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1.  Candida-specific cell-mediated immunity is demonstrable in mice with experimental vaginal candidiasis. 
Infection and Immunity  1993;61(5):1990-1995.
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.
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PMCID: PMC280793  PMID: 8097493
2.  Candida-specific Th1-type responsiveness in mice with experimental vaginal candidiasis. 
Infection and Immunity  1993;61(10):4202-4207.
The role of systemic cell-mediated immunity (CMI) as a host defense mechanism in the vagina is poorly understood. Using a murine pseudoestrus model of experimental vaginal candidiasis, we previously found that animals given a vaginal inoculum of viable Candida albicans blastoconidia acquired a persistent vaginal infection and developed Candida-specific delayed-type hypersensitivity (DTH) responses. The present study was designed to characterize the peripheral CMI reactivity generated from the vaginal infection in mice and to determine whether pseudoestrus is a prerequisite for the induction of peripheral CMI reactivity. Mice treated or not treated with estrogen and given a vaginal inoculum of C. albicans blastoconidia were examined for 4 weeks for their vaginal Candida burden and peripheral CMI reactivity, including DTH responsiveness and in vitro Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma]/Th2 (IL-4, IL-10)-type lymphokine production in response to Candida antigens. Results showed that although mice not treated with estrogen before being given a vaginal inoculum of C. albicans blastoconidia developed only a short-lived vaginal infection and harbored significantly fewer Candida CFU in the vagina compared with those given estrogen and then infected; DTH reactivity was equivalent in both groups. In vitro measurement of CMI reactivity further showed that lymph node cells from both estrogen- and non-estrogen-treated infected mice produced elevated levels of IL-2 and IFN-gamma in response to Candida antigens during the 4 weeks after vaginal inoculation. In contrast, lymph node cells from the same vaginally infected mice showed no IL-10 production and only small elevations of IL-4 during week 4 of infection. These results suggest that mice with experimental vaginal candidiasis develop predominantly Th1-type Candida-specific peripheral CMI reactivity and that similar patterns of Th1-type reactivity occur in mice regardless of the persistence of infection and the estrogen status of the infected mice.
PMCID: PMC281145  PMID: 8406809
3.  Mice immunized by primary vaginal Candida albicans infection develop acquired vaginal mucosal immunity. 
Infection and Immunity  1995;63(2):547-553.
It has been postulated that systemic cell-mediated immunity (CMI) is an important host defense mechanism against Candida infections of the vagina. However, in an estrogen-dependent murine model of experimental vaginal candidiasis, we recently showed that systemic Candida-specific Th1-type CMI induced by immunization with Candida culture filtrate antigen had no effect on vaginal Candida population levels during the course of a vaginal infection. In the present study, mice given a second vaginal inoculation in the presence of peripheral Candida-specific Th1-type CMI induced by prior vaginal infection had anamnestic-type increased delayed-type hypersensitivity (DTH) responses, concomitant with significantly fewer Candida organisms in the vagina than in primary-infected mice. In addition, organisms in secondary-infected mice were fragmented and superficial penetration into the epithelium was reduced. The systemic presence of Candida-specific T suppressor (Ts) cells that significantly suppressed the infection-derived anamnestic DTH reactivity did not abrogate the protective effect in the vagina. Additional experiments showed that vaginally immunized mice were not protected from gastrointestinal or systemic candidiasis and, in contrast to mice with a second vaginal infection, did not demonstrate anamnestic DTH reactivity. These results suggest that a moderate level of local protection against a Candida vaginal infection can be achieved by vaginal immunization but that the protective role of acquired peripheral Candida-specific Th1-type reactivity at the vaginal mucosa appears to be limited.
PMCID: PMC173030  PMID: 7822020
4.  Circulating CD4 and CD8 T cells have little impact on host defense against experimental vaginal candidiasis. 
Infection and Immunity  1995;63(7):2403-2408.
The etiology of recurrent vulvovaginal candidiasis in otherwise healthy women of child-bearing age remains an enigma. To date, results from both clinical studies and a murine model of vaginal candidiasis indicate that Candida vaginitis can occur in the presence of Candida-specific Th1-type cell-mediated immunity expressed in the peripheral circulation. The present study was designed to determine the role of circulating CD4 and CD8 cells in primary and secondary vaginal infections with Candida albicans. Vaginal fungal burden, Candida-specific delayed-type hypersensitivity (DTH), and lymph node cell Th1/Th2 cytokine production were monitored in CD4 and/or CD8 cell-depleted mice during persistent primary vaginal infections and secondary vaginal infections against which partial protection was observed. Treatment of mice with anti-CD4 or anti-CD8 antibodies resulted in 90% or greater depletion of the respective cell populations. Mice depleted of CD4 cells had significantly reduced Candida-specific DTH and lymph node cell Th1-type cytokine production during a primary vaginal infection, as well as reduced anamnestic DTH during a secondary vaginal infection. In contrast, mice depleted of CD8 cells showed only reduced gamma interferon production during a primary infection; no alterations in DTH were observed. Despite reductions in DTH and cytokine production, however, CD4 and/or CD8 cell depletion had no effect on vaginal C. albicans burden in mice after a primary or secondary vaginal inoculation. Taken together, these results suggest that while circulating CD4 and CD8 cells contribute to systemic Candida-specific cell-mediated immunity in vaginally infected mice, neither CD4 nor CD8 circulating T cells appear to provide significant host defenses against C. albicans at the vaginal mucosa.
