A C. albicans TRAPP Complex-Associated Gene Contributes to Cell Wall Integrity, Hyphal and Biofilm Formation, and Tissue Invasion.

While endocytic and secretory pathways are well-studied cellular processes in the model yeast Saccharomyces cerevisiae, they remain understudied in the opportunistic fungal pathogen Candida albicans. We previously found that null mutants of C. albicans homologs of the S. cerevisiae early endocytosis genes ENT2 and END3 not only exhibited delayed endocytosis but also had defects in cell wall integrity, filamentation, biofilm formation, extracellular protease activity, and tissue invasion in an in vitro model. In this study, we focused on a potential C. albicans homolog to S. cerevisiae TCA17, which was discovered in our whole-genome bioinformatics approach aimed at identifying genes involved in endocytosis. In S. cerevisiae, TCA17 encodes a transport protein particle (TRAPP) complex-associated protein. Using a reverse genetics approach with CRISPR-Cas9-mediated gene deletion, we analyzed the function of the TCA17 homolog in C. albicans. Although the C. albicans tca17Δ/Δ null mutant did not have defects in endocytosis, it displayed an enlarged cell and vacuole morphology, impaired filamentation, and reduced biofilm formation. Moreover, the mutant exhibited altered sensitivity to cell wall stressors and antifungal agents. When assayed using an in vitro keratinocyte infection model, virulence properties were also diminished. Our findings indicate that C. albicans TCA17 may be involved in secretion-related vesicle transport and plays a role in cell wall and vacuolar integrity, hyphal and biofilm formation, and virulence. IMPORTANCE The fungal pathogen Candida albicans causes serious opportunistic infections in immunocompromised patients and has become a major cause of hospital-acquired bloodstream infections, catheter-associated infections, and invasive disease. However, due to a limited understanding of Candida molecular pathogenesis, clinical approaches for the prevention, diagnosis, and treatment of invasive candidiasis need significant improvement. In this study, we focus on identifying and characterizing a gene potentially involved in the C. albicans secretory pathway, as intracellular transport is critical for C. albicans virulence. We specifically investigated the role of this gene in filamentation, biofilm formation, and tissue invasion. Ultimately, these findings advance our current understanding of C. albicans biology and may have implications for the diagnosis and treatment of candidiasis.

The Early Endocytosis Gene PAL1 Contributes to Stress Tolerance and Hyphal Formation in Candida albicans.

The endocytic and secretory pathways of the fungal pathogen Candida albicans are fundamental to various key cellular processes such as cell growth, cell wall integrity, protein secretion, hyphal formation, and pathogenesis. Our previous studies focused on several candidate genes involved in early endocytosis, including ENT2 and END3, that play crucial roles in such processes. However, much remains to be discovered about other endocytosis-related genes and their contributions toward Candida albicans secretion and virulence. In this study, we examined the functions of the early endocytosis gene PAL1 using a reverse genetics approach based on CRISPR-Cas9-mediated gene deletion. Saccharomyces cerevisiae Pal1 is a protein in the early coat complex involved in clathrin-mediated endocytosis that is later internalized with the coat. The C. albicans pal1Δ/Δ null mutant demonstrated increased resistance to the antifungal agent caspofungin and the cell wall stressor Congo Red. In contrast, the null mutant was more sensitive to the antifungal drug fluconazole and low concentrations of SDS than the wild type (WT) and the re-integrant (KI). While pal1Δ/Δ can form hyphae and a biofilm, under some hyphal-inducing conditions, it was less able to demonstrate filamentous growth when compared to the WT and KI. The pal1Δ/Δ null mutant had no defect in clathrin-mediated endocytosis, and there were no changes in virulence-related processes compared to controls. Our results suggest that PAL1 has a role in susceptibility to antifungal agents, cell wall integrity, and membrane stability related to early endocytosis.

Candida albicans END3 Mediates Endocytosis and Has Subsequent Roles in Cell Wall Integrity, Morphological Switching, and Tissue Invasion.

