD-1 outbred mice were markedly resistant, with day 6 cfu levels showing a 74- to 87-fold decrease with respect to day 1 levels, whereas other outbred strains (CF-1, SW, ICR) were susceptible. A BALB/c substrain (ByJ) was also susceptible. With exception of CBA/J, which showed modest resistance, all inbred strains were similarly susceptible, including DBA/2, AKR/J, C3H/HeN, A/J and C57BL/6. The differences between CD-1 and BALB/c mice were also seen with a second C. albicans isolate. Our results show susceptibility to vaginal candidiasis is independent of the major histocompatibility locus H2 haplotype and any effect ascribable to use of particular commercial mouse suppliers. Differences among mouse strains in susceptibility to C. albicans, as seen in previous studies involving nonvaginal challenge routes, are not reflected in this vaginal candidiasis model; in general, such resistance patterns appear specific to the route of challenge administration. The resistance seen in mouse strain CD-1 is of particular interest in that CD-1 is known to be resistant to endocrine disruption by estrogen. Our results suggest this estrogen insensitivity may have broad-ranging effects on processes other than gametogenesis, including vaginal susceptibility to candidiasis.     

Circulating Candida-specific anti-mannan antibodies precede invasive candidiasis in patients undergoing myelo-ablative chemotherapy

The kinetics of circulating Candida mannan and anti-mannan antibodies were studied in consecutive plasma samples, obtained upon hospital admission, of 21 patients with microbiologically proven invasive candidiasis and 30 control patients who underwent myelo-ablative chemotherapy. The detection of Candida anti-mannan antibodies preceded the diagnosis of invasive candidiasis in infected patients, and the antibodies were detected significantly more often in patients who had experienced multiple episodes of neutropenia than in the control group (OR 8.9, 95% CI  5.6–14.3; p <0.05). Mannan was predominantly detected in patients who developed invasive candidiasis during their first episode of neutropenia (OR 3.7, 95% CI  1.4–9.7; p <0.05). This observation suggests that patients with multiple episodes of neutropenia have been previously exposed to Candida and that the presence of anti-mannan antibodies in these patients might be associated with an increased risk of developing clinically manifest invasive candidiasis.     

Impact of interleukin-13 responsiveness on the synthetic and proliferative properties of Th1- and Th2-type pulmonary granuloma fibroblasts

Interleukin-13 (IL-13) has emerged as a major cytokine mediator of fibroblast activation and pulmonary fibrosis. Normal (from noninflamed lung), Th1-type (induced by the pulmonary embolization of purified peptide derivative-coated beads in mice sensitized to purified peptide derivative), and Th2-type (induced by the pulmonary embolization of Schistosoma mansoni egg antigen-coated beads in mice sensitized with S. mansoni eggs) primary fibroblast cell lines all exhibited constitutive gene expression of two receptor chains that bind and signal IL-13-mediated cellular events: IL-4Ralpha and IL-13Ralpha1. However, all three fibroblast cell lines exhibited divergent synthetic and proliferative responses to the exogenous addition of either recombinant IL-13 or a chimeric protein comprised of IL-13 and a truncated version of Pseudomonas exotoxin (IL13-PE), which targets and kills IL-13 receptor overexpressing cells. The exogenous addition of IL-13 to Th1-type and Th2-type fibroblast cultures significantly increased the cellular expression of IL-13Ralpha2, which may function as an IL-13 decoy receptor. After a 24-hour exposure to IL-13, the total collagen generation and cellular proliferation by Th2-type fibroblasts were significantly higher than that observed in similar numbers of normal and Th1-type fibroblasts. In addition IL13-PE, which binds with highest affinity to IL-13Ralpha2, exhibited down-regulatory effects on proliferation and matrix generation expression by Th1- and Th2-type, but not normal, fibroblasts. Thus, these data demonstrate that fibroblasts derived from murine pulmonary granulomas exhibit divergent expression of functional IL-13 receptor and this expression dictates the responsiveness and susceptibility to recombinant IL-13 and IL-13 immunotoxin, respectively.     

Hormonal factors in vaginal candidiasis in rats.

The hormonal status of rats affected vaginal infection with Candida albicans. Four hours after infection viable counts were higher and germ tubes were longer in those animals in estrous than in other animals. However, the infection was not maintained with the change in epithelial cell type which occurred as part of the estrous cycle. Estrogen dosing following ovariectomy predisposed toward infection, while progesterone dosing did not. In rats injected with progesterone, germ tube clumping was seen, leukocytes were present, and elimination occurred before hyphal growth was evident. In rats injected with estrogen, however, infection was maintained, with hyphal growth extending throughout the cornified epithelial layer. Vaginal washings from rats dosed with estrogen promoted elongation of germ tubes in vitro to a greater extent than washings from other rats. Preincubation of blastospores in progesterone and subsequent infection of rats in pseudoestrous promoted clumping of germ tubes in the vagina. Strains of C. albicans varied in their virulence, which correlated with their ability to produce germ tubes in vitro. Loss of virulence occurred on subculture of a clinical isolate.     

High Th1-type cytokine serum levels in patients with infectious mononucleosis

Most adults are asymptomatically infected with Epstein-Barr virus (EBV). Primary EBV infection is commonly associated with acute infectious mononucleosis (IM). T cell immune activation plays an important role in EBV-associated diseases. IM shows a mainly Th1-type profile, so Th1-type cytokines such as interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and lymphotoxin (LT) are moderately enhanced. We measured IL-2 and IFN-gamma in serum during acute phase of the disease and during convalescence. Sera were collected from 23 IM patients, 13 patients with similar clinical manifestations but without IM, and 10 healthy donors. The levels of IL-2, IFN-gamma and IL-12 were significantly higher in patients with acute IM than in healthy individuals. IL-2, IFN-gamma and IL-12 decreased during convalescence. These three cytokines may be useful as sensitive markers of IM during severe illness and its later phases.     

Downregulation of IFN-gamma production in patients with recurrent vaginal candidiasis.

BACKGROUND: Recurrent vaginal candidiasis (RVC) is an important health problem with unknown pathogenesis. Although impairment of the T-cell response is associated with persistent or recurrent candidiasis, data on immunologic responses in patients with RVC are controversial. OBJECTIVES: To evaluate the T-cell response in patients with RVC and the ability of cytokines and cytokine antagonists to modulate IFN-gamma production in cultures stimulated with Candida albicans antigens. METHODS: Participants in the study included 13 patients with RVC and 7 control women with sporadic candidiasis. Cytokines were determined by ELISA in supernatants of mononuclear cells with C albicans, purified protein derivative, or tetanus toxoid antigen. RESULTS: IFN-gamma production was absent or low in 11 of 13 women (84.6%) with RVC. Absent or low IFN-gamma production was specific to C albicans antigens (189 +/- 389 pg/mL), because high IFN-gamma levels were found in cultures stimulated with purified protein derivative (739 +/- 774 pg/mL) or tetanus toxoid antigens (1085 +/- 546 pg/mL). Monoclonal antibody anti-IL-10 enhanced IFN-gamma levels (750 +/- 753 pg/mL), and IL-10 suppressed this cytokine production in patients with sporadic candidiasis. CONCLUSIONS: Mononuclear cells from patients with RVC stimulated with C albicans antigen have low or absent IFN-gamma production. IL-10 plays an important role in downregulation of the T-cell response in these patients.