PMCID: PMC173321  PMID: 7790050
5.  Local Production of Chemokines during Experimental Vaginal Candidiasis 
Infection and Immunity  1999;67(11):5820-5826.
Recurrent vulvovaginal candidiasis, caused by Candida albicans, is a significant problem in women of childbearing age. Although cell-mediated immunity (CMI) due to T cells and cytokines is the predominant host defense mechanism against C. albicans at mucosal tissue sites, host defense mechanisms against C. albicans at the vaginal mucosa are poorly understood. Based on an estrogen-dependent murine model of vaginal candidiasis, our data suggest that systemic CMI is ineffective against C. albicans vaginal infections. Thus, we have postulated that local immune mechanisms are critical for protection against infection. In the present study, the kinetic production of chemokines normally associated with the chemotaxis of T cells, macrophages (RANTES, MIP-1α, MCP-1), and polymorphonuclear neutrophils (MIP-2) was examined following intravaginal inoculation of C. albicans in estrogen-treated or untreated mice. Results showed significant increases in MCP-1 protein and mRNA in vaginal tissue of infected mice as early as 2 and 4 days postinoculation, respectively, that continued through a 21-day observation period, irrespective of estrogen status. No significant changes were observed with RANTES, MIP-1α, or MIP-2, although relatively high constitutive levels of RANTES mRNA and MIP-2 protein were observed. Furthermore, intravaginal immunoneutralization of MCP-1 with anti-MCP-1 antibodies resulted in a significant increase in vaginal fungal burden early during infection, suggesting that MCP-1 plays some role in reducing the fungal burden during vaginal infection. However, the lack of changes in leukocyte profiles in vaginal lavage fluids collected from infected versus uninfected mice suggests that MCP-1 functions to control vaginal C. albicans titers in a manner independent of cellular chemotactic activity.
PMCID: PMC96961  PMID: 10531235
6.  Cell Adhesion Molecule and Lymphocyte Activation Marker Expression during Experimental Vaginal Candidiasis 
Infection and Immunity  2001;69(8):5072-5079.
Cell-mediated immunity by Th1-type CD4+ T cells is the predominant host defense mechanism against mucosal candidiasis. However, studies using an estrogen-dependent murine model of vaginal candidiasis have demonstrated little to no change in resident vaginal T cells during infection and no systemic T-cell infiltration despite the presence of Candida-specific systemic Th1-type responses in infected mice. The present study was designed to further investigate these observations by characterizing T-cell activation and cell adhesion molecule expression during primary and secondary C. albicans vaginal infections. While flow cytometry analysis of activation markers showed some evidence for activation of CD3+ draining lymph node and/or vaginal lymphocytes during both primary and secondary vaginal Candida infection, CD3+ cells expressing the homing receptors and integrins α4β7, αM290β7, and α4β1 in draining lymph nodes of mice with primary and secondary infections were reduced compared to results for uninfected mice. At the local level, few vaginal lymphocytes expressed integrins, with only minor changes observed during both primary and secondary infections. On the other hand, immunohistochemical analysis of vaginal cell adhesion molecule expression showed increases in mucosal addressin cell adhesion molecule 1 and vascular cell adhesion molecule 1 expression during both primary and secondary infections. Altogether, these data suggest that although the vaginal tissue is permissive to cellular infiltration during a vaginal Candida infection, the reduced numbers of systemic cells expressing the reciprocal cellular adhesion molecules may preempt cellular infiltration, thereby limiting Candida-specific T-cell responses against infection.
doi:10.1128/IAI.69.8.5072-5079.2001
PMCID: PMC98602  PMID: 11447188
7.  Effects of Reproductive Hormones on Experimental Vaginal Candidiasis 
Infection and Immunity  2000;68(2):651-657.
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.
PMCID: PMC97188  PMID: 10639429
8.  Candida-Specific Antibodies during Experimental Vaginal Candidiasis in Mice  
Infection and Immunity  2002;70(10):5790-5799.