The role of endocytosis in Candida albicans secretion, filamentation, and virulence remains poorly understood, despite its importance as a fundamental component of intracellular trafficking. Given that secretory mutants display defects in endocytosis, we have focused our attention on endocytic mutants to understand the interconnection between endocytosis and other secretory pathways. Using a reverse-genetic approach based upon CRISPR-Cas9 mediated gene deletion, we studied the functions of the gene END3, which plays a key role in clathrin-based endocytosis. In the end3Δ/Δ null mutant, clathrin-mediated endocytosis was substantially reduced. While in vitro growth, cell morphology, and vacuoles appeared normal, the mutant was impaired in actin patch formation, filamentous growth, biofilm formation, cell wall integrity, and extracellular protease secretion. In addition, susceptibility to various antifungal agents was altered. Consistent with the inability to form hyphae, in an in vitro keratinocyte infection model, the null mutant displayed reduced damage of mammalian adhesion zippers and host cell death. Thus, C. albicans END3 has a role in efficient endocytosis that is required for cell wall integrity, protein secretion, hyphal formation, and virulence-related processes. These findings suggest that impaired endocytosis subsequently affects other secretory pathways, providing evidence of the interconnection between these processes. IMPORTANCE Candida albicans is a fungal commensal organism that can cause serious opportunistic infections in immunocompromised patients leading to substantial complications and mortality. A better understanding of the microbe's biology to develop more effective therapeutic and diagnostic tools is required as invasive candidiasis is a problem of continued clinical importance. This study focuses on endocytosis, an important but incompletely understood cellular mechanism needed to uptake nutrients and communicate with a cell's environment. In this study, we have assessed the role of endocytosis in cell wall integrity, biofilm formation, and tissue invasion in C. albicans. These findings will improve our understanding of cellular mechanisms underlying endocytosis and will inform us of the interconnection with other intracellular transport processes.

Candida albicans ENT2 Contributes to Efficient Endocytosis, Cell Wall Integrity, Filamentation, and Virulence.

Epsins play a pivotal role in the formation of endocytic vesicles and potentially provide a linkage between endocytic and other trafficking pathways. We identified a Candida albicans epsin, ENT2, that bears homology to the Saccharomyces cerevisiae early endocytosis genes ENT1 and ENT2 and studied its functions by a reverse genetic approach utilizing CRISPR-Cas9-mediated gene deletion. The C. albicans ent2Δ/Δ null mutant displayed cell wall defects and altered antifungal drug sensitivity. To define the role of C. albicans ENT2 in endocytosis, we performed assays with the lipophilic dye FM4-64 that revealed greatly reduced uptake in the ent2Δ/Δ mutant. Next, we showed that the C. albicans ent2Δ/Δ mutant was unable to form hyphae and biofilms. Assays for virulence properties in an in vitro keratinocyte infection model demonstrated reduced damage of mammalian adhesion zippers and host cell death from the ent2Δ/Δ mutant. We conclude that C. albicans ENT2 has a role in efficient endocytosis, a process that is required for maintaining cell wall integrity, hyphal formation, and virulence-defining traits. IMPORTANCE The opportunistic fungal pathogen Candida albicans is an important cause of invasive infections in hospitalized patients and a source of considerable morbidity and mortality. Despite its clinical importance, we still need to improve our ability to diagnose and treat this common pathogen. In order to support these advancements, a greater understanding of the biology of C. albicans is needed. In these studies, we are focused on the fundamental biological process of endocytosis, of which little is directly known in C. albicans. In addition to studying the function of a key gene in this process, we are examining the role of endocytosis in the virulence-related processes of filamentation, biofilm formation, and tissue invasion. These studies will provide greater insight into the role of endocytosis in causing invasive fungal infections.

Stimulation of cell motility and expression of late markers of differentiation in human oral keratinocytes by Candida albicans.

A hallmark of the mucosa of immunocompromized hosts in oral candidiasis is a hyperkeratinized region heavily colonized with fungi at the surface of the terminally differentiated epithelium. To gain insight into the processes important for promoting mucosal invasion by fungi, we characterized the response of keratinocytes to the presence of Candida albicans. Indirect immunofluorescence and kymographic analyses revealed a multifaceted keratinocyte response of OKF6/TERT-2 cells to C. albicans that consisted of: cytoskeletal reorganization within 3 h, motility and cell expansion with formation of E-cadherin-mediated cell-cell adhesions within 6 h, increased expression of late differentiation markers and decreased expression of calprotectin. The initial expansive phase was followed by dissolution of cell-cell adhesions and a decrease in cell size accompanied by loss of E-cadherin. The keratinocyte response depended on soluble factors associated with hyphal growth as demonstrated using the efg1Delta/efg1Delta, cap1Delta/cap1Delta, als3Delta/als3Delta, hwp1Delta/hwp1Deltaand sap4-6Delta/sap4-6Delta mutants and was not observed in the presence of the non-pathogenic yeast, Saccharomyces cerevisiae. These studies show the potential for C. albicans to manipulate the stratified epithelial cells to a state of differentiation that is more permissive of fungal colonization of oral tissue, which is likely to play an important role in the pathogenesis of candidiasis.