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 naïve 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.
doi:10.1128/IAI.70.10.5790-5799.2002
PMCID: PMC128320  PMID: 12228309
9.  Effects of systemic cell-mediated immunity on vaginal candidiasis in mice resistant and susceptible to Candida albicans infections. 
Infection and Immunity  1995;63(10):4191-4194.
Studies to date with CBA/J mice suggest a limited role for systemic cell-mediated immunity (CMI) against vaginal Candida albicans infections. The results of the present study show that preinduced Candida-specific systemic CMI was equally nonprotective against C. albicans vaginal infections in mice with high (BALB/cJ), low (DBA/2), or intermediate (CBA/J) resistance to C. albicans infections. Similarly, the locally acquired partial protection against a second C. albicans vaginal infection was equally observed with BALB/cJ, DBA/2, and CBA/J mice. These results indicate that observations made previously with CBA/J mice were not murine strain specific and provide additional support for the hypothesis that systemic CMI does not represent a dominant host defense mechanism at the vaginal mucosa.
PMCID: PMC173593  PMID: 7558342
10.  Analysis of Vaginal Cell Populations during Experimental Vaginal Candidiasis 
Infection and Immunity  1999;67(6):3135-3140.
Studies with an estrogen-dependent murine model of vaginal candidiasis suggest that local cell-mediated immunity (CMI) is more important than systemic CMI for protection against vaginitis. The present study, however, showed that, compared to uninfected mice, little to no change in the percentage or types of vaginal T cells occurred during a primary vaginal infection or during a secondary vaginal infection where partial protection was observed. Furthermore, depletion of polymorphonuclear leukocytes (PMN) had no effect on infection in the presence or absence of pseudoestrus. These results indicate a lack of demonstrable effects by systemic CMI or PMN against vaginitis and suggest that if local T cells are important, they are functioning without showing significant increases in numbers within the vaginal mucosa during infection.
PMCID: PMC96633  PMID: 10338532
11.  Chlamydia trachomatis Infection Does Not Enhance Local Cellular Immunity against Concurrent Candida Vaginal Infection 
Infection and Immunity  2001;69(5):3451-3454.
Although Th1-type cell-mediated immunity (CMI) is the predominant host defense mechanism against mucosal Candida albicans infection, CMI against a vaginal C. albicans infection in mice is limited at the vaginal mucosa despite a strong Candida-specific Th1-type response in the draining lymph nodes. In contrast, Th1-type CMI is highly effective against an experimental Chlamydia trachomatis genital tract infection. This study demonstrated through two independent designs that a concurrent Candida and Chlamydia infection could not accelerate or modulate the anti-Candida CMI response. Together, these results suggest that host responses to these genital tract infections are independent and not influenced by the presence of the other.
doi:10.1128/IAI.69.5.3451-3454.2001
PMCID: PMC98310  PMID: 11292774
12.  A Vaccine and Monoclonal Antibodies That Enhance Mouse Resistance to Candida albicans Vaginal Infection 
Infection and Immunity  1998;66(12):5771-5776.
We previously reported that a vaccine composed of liposome-mannan complexes of Candida albicans (L-mann) stimulates mice to produce protective antibodies against disseminated candidiasis. An immunoglobulin M (IgM) monoclonal antibody (MAb), B6.1, specific for a β-1,2-mannotriose in the complexes protects against the disease, whereas MAb B6 does not. In the present study, the vaccine and MAbs B6.1 and B6 were tested for the ability to protect against Candida vaginal infection, established by intravaginal (i.vg.) inoculation of yeast cells in mice maintained in pseudoestrus. Fungal CFU in each vagina was determined to assess the severity of infection. Mice vaccinated before infection developed about 62% fewer vaginal CFU than nonimmunized controls. Naive mice that received polyclonal antiserum (from vaccinated mice) i.vg. before infection had 60% fewer CFU than controls. The serum protective factor was stable at 56°C, but C. albicans cells absorbed this factor. Mice given MAb B6.1 i.vg. after infection was established had fewer Candida CFU in vaginal tissue than control mice given buffer instead of antibody. MAbs B6.1 and B6 given intraperitoneally before infection protected mice, but MAbs preabsorbed with yeast cells did not. MAb B6.1 also protected against C. tropicalis vaginal infection, but MAb B6 did not. The protective activities of MAbs B6.1 and B6 appeared to be specific because an irrelevant IgM carbohydrate-specific MAb and an irrelevant IgG protein-specific MAb were not protective; also, MAb B6.1 did not affect development of vaginal chlamydial infection. These studies show that an appropriate antibody response, or administration of protective antibodies, can help the host to resist Candida vaginal infection.
PMCID: PMC108729  PMID: 9826353
13.  Immunoregulation in experimental murine candidiasis: specific suppression induced by Candida albicans cell wall glycoprotein. 
Infection and Immunity  1985;49(1):172-181.
Immune regulation in candidiasis is inferred from studies of both human and animal infection, with a suppressive role suggested for cell wall polysaccharide. To study the immunosuppressive potential of Candida albicans in a murine model, whole blastoconidia or purified cell wall components of C. albicans were tested for their effects on the development of acquired immune responses by superimposing a pretreatment regimen upon an established immunization protocol. CBA/J or BALB/cByJ mice were pretreated twice intravenously with 100 micrograms of mannan (MAN), 100 or 200 micrograms of glycoprotein (GP), or 5 X 10(7) heat-killed C. albicans blastoconidia, followed 1 week later by an immunization protocol of two cutaneous inoculations of viable C. albicans blastoconidia given 2 weeks apart. Delayed hypersensitivity (DTH) to GP or to a membrane-derived antigen, B-HEX, was tested 7 days after the second inoculation, and lymphocyte stimulation was tested with mitogens and Candida antigens after 12 days. To assess protection, mice were challenged intravenously with viable C. albicans blastoconidia 14 days after the second cutaneous inoculation and sacrificed 28 days later for quantitative culture of kidneys and brains. Sera were obtained for enzyme-linked immunosorbent assays at selected intervals. Pretreatment with GP resulted in specific in vivo suppression of DTH to GP but not to B-HEX antigen and specific in vitro suppression of lymphocyte stimulation to GP but not to other Candida antigens or mitogens. MAN and heat-killed C. albicans blastoconidia had no such effects. GP pretreatment also diminished the protective effect of immunization against challenge, demonstrable in the brain, while not altering significantly the production of antibody in response to infection. Contrary to clinical evidence, MAN was not immunosuppressive in this model, and in fact, the immunosuppressive potential of GP, which is composed largely of MAN, was found to be dependent upon the presence of its heat-labile protein moiety.
PMCID: PMC262075  PMID: 4008047
14.  Vaginal yeast colonisation, prevalence of vaginitis, and associated local immunity in adolescents 
Objectives: To evaluate point prevalence vaginal yeast colonisation and symptomatic vaginitis in middle adolescents and to identify relation of these yeast conditions with reproductive hormones, sexual activity, sexual behaviours, and associated local immunity.
Methods: Middle adolescent females (n = 153) were evaluated for sexually transmitted infections (STIs), asymptomatic yeast colonisation, and symptomatic vulvovaginal candidiasis (VVC) by standard criteria. Also evaluated were local parameters, including vaginal associated cytokines, chemokines, and antibodies, vaginal epithelial cell antifungal activity, and Candida specific peripheral blood lymphocyte responses. Correlations between yeast colonisation/vaginitis and local immunomodulators, reproductive hormones, douching, sexual activity, condom use, and STIs were identified.
Results: Rates of point prevalence asymptomatic yeast colonisation (22%) were similar to adults and similarly dominated by Candida albicans, but with uncharacteristically high vaginal yeast burden. In contrast with the high rate of STIs (18%), incidence of symptomatic VVC was low (<2%). Immunological properties included high rates of Candida specific systemic immune sensitisation, a Th2 type vaginal cytokine profile, total and Candida specific vaginal antibodies dominated by IgA, and moderate vaginal epithelial cell anti-Candida activity. Endogenous reproductive hormones were in low concentration. Sexual activity positively correlated with vaginal yeast colonisation, whereas vaginal cytokines (Th1, Th2, proinflammatory), chemokines, antibodies, contraception, douching, or condom use did not.
Conclusion: Asymptomatic vaginal yeast colonisation in adolescents is distinct in some ways with adults, and positively correlates with sexual activity, but not with local immunomodulators or sexual behaviours. Despite several factors predictive for VVC, symptomatic VVC was low compared to STIs.
doi:10.1136/sti.2002.003855
PMCID: PMC1758371  PMID: 14755036
15.  Vaginal and Oral Epithelial Cell Anti-Candida Activity  
Infection and Immunity  2002;70(12):7081-7088.