The Role of Secretory Pathways in Candida albicans Pathogenesis.

Candida albicans is a fungus that is a commensal organism and a member of the normal human microbiota. It has the ability to transition into an opportunistic invasive pathogen. Attributes that support pathogenesis include secretion of virulence-associated proteins, hyphal formation, and biofilm formation. These processes are supported by secretion, as defined in the broad context of membrane trafficking. In this review, we examine the role of secretory pathways in Candida virulence, with a focus on the model opportunistic fungal pathogen, Candida albicans.

Vitamin D Status Impacts Genital Mucosal Immunity and Markers of HIV-1 Susceptibility in Women.

While vitamin D insufficiency is known to impact a multitude of health outcomes, including HIV-1, little is known about the role of vitamin D-mediated immune regulation in the female reproductive tract (FRT). We performed a pilot clinical study of 20 women with circulating 25(OH)D levels <62.5 nmol/L. Participants were randomized into either weekly or daily high-dose oral vitamin D supplementation groups. In addition to serum vitamin D levels, genital mucosal endpoints, including soluble mediators, immune cell populations, gene expression, and ex vivo HIV-1 infection, were assessed. While systemic vitamin D levels showed a significant increase following supplementation, these changes translated into modest effects on the cervicovaginal factors studied. Paradoxically, post-supplementation vitamin D levels were decreased in cervicovaginal fluids. Given the strong correlation between vitamin D status and HIV-1 infection and the widespread nature of vitamin D deficiency, further understanding of the role of vitamin D immunoregulation in the female reproductive tract is important.

The Effect of Hormonal Contraception on Cervicovaginal Mucosal End Points Associated with HIV Acquisition.

Reproductive age women may choose to concurrently use topical antiretrovirals and hormonal contraceptives (HCs) to simultaneously prevent HIV-1 infection and unintended/mistimed pregnancy. There are conflicting data on the effect of HCs on mucosal susceptibility to HIV-1. The objective of this study was to evaluate cervicovaginal (CV) mucosal data from healthy women before and after initiation of either oral contraceptive pills (OCPs) or depot medroxyprogesterone acetate (DMPA) injection. CONRAD A10-114 was a prospective, open-label, parallel cohort study. We enrolled 74 women and 62 completed the visits (32 and 30 who selected OCPs and DMPA, respectively). Participants provided CV lavage, vaginal biopsies, and CV swabs at baseline in the luteal phase and then ∼6 weeks after initiating HCs. After contraceptive initiation, there were significant increases in vaginal immune cell density among both DMPA and OCP users. Changes for OCP users were concentrated in the subepithelial lamina propria, whereas for DMPA users, they were distributed throughout the vaginal tissue, including the epithelium (CD45+, CD3+, CD4+, and CD1a+). Contraceptive use altered concentrations of soluble CV inflammatory and immune mediators, with significant reductions in some proinflammatory cytokines and secretory leukoprotease inhibitor. Compared with baseline, p24 antigen production after ex vivo HIV-1 infection of vaginal biopsies doubled after DMPA use, but all p-values were >.05. HIV-1 replication was significantly higher in DMPA-exposed tissues compared with those from the OCP group at the end of the tissue culture (p = .01). Although not statistically significant, median in vitro inhibition of HIV-1 by CV fluid (innate antiviral activity), was reduced by ∼50% with HCs (p > .21). Exposure to exogenous contraceptive hormones significantly increased vaginal immune cells and reduced CV proinflammatory cytokines and antimicrobial peptides. DMPA users showed higher susceptibility to HIV-1 ex vivo infection.

Randomized, placebo controlled phase I trial of safety, pharmacokinetics, pharmacodynamics and acceptability of tenofovir and tenofovir plus levonorgestrel vaginal rings in women.