Candida albicans is the causative agent of acute and recurrent vulvovaginal candidiasis (VVC), a common mucosal infection affecting significant numbers of women in their reproductive years. While any murine host protective role for cell-mediated immunity (CMI), humoral immunity, and innate resistance by neutrophils against the vaginal infection appear negligible, significant in vitro growth inhibition of Candida species by vaginal and oral epithelial cell-enriched cells has been observed. Both oral and vaginal epithelial cell anti-Candida activity has a strict requirement for cell contact to C. albicans with no role for soluble factors, and oral epithelial cells inhibit C. albicans through a cell surface carbohydrate moiety. The present study further evaluated the inhibitory mechanisms by murine vaginal epithelial cells and the fate of C. albicans by oral and vaginal epithelial cells. Similar to human oral cells, anti-Candida activity produced by murine vaginal epithelial cells is unaffected by enzymatic cleavage of cell surface proteins and lipids but sensitive to periodic acid cleavage of surface carbohydrates. Analysis of specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose and mannose-containing carbohydrates, also similar to oral cells. Staining for live and dead Candida in the coculture with fluorescein diacetate (FDA) and propidium iodide (PI), respectively, showed a clear predominance of live organisms, suggesting a static rather than cidal action. Together, the results suggest that oral and vaginal epithelial cells retard or arrest the growth rather than kill C. albicans through an as-yet-unidentified carbohydrate moiety in a noninflammatory manner.
doi:10.1128/IAI.70.12.7081-7088.2002
PMCID: PMC133056  PMID: 12438389
16.  A mannoprotein constituent of Candida albicans that elicits different levels of delayed-type hypersensitivity, cytokine production, and anticandidal protection in mice. 
Infection and Immunity  1994;62(12):5353-5360.
To identify major immunogenic constituents of Candida albicans, the effect of a mannoprotein fraction (MP-F2) on the elicitation of a delayed-type hypersensitivity (DTH) reaction, cytokine production, and protection from a virulent Candida challenge in a mouse candidiasis model was studied. In mice immunized with whole cells of a low-virulence strain of C. albicans and thus protected against a challenge with a highly virulent strain of this fungus, MP-F2 was able to elicit a strong DTH response that was accompanied by splenocyte proliferation in vitro in the presence of Candida antigen. The supernatants of MP-F2-stimulated splenocyte cultures contained gamma interferon (IFN-gamma, a typical CD4+ T helper-1 (Th1) cytokine, but no interleukin-4, (IL-4), a typical CD4+ Th2 cytokine. IFN-gamma was produced by CD4+ cells, and its level could be greatly increased by the addition of anti-IL-4 or, mostly, anti-IL-10 antibodies to the CD4+ cell cultures. Upon a suitable schedule of immunization, MP-F2 was also able to induce a vigorous DTH response in Candida-uninfected mice, a response that could be efficiently transferred into naive recipients by CD4+ cells from the spleens of MP-F2-immunized mice. The immunization described above also conferred to mice a low degree of protection against a virulent Candida challenge, both in terms of median survival time and in the number of Candida cells in the kidney. However, while DTH induction by MP-F2 was as strong as that induced by whole cells, MP-F2-induced protection was significantly weaker than that conferred by Candida whole-cell immunization. Mice immunized with either MP-F2 or Candida whole cells had an inverted ratio between the number of CD4+ splenocytes producing IFN-gamma and that of cells producing IL-4, compared with nonimmunized animals. However, the number of IL-4-producing CD4+ cells was significantly higher in MP-F2-vaccinated, weakly protected mice than in Candida whole-cell-vaccinated, highly protected animals. Overall, our data suggest that the MP-F2 fraction contains one or more major immunogens of C. albicans which are capable of interfering with the balance of CD4+ Th1 and Th2 responses that is so critical in the outcome of host-Candida relationship and are thus potentially relevant in the mechanisms of Candida-specific DTH regulation and protection.
PMCID: PMC303275  PMID: 7960114
17.  Local Anticandidal Immune Responses in a Rat Model of Vaginal Infection by and Protection against Candida albicans 
Infection and Immunity  2000;68(6):3297-3304.
Humoral (antibody [Ab]) and cellular Candida-specific immune responses in the vaginas of pseudoestrus rats were investigated during three successive infections by Candida albicans. After the first, protective infection, Abs against mannan and aspartyl proteinase antigens were present in the vaginal fluid, and their titers clearly increased during the two subsequent, rapidly healing infections. In all animals, about 65 and 10% of vaginal lymphocytes (VL) were CD3+ (T cells) and CD3− CD5+ (B cells), respectively. Two-thirds of the CD3+ T cells expressed the α/β and one-third expressed the γ/δ T-cell receptor (TCR). This proportion slightly fluctuated during the three rounds of C. albicans infection, but no significant differences between infected and noninfected rats were found. More relevant were the changes in the CD4+/CD8+ T-cell ratio, particularly for cells bearing the CD25 (interleukin-2 receptor α) marker. In fact, a progressively increased number of both CD4+ α/β TCR and CD4+ CD25+ VL was observed after the second and third Candida challenges, reversing the high initial CD8+ cell number of controls (estrogenized but uninfected rats). The CD3− CD5+ cells also almost doubled from the first to the third infection. Analysis of the cytokines secreted in the vaginal fluid of Candida-infected rats showed high levels of interleukin 12 (IL-12) during the first infection, followed by progressively increasing amounts of IL-2 and gamma interferon during the subsequent infections. No IL-4 or IL-5 was ever detected. During the third infection, VL with in vitro proliferative activity in response to an immunodominant mannoprotein antigen of C. albicans were present in the vaginal tissue. No response to this antigen by mitogen-responsive blood, lymph node, and spleen cells was found. In summary, the presence of protective Ab and T helper type 1 cytokines in the vaginal fluids, the in vitro proliferation of vaginal lymphocytes in response to Candida antigenic stimulation, and the increased number of activated CD4+ cells and some special B lymphocytes after C. albicans challenge constitute good evidence for induction of locally expressed Candida-specific Ab and cellular responses which are potentially involved in anticandidal protection at the vaginal level.
PMCID: PMC97585  PMID: 10816477
18.  T lymphocytes in the murine vaginal mucosa are phenotypically distinct from those in the periphery. 
Infection and Immunity  1996;64(9):3793-3799.
The results from both clinical studies of women with recurrent vulvovaginal candidiasis and a murine model of experimental vaginitis indicate that systemic cell-mediated immunity may not represent a dominant host defense mechanism against vaginal infections by Candida albicans. Recent experimental evidence indicates the presence of local vaginal immune reactivity against C. albicans. The present study was designed to examine T-lymphocyte subpopulations in the vaginal mucosae of naive CBA/J mice. Vaginal lymphocytes (VL) were isolated by collagenase digestion of whole vaginal tissues. Cell populations were identified by flow cytometry, and the results were compared with those for both lymph node cells (LNC) and peripheral blood lymphocytes (PBL). The results of flow cytometry showed that 45% +/- 10% of lymphocytes in the vaginal mucosa are CD3+ compared with 75% +/- 5% in LNC and 50% +/- 5% in PBL. The majority (85%) of CD3+ VL are CD4+ and express the alpha/beta T-cell receptor (TCR), similar to the results for LNC and PBL. In contrast to LNC and PBL, VL contain a significantly higher percentage (15 to 20%) of gamma/delta TCR+ cells, 80% or more of which appear to express CD4. In addition, while CD4-CD8 cell ratios in LNC and PBL were 3:1 and 6:1, respectively, only 1% of VL expressed CD8, resulting in a CD4-CD8 cell ratio of > 100:1. Finally, while LNC and PBL recognized two epitope-distinct (GK 1.5 and 2B6) anti-CD4 antibodies, VL recognized only 2B6 anti-CD4 antibodies. Further analysis of VL showed that Thy-1 cells, but not CD4 cells, were reduced after intravaginal injection of complement-fixing anti-Thy-1.2 and GK 1.5 anti-CD4 antibodies, respectively. Taken together, these data suggest that T lymphocytes in the vaginal mucosae of mice are phenotypically distinct from those in the periphery and that CD4+ VL have an uncharacteristic or atypical expression of the CD4 receptor.
PMCID: PMC174295  PMID: 8751931
19.  Role for Dendritic Cells in Immunoregulation during Experimental Vaginal Candidiasis  
Infection and Immunity  2006;74(6):3213-3221.
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.
doi:10.1128/IAI.01824-05
PMCID: PMC1479243  PMID: 16714548
20.  The Acute Neutrophil Response Mediated by S100 Alarmins during Vaginal Candida Infections Is Independent of the Th17-Pathway 
PLoS ONE  2012;7(9):e46311.
Vulvovaginal candidiasis (VVC) caused by Candida albicans affects a significant number of women during their reproductive ages. Clinical observations revealed that a robust vaginal polymorphonuclear neutrophil (PMN) migration occurs in susceptible women, promoting pathological inflammation without affecting fungal burden. Evidence to date in the mouse model suggests that a similar acute PMN migration into the vagina is mediated by chemotactic S100A8 and S100A9 alarmins produced by vaginal epithelial cells in response to Candida. Based on the putative role for the Th17 response in mucosal candidiasis as well as S100 alarmin induction, this study aimed to determine whether the Th17 pathway plays a role in the S100 alarmin-mediated acute inflammation during VVC using the experimental mouse model. For this, IL-23p19−/−, IL-17RA−/− and IL-22−/− mice were intravaginally inoculated with Candida, and vaginal lavage fluids were evaluated for fungal burden, PMN infiltration, the presence of S100 alarmins and inflammatory cytokines and chemokines. Compared to wild-type mice, the cytokine-deficient mice showed comparative levels of vaginal fungal burden and PMN infiltration following inoculation. Likewise, inoculated mice of all strains with substantial PMN infiltration exhibited elevated levels of vaginal S100 alarmins in both vaginal epithelia and secretions in the vaginal lumen. Finally, cytokine analyses of vaginal lavage fluid from inoculated mice revealed equivalent expression profiles irrespective of the Th17 cytokine status or PMN response. These data suggest that the vaginal S100 alarmin response to Candida does not require the cells or cytokines of the Th17 lineage, and therefore, the immunopathogenic inflammatory response during VVC occurs independently of the Th17-pathway.