To prevent the global health burdens of human immunodeficiency virus [HIV] and unintended/mistimed pregnancies, we developed an intravaginal ring [IVR] that delivers tenofovir [TFV] at ~10mg/day alone or with levonorgestrel [LNG] at ~20µg/day for 90 days. We present safety, pharmacokinetics, pharmacodynamics, acceptability and drug release data in healthy women. CONRAD A13-128 was a randomized, placebo controlled phase I study. We screened 86 women; 51 were randomized to TFV, TFV/LNG or placebo IVR [2:2:1] and 50 completed all visits, using the IVR for approximately 15 days. We assessed safety by adverse events, colposcopy, vaginal microbiota, epithelial integrity, mucosal histology and immune cell numbers and phenotype, cervicovaginal [CV] cytokines and antimicrobial proteins and changes in systemic laboratory measurements, and LNG and TFV pharmacokinetics in multiple compartments. TFV pharmacodynamic activity was measured by evaluating CV fluid [CVF] and tissue for antiviral activity using in vitro models. LNG pharmacodynamic assessments were timed based on peak urinary luteinizing hormone levels. All IVRs were safe with no significant colposcopic, mucosal, immune and microbiota changes and were acceptable. Among TFV containing IVR users, median and mean CV aspirate TFV concentrations remained above 100,000 ng/mL 4 hours post IVR insertion and mean TFV-diphosphate [DP] concentrations in vaginal tissue remained above 1,000 fmol/mg even 3 days post IVR removal. CVF of women using TFV-containing IVRs completely inhibited [94-100%] HIV infection in vitro. TFV/LNG IVR users had mean serum LNG concentrations exceeding 300 pg/mL within 1 hour, remaining high throughout IVR use. All LNG IVR users had a cervical mucus Insler score <10 and the majority [95%] were anovulatory or had abnormal cervical mucus sperm penetration. Estimated in vivo TFV and LNG release rates were within expected ranges. All IVRs were safe with the active ones delivering sustained high concentrations of TFV locally. LNG caused changes in cervical mucus, sperm penetration, and ovulation compatible with contraceptive efficacy. The TFV and TFV/LNG rings are ready for expanded 90 day clinical testing. Trial registration ClinicalTrials.gov #NCT02235662.

The nuclear pore complex and the DEAD box protein Rat8p/Dbp5p have nonessential features which appear to facilitate mRNA export following heat shock.

Nuclear pore complexes (NPCs) play an essential role in RNA export. Nucleoporins required for mRNA export in Saccharomyces cerevisiae are found in the Nup84p and Nup82p subcomplexes of the NPC. The Nup82p subcomplex contains Nup82p, Rat7p/Nup159p, Nsp1p, Gle1p/Rss1p, and Rip1p/Nup42p and is found only on the cytoplasmic face of NPCs. Both Rat7p and Gle1p contain binding sites for Rat8p/Dbp5p, an essential DEAD box protein and putative RNA helicase. Rip1p interacts directly with Gle1p and is the only protein known to be essential for mRNA export after heat shock but not under normal growth conditions. We report that in cells lacking Rip1p, both Gle1p and Rat8p dissociate from NPCs following heat shock at 42 degrees C. Rat8p but not Gle1p was retained at NPCs if rip1Delta cells were first shifted to 37 degrees C and then to 42 degrees C, and this was correlated with preserving mRNA export in heat-shocked rip1Delta cells. Export following ethanol shock was less dependent on the presence of Rip1p. Exposure to 10% ethanol led to dissociation of Rat8p from NPCs in both wild-type and rip1Delta cells. Following this treatment, Rat8p was primarily nuclear in wild-type cells but primarily cytoplasmic in rip1Delta cells. We also determined that efficient export of heat shock mRNA after heat shock depends upon a novel 6-amino-acid element within Rat8p. This motif is not required under normal growth conditions or following ethanol shock. These studies suggest that the molecular mechanism responsible for the defect in export of heat shock mRNAs in heat-shocked rip1Delta cells is dissociation of Rat8p from NPCs. These studies also suggest that both nuclear pores and Rat8p have features not required for mRNA export in growing cells but which enhance the ability of mRNAs to be exported following heat shock.

Importin beta-depending nuclear import pathways: role of the adapter proteins in the docking and releasing steps.

Nuclear imports of uridine-rich small nuclear ribonucleoprotein (U1 snRNP) and proteins with classical nuclear localization signal (cNLS-protein) are mediated by importin beta. However, due to the presence of different import signals, the adapter protein of the imported molecules and importin beta is different for each pathway. Although the adapter for cNLS-protein is importin alpha, the adapter for U1 snRNP is snurportin1 (SPN1). Herein, we show that the use of distinct adapters by importin beta results in differences at the docking and releasing step for these two import pathways. Nuclear pore complex (NPC) docking of U1 snRNP but not of cNLS-protein was inhibited by an anti-CAN/Nup214 antibody. Thus, the initial NPC-binding site is different for each pathway. Pull-down assays between immobilized SPN1 and two truncated forms of importin beta documented that SPN1 and importin alpha have different binding sites on importin beta. Importin beta fragment 1-618, which binds to SPN1 but not to importin alpha, was able to support the nuclear import of U1 snRNPs. After the translocation through the NPC, both import complexes associated with the nuclear side of the NPC. However, we found that the nature of the importin beta-binding domain of the adapters influences the release of the cargo into the nucleoplasm.

Comparison of Mucosal Markers of Human Immunodeficiency Virus Susceptibility in Healthy Premenopausal Versus Postmenopausal Women.

The objective of this study was to characterize cervicovaginal (CV) mucosal factors modulating susceptibility to human immunodeficiency virus (HIV) acquisition in healthy premenopausal (PRE) and postmenopausal (POST) women before and after treatment with estradiol (E2). We compared CV mucosal epithelial histology and immune cells, vaginal microbiota, antimicrobial activity of and soluble mucosal protein concentrations in the CV fluid lavage (CVL), and p24 antigen production after ex vivo infection of ectocervical tissues with HIV-1BaL among PRE women (n = 20) in the follicular and luteal phases of the menstrual cycle and POST women (n = 17) at baseline and after ∼1 month of treatment with 0.01% vaginal E2 cream. Compared to PRE women, we measured higher levels of p24 antigen after ex vivo infection in tissues from POST women. POST women had a significantly thinner vaginal epithelium with decreased tight junction proteins and a higher density of mucosal immune T cells and lower levels of CD1a antigen-presenting cells, antimicrobial peptides, and inflammatory cytokines in the CVL (p values <.05). POST women had higher vaginal pH and lower vaginal Lactobacilli (p values <.05) than PRE women. After vaginal E2 therapy, CV endpoints and ex vivo HIV replication in POST tissues were similar to those observed in PRE tissues. The CV mucosa in POST women is thinned and compromised, with increased HIV-target immune cells and decreased antimicrobial factors, being more susceptible to HIV infection. After POST women receive topical E2 treatment, mucosal endpoints are similar to PRE levels.

Blocking CXCL9 Decreases HIV-1 Replication and Enhances the Activity of Prophylactic Antiretrovirals in Human Cervical Tissues.

OBJECTIVES: The interferon-gamma-induced chemokine CXCL9 is expressed in a wide range of inflammatory conditions including those affecting the female genital tract. CXCL9 promotes immune cell recruitment, activation, and proliferation. The role of CXCL9 in modulating HIV-1 infection of cervicovaginal tissues, a main portal of viral entry, however, has not been established. We report a link between CXCL9 and HIV-1 replication in human cervical tissues and propose CXCL9 as a potential target to enhance the anti-HIV-1 activity of prophylactic antiretrovirals. DESIGN: Using ex vivo infection of human cervical tissues as a model of mucosal HIV-1 acquisition, we described the effect of CXCL9 neutralization on HIV-1 gene expression and mucosal CD4 T-cell activation. The anti-HIV-1 activity of tenofovir, the leading mucosal pre-exposure prophylactic microbicide, alone or in combination with CXCL9 neutralization was also studied. METHODS: HIV-1 replication was evaluated by p24 ELISA. HIV-1 DNA and RNA, and CD4, CCR5, and CD38 transcription were evaluated by quantitative real-time polymerase chain reaction. Frequency of activated cervical CD4 T cells was quantified using fluorescence-activated cell sorting. RESULTS: Antibody blocking of CXCL9 reduced HIV-1 replication by decreasing mucosal CD4 T-cell activation. CXCL9 neutralization in combination with suboptimal concentrations of tenofovir, possibly present in the cervicovaginal tissues of women using the drug inconsistently, demonstrated an earlier and greater decrease in HIV-1 replication compared with tissues treated with tenofovir alone. CONCLUSIONS: CXCL9 neutralization reduces HIV-1 replication and may be an effective target to enhance the efficacy of prophylactic antiretrovirals.

Comparison of Follicular and Luteal Phase Mucosal Markers of HIV Susceptibility in Healthy Women.