doi:10.1371/journal.pone.0046311
PMCID: PMC3457984  PMID: 23050010
21.  Immunopathogenesis of recurrent vulvovaginal candidiasis. 
Clinical Microbiology Reviews  1996;9(3):335-348.
Recurrent vulvovaginal candidiasis (RVVC) is a prevalent opportunistic mucosal infection, caused predominantly by Candida albicans, which affects a significant number of otherwise healthy women of childbearing age. Since there are no known exogenous predisposing factors to explain the incidence of symptomatic vaginitis in most women with idiopathic RVVC, it has been postulated that these particular women suffer from an immunological abnormality that prediposes them to RVVC. Because of the increased incidence of mucosal candidiasis in individuals with depressed cell-mediated immunity (CMI), defects in CMI are viewed as a possible explanation for RVVC. In this review, we attempt to place into perspective the accumulated information regarding the immunopathogenesis of RVVC, as well as to provide new immunological perspectives and hypotheses regarding potential immunological deficiencies that may predispose to RVVC and potentially other mucosal infections by the same organism. The results of both clinical studies and studies in an animal model of experimental vaginitis suggest that systemic CMI may not be the predominant host defense mechanism against C. albicans vaginal infections. Rather, locally acquired mucosal immunity, distinct from that in the peripheral circulation, is now under consideration as an important host defense at the vaginal mucosa, as well as the notion that changes in local CMI mechanism(s) may predispose to RVVC.
PMCID: PMC172897  PMID: 8809464
22.  Feasibility of Histological Scoring and Colony Count for Evaluating Infective Severity in Mouse Vaginal Candidiasis 
Experimental Animals  2013;62(3):205-210.
Qualitative measurement of the infective level is relatively difficult in experimental vaginal candidiasis. Female BALB/c mice aged 8 to 10 weeks were randomly divided into E1, E2 and E0 groups, which received subcutaneous injection of 0.05 mg, 0.1 mg of estradiol benzoate or 0.1 ml soybean oil 3 days before vaginal inoculation, respectively, and hormone treatment continued every other day thereafter. Each group was further divided into infected and noninfected subgroups. The infected mice were inoculated intravaginally with 10 µl (5 × 104 conidia) of Candida albicans suspension, while the noninfected mice were inoculated with 10 µl phosphate-buffered saline. Direct microscopic examination, colony count and vaginal histopathology including infection degree and inflammation extent were performed at 3, 7 and 14 days post inoculation. Estrogen treatment increased the vaginal fungal burden and extent of infection and inflammation compared with the control group, and 0.3 mg/week estrogen generally induced more severe infection and inflammation than 0.15 mg/week estrogen did. Colony count peaked on day 3 and decreased remarkably after 7 days. Infection score increased gradually during the first 7 days and decreased on day 14, while inflammation extent exacerbated progressively over the course of 14 days. This study demonstrates that the modified histological scoring system might be more feasible than colony count for evaluation of infectivity and dynamic change in experimental vaginal candidiasis.
doi:10.1538/expanim.62.205
PMCID: PMC4160942  PMID: 23903055
Candida albicans; estrogen; mice; vaginal candidiasis
23.  Characterization of cellular infiltrates and cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response. 
Infection and Immunity  1993;61(7):2854-2865.