The purpose of this study was to evaluate differences in vaginal immune cell populations, vaginal tissue gene expression, antimicrobial activity of the cervicovaginal (CV) lavage (CVL), vaginal flora, and p24 antigen production from CV tissues after ex vivo human immunodeficiency virus (HIV) infection between follicular (FOL) and luteal (LUT) phases of the menstrual cycle. CV tissue biopsies, CV secretions, and blood samples were obtained as part of two longitudinal clinical trials of healthy women (CONRAD D11-119 and A12-124 studies). Participants (n = 39) were HIV-seronegative women not using exogenous hormone supplementation, with normal menstrual cycles, who were screened to exclude sexually transmitted and reproductive tract infections. Serum levels of estradiol and progesterone were significantly higher in the LUT versus the FOL phase of the menstrual cycle. Controlling for race, reported contraceptive use/sexual practices, and clinical trial, we found no differences in vaginal tissue immune cell populations and activation status, transcriptomes, inhibition of HIV, herpes simplex virus type 2 and Escherichia coli by the CVL, vaginal pH or Nugent score, or production of p24 antigen after ex vivo infection by HIV-1BaL between CV samples obtained in the FOL phase versus the LUT phase of the menstrual cycle. There were no significant correlations between serum estradiol and progesterone levels and CV endpoints. The hypothesis that the LUT phase of the menstrual cycle represents a more vulnerable stage for mucosal infection with HIV was not supported by data from samples obtained from the lower genital tract (ectocervix and vagina) from these two clinical trials.

Enhancing Interferon Regulatory Factor 7 Mediated Antiviral Responses and Decreasing Nuclear Factor Kappa B Expression Limit HIV-1 Replication in Cervical Tissues.

Establishment of a productive HIV-1 infection in the female reproductive tract likely depends on the balance between anti-viral and pro-inflammatory responses leading to activation and proliferation of HIV target cells. Immune modulators that boost anti-viral and depress pro-inflammatory immune responses may decrease HIV-1 infection or replication. Polyinosinic:polycytidylic [Poly (I:C)] has been reported to down-regulate HIV-1 replication in immune cell subsets and lymphoid tissues, yet the scope and mechanisms of poly (I:C) regulation of HIV-1 replication in the cervicovaginal mucosa, the main portal of viral entry in women remain unknown. Using a relevant, underexplored ex vivo cervical tissue model, we demonstrated that poly (I:C) enhanced Interferon Regulatory Factor (IRF)7 mediated antiviral responses and decreased tissue Nuclear Factor Kappa B (NFκB) RNA expression. This pattern of cellular transcription factor expression correlated with decreased HIV-1 transcription and viral release. Reducing IRF7 expression up-regulated HIV-1 and NFκB transcription, providing proof of concept for the critical involvement of IRF7 in cervical tissues. By combining poly (I:C) with a suboptimal concentration of tenofovir, the leading anti-HIV prophylactic microbicide candidate, we demonstrated an earlier and greater decrease in HIV replication in poly (I:C)/tenofovir treated tissues compared with tissues treated with tenofovir alone, indicating overall improved efficacy. Poly (I:C) decreases HIV-1 replication by stimulating IRF7 mediated antiviral responses while reducing NFκB expression. Early during the infection, poly (I:C) improved the anti-HIV-1 activity of suboptimal concentrations of tenofovir likely to be present during periods of poor adherence i.e. inconsistent or inadequate drug use. Understanding interactions between anti-viral and pro-inflammatory immune responses in the genital mucosa will provide crucial insights for the identification of targets that can be harnessed to develop preventative combination strategies to improve the efficacy of topical or systemic antiviral prophylactic agents and protect women from HIV-1 and other sexually transmitted infections.

Human oral keratinocytes: a model system to analyze host-pathogen interactions.

Host-pathogen interactions are complex and dynamic processes that result in a variety of responses. The ability of the host to respond appropriately to the presence of a microbial agent defines the outcome of these interactions. Fungal infections are a problem of growing clinical importance and are responsible for serious health problems in multimorbid patients. Different model systems, including primary cells and cell lines derived from different tissues, are used to study several processes that contribute to the virulence of pathogenic fungi. In this chapter, we describe an in vitro assay to characterize the response of human oral keratinocytes (OKF6/TERT-2) to the presence of the human pathogenic fungus, Candida albicans. The dynamic cellular changes such as expression of differentiation markers can be monitored by epifluorescence deconvolution microscopy. Analyses of immunofluorescence data by linescan analysis and fluorescence intensity measurements are described to identify changes in protein expression levels. The use of this in vitro model system will also provide new information about host cell behavior and identify potential drug targets in the future.