Cryptococcosis, an increasingly important opportunistic infection caused by the encapsulated yeast-like organism Cryptococcus neoformans, is limited by an anticryptococcal cell-mediated immune (CMI) response. Gaining a thorough understanding of the complex anticryptococcal CMI response is essential for developing means of controlling infections with C. neoformans. The murine cryptococcosis model utilizing footpad swelling to cryptococcal antigen (delayed-type hypersensitivity [DTH]) has proven to be a valuable tool for studying the induction and regulation of the anticryptococcal CMI response, but this technique has limitations with regard to evaluating the role of the final effector cells recruited by an ongoing CMI response. The purpose of this study was to assess the types of cells and cytokines induced into the site of cryptococcal antigen deposition in C. neoformans-infected and -immunized mice compared with those for control mice. We used a gelatin sponge implant model to examine the cells and cytokines present at the site of an anticryptococcal DTH response. Sponges implanted in infected mice and injected with cryptococcal culture filtrate antigen (CneF) 24 h before assessment had significantly increased numbers of infiltrating leukocytes compared with saline-injected sponges in the same animals. Exaggerated influxes of neutrophils and mononuclear cells were the major contributors to the increase in total numbers of cells in the DTH-reactive sponges. The numbers of CD4+ and LFA-1+ cells were found to be significantly increased in the CneF-injected sponges of infected and immunized mice over the numbers in control sponges. The numbers of large granular lymphocytes were also increased in DTH-reactive sponges compared with control sponges. Gamma interferon, interleukin 2 (IL-2), and IL-5 are clearly relevant cytokines in the anticryptococcal CMI response, since they were produced in greater amounts in the CneF-injected sponges from C. neoformans-infected and -immunized mice than in control sponges. IL-4 was not associated with the expression of DTH to cryptococcal antigen. The gelatin sponge model is an excellent tool for studying cells and cytokines involved in specific CMI responses.
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PMCID: PMC280931  PMID: 8514388
24.  CTLA-4 Down-Regulates the Protective Anticryptococcal Cell-Mediated Immune Response 
Infection and Immunity  2000;68(8):4624-4630.
Cell-mediated immune (CMI) responses defined by delayed-type hypersensitivity (DTH) reactivity to cryptococcal culture filtrate antigen (CneF) can be either protective or nonprotective against an infection with Cryptococcus neoformans. The protective and nonprotective anticryptococcal DTH responses are induced by different immunogens and have differing activated-T-cell profiles. This study examined the effects of blockade of the interaction between cytotoxic T lymphocyte antigen 4 (CTLA-4) and its ligands B7-1 (CD80) and B7-2 (CD86) on the anticryptococcal DTH responses and protection. We found that CTLA-4 blockade at the time of immunization with the immunogen that induces the protective response, CneF, in complete Freund's adjuvant (CFA) or the immunogen that induces the nonprotective response, heat-killed cryptococcal cells (HKC), enhanced anticryptococcal DTH reactivity. In contrast, blocking CTLA-4 after the immune response was induced failed to enhance responses. Blockade of CTLA-4 in an infection model resulted in earlier development of the anticryptococcal CMI response than in control mice. Concomitant with increases in DTH reactivity in mice treated with anti-CTLA-4 Fab fragments at the time of immunization, there were decreases in cryptococcal CFU in lungs, spleens, and brains compared to controls. Blockade of CTLA-4 resulted in long-term protection, as measured by significantly increased survival times, only in mice given the protective immunogen, CneF-CFA. Anti-CTLA-4 treatment did not shift the response induced by the nonprotective immunogen, HKC, to a long-term protective one. Our data indicate that blockade of CTLA-4 interactions with its ligands may be useful in enhancing host defenses against C. neoformans.
PMCID: PMC98393  PMID: 10899865
25.  Differences in Components at Delayed-Type Hypersensitivity Reaction Sites in Mice Immunized with Either a Protective or a Nonprotective Immunogen of Cryptococcus neoformans 
Infection and Immunity  2002;70(2):591-600.
Cell-mediated immunity is the major protective mechanism against Cryptococcus neoformans. Delayed swelling reactions, i.e., delayed-type hypersensitivity (DTH), in response to an intradermal injection of specific antigen are used as a means of detecting a cell-mediated immune (CMI) response to the antigen. We have found previously that the presence of an anticryptococcal DTH response in mice is not always indicative of protection against a cryptococcal infection. Using one immunogen that induces a protective anticryptococcal CMI response and one that induces a nonprotective response, we have shown that mice immunized with the protective immunogen undergo a classical DTH response characterized by mononuclear cell and neutrophil infiltrates and the presence of gamma interferon and NO. In contrast, immunization with the nonprotective immunogen results in an influx of primarily neutrophils and production of tumor necrosis factor alpha (TNF-α) at the DTH reaction site. Even when the anticryptococcal DTH response was augmented by blocking the down-regulator, CTLA-4 (CD152), on T cells in the mice given the nonprotective immunogen, the main leukocyte population infiltrating the DTH reaction site is the neutrophil. Although TNF-α is increased at the DTH reaction site in mice immunized with the nonprotective immunogen, it is unlikely that TNF-α activates the neutrophils, because the density of TNF receptors on the neutrophils is reduced below control levels. Uncoupling of DTH reactivity and protection has been demonstrated in other infectious-disease models; however, the mechanisms differ from our model. These findings stress the importance of defining the cascade of events occurring in response to various immunogens and establishing the relationships between protection and DTH reactions.
PMCID: PMC127722  PMID: 11796587

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