IL-8 decreases HIV-1 transcription in peripheral blood lymphocytes and ectocervical tissue explants.

IL-8 is enhanced in the peripheral blood and lymphoid tissue of HIV-infected individuals, suggesting that IL-8 is important in the pathogenesis of HIV-1 infection and progression to AIDS. Characterizing the mechanisms of IL-8 regulation of HIV-1 replication may be relevant in addressing the role of IL-8 as a therapeutic target in HIV-1 infection. We evaluated replication of primary R5-tropic HIV-1 in peripheral blood lymphocytes and ectocervical tissue explants infected in vitro in the presence of physiological concentrations of IL-8 found in the serum and genital tract secretions of HIV-infected individuals. To identify the specific stages of the viral life cycle targeted by IL-8, we performed real-time polymerase chain reaction to detect HIV-1 reverse transcription, integration, and transcription. Early during the infection, IL-8 decreased HIV-1 reverse transcription and viral integration. This effect was transient, as on day 4 after infection, we detected no differences on HIV-1 DNA or proviral DNA in peripheral blood lymphocyte. IL-8 decreased HIV-1 transcription in both lymphocytes and ectocervical tissue explants. The decrease in viral RNA expression was associated with reduced HIV-1 replication, as measured by viral p24 release in the culture supernatants. This is the first report to suggest that IL-8 decreases replication of primary R5-tropic HIV-1 by transcriptional mechanisms.

HIV type 1 infection in women: increased transcription of HIV type 1 in ectocervical tissue explants.

BACKGROUND: Mucosal surfaces of the female reproductive tract are the main routes of heterosexual transmission of human immunodeficiency virus type 1 (HIV-1), but the contribution of each of the reproductive sites to mucosal transmission is unknown. METHODS: We compared levels of HIV-1 transcription between ectocervical and endometrial tissue explants infected ex vivo with HIV-1. RESULTS: We detected higher levels of HIV-1 transcription in the ectocervix. Although CD45 expression was also increased at this site, higher levels of HIV-1 transcription could not be accounted for exclusively by differences in CD45 expression. This suggests that factors other than CD45 levels regulate HIV-1 transcription within the ectocervix. We detected higher levels of interleukin (IL)-6 at this site. Furthermore, addition of recombinant IL-6 to tissue explants enhanced HIV-1 transcription to a much greater degree in the ectocervix than in the endometrium. CONCLUSIONS: This is, to our knowledge, the first study to compare ectocervix and endometrium in a tissue explant model of HIV-1 infection and to demonstrate greater HIV-1 transcription in the ectocervix. Our results suggest that the ectocervix is more conducive to HIV-1 replication than is the endometrium and that IL-6 enhances HIV-1 transcription at this site. Thus, the ectocervix is an important site to be considered in heterosexual transmission of HIV-1.

Estradiol and progesterone regulate HIV type 1 replication in peripheral blood cells.

Endogenous levels of estradiol and progesterone fluctuate in the peripheral blood of premenopausal women during the reproductive cycle. We studied the effects of these sex hormones on HIV-1 replication in peripheral blood mononuclear cells (PBMCs). We compared HIV-1 replication in PBMCs infected in the presence of mid-secretory (high concentrations) and mid-proliferative (low concentrations) or in the absence of sex hormones. With PBMCs from men, we used concentrations of estradiol and progesterone that are normally present in their plasma. Our findings demonstrate that mid-proliferative phase conditions increased, and mid-secretory phase conditions decreased, HIV-1 replication. To determine if sex hormones affect specific stages of the viral life cycle we performed real-time PCR assays and found decreased levels of HIV-1 integration in the mid-secretory phase and increased levels viral transcription in the mid-proliferative phase. No significant effects on HIV-1 reverse transcription or on CCR5 expression were found. In addition, we assessed hormonal regulation of the HIV-1 LTR in the absence of the viral regulatory protein Tat. We observed that mid-proliferative hormone levels enhanced, whereas mid-secretory hormone concentrations reduced, the activity of the LTR. These findings demonstrate that in HIV-1-infected cells, estradiol and progesterone regulate HIV-1 replication most likely by directly altering HIV-1 transcriptional activation. An additional indirect mechanism of sex hormone regulation of cytokine and chemokine secretion cannot be excluded.