Prevention of sexual acquisition of HIV in women requires a substantial increase in our knowledge about HIV-target cell availability and regulation in the female reproductive tract (FRT). In this study, we analyzed the phenotype and susceptibility to HIV infection of CD4(+) Tcell in the endometrium (EM), endocervix (END), and ectocervix (ECT) of the FRT. We found that T helper type 17 (Th17) cells represent a major subset in FRT tissues analyzed and that Th17 cells were the main CD4(+) T-cell population expressing C-C motif chemokine receptor 5 (CCR5) and CD90. In premenopausal women, CD4(+) Tcells and Th17 cells, in particular, were significantly lower in EM relative to END and ECT. Th17 cells were elevated in EM from postmenopausal women relative to premenopausal tissues but not changed in END and ECT. Susceptibility of CD4(+) Tcells to HIV infection measured as intracellular p24 was lowest in the EM and highest in the ECT. Additionally, we found that Th17 cells co-expressing CCR5 and CD90 were the most susceptible to HIV infection. Our results provide valuable information for designing preventive strategies directed at targeting highly susceptible target cells in the FRT.

A steam generator (SG) tube rupture (SGTR) model is studied in this paper. This model based on a experimental facility called Aerosol Trapping In a Steam Generator (ARTIST), which is a model of a scaled steam generator tube bundle consisting of 270 tubes and a guillotine tube to address aerosol deposition phenomena on two different scales: near the tube break, where the gas velocities and turbulence are very intensive, and far away from the break, where the flow velocities are three orders of magnitude lower.Owing to complexity of the flow, 3D simulations with highly resolved computational mesh near the break were done. First, the flow inside an isolated tube with a guillotine tube break has been studied in the framework of Reynolds Averaged Navier Stokes (RANS) approach. The next part is devoted to the simulation of an inclined gas jet entering the SG tube bundle via the guillotine tube breach with more advanced CFD tools. In particular, Detached Eddy Simulation (DES) and RANS are applied to tackle the wide range of flow scales. Flow field velocity comparison showed that DES results are reproducing wavy structure of the flow field in far field from the break observed in experiment.Particle transport and deposition is modelled by Lagrangian continuous random walk (CRW) model, which has been developed and validated previously. It is found that the DES combined with the CRW to supply fluctuating velocity components predicts deposition rates that are generally within the scatter of the measured data. Monodisperse, spherical SiO2 particles with AMMD = 1.4 μm were used as aerosol particles in simulations.To be economically feasible, the computations were made with the open source CFD code OpenFOAM. Comparison of the calculated flow with the experimental axial velocity distribution data at different vertical levels has been performed.

This review examines the multiple levels of pre-existing immunity in the upper and lower female reproductive tract. In addition, we highlight the need for further research of innate and adaptive immune protection of mucosal surfaces in the female reproductive tract. Innate mechanisms include the mucus lining, a tight epithelial barrier and the secretion of antimicrobial peptides and cytokines by epithelial and innate immune cells. Stimulation of the innate immune system also serves to bridge the adaptive arm resulting in the generation of pathogen-specific humoral and cell-mediated immunity. Less understood are the multiple components that act in a coordinated way to provide a network of ongoing protection. Innate and adaptive immunity in the human female reproductive tract are influenced by the stage of menstrual cycle and are directly regulated by the sex steroid hormones, progesterone and estradiol. Furthermore, the effect of hormones on immunity is mediated both directly on immune and epithelial cells and indirectly by stimulating growth factor secretion from stromal cells. The goal of this review is to focus on the diverse aspects of the innate and adaptive immune systems that contribute to a unique network of protection throughout the female reproductive tract.

Female reproductive tract (FRT) epithelial cells protect against potential pathogens and sexually transmitted infections. The purpose of this study was to determine if epithelial cells from the upper FRT secrete antimicrobials that inhibit reproductive tract pathogens that threaten women's health. Apical secretions from primary cultures of Fallopian tube, uterine, cervical, and ectocervical epithelial cells were incubated with Neisseria gonorrhoeae, Candida albicans (yeast and hyphal forms), human immunodeficiency virus 1 (HIV-1), and Lactobacillus crispatus before being tested for their ability to grow and/or infect target cells. Epithelial cell secretions from the upper FRT inhibit N. gonorrhoeae and both forms of Candida, as well as reduce HIV-1 (R5) infection of target cells. In contrast, none had an inhibitory effect on L. crispatus. An analysis of cytokines and chemokines in uterine secretions revealed several molecules that could account for pathogen inhibition. These findings provide definitive evidence for the critical role of epithelial cells in protecting the FRT from infections, without comprising the beneficial presence of L. crispatus, which is part of the normal vaginal microflora of humans.

Pattern recognition receptors (PRRs) play an important role in host anti-donor responses to transplanted tissue. A key trigger of the host alloresponse involves recognition of foreign antigen presented on activated antigen presenting cells by the host T cells. Emerging data suggest that PRR blockade can abrogate host anti-donor responses by interfering with activation of antigen presenting cells, particularly activation of dendritic cells. Our study asked whether blockade of a well-characterized family of intracellular PRRs, the NOD family, interfered with alloantigen recognition and allograft rejection. We found that deletion of either NOD1 or NOD2 in antigen presenting cells (APCs) had no effect on induction of T cell proliferation to alloantigen, but that simultaneous deletion of NOD1 and NOD2 significantly inhibited T cell responses. There was however no effect of the NOD deletion on skin graft rejection when NOD1 × NOD2 skin was transplanted onto allogeneic hosts or when WT skin was transplanted onto NOD1 × NOD2 deficient recipients. The conclusion of this study is that in vitro alloresponses are negatively impacted by the simultaneous deletion of NOD1 and NOD2, but that allograft rejection across a stringent allo barrier is not affected. Our results suggest that the NOD family members, NOD1 and NOD2, play a collaborative role in T cell activation by alloantigen and that their blockade in vitro can inhibit T cell responses.

Infection of mesangial cells with HIV and SIV: Identification of GPR1 as a coreceptor.Mesangial cells are an important component of the glomerulus. Dysfunction of mesangial cells is thought to be involved in the development of human immunodeficiency virus type 1 (HIV-1)-associated nephropathy (HIVAN). HIVAN is a structural renal failure frequently observed in patients with acquired immune deficiency syndrome. However, the susceptibility of mesangial cells to HIV-1 is disputable. More than ten G protein-coupled receptors, including chemokine receptors, have been shown to act as HIV-1 coreceptors that determine the susceptibilities of cells to HIV-1 strains with specific cell tropisms.We examined the susceptibility of mesangial cells to various HIV-1, HIV type 2 (HIV-2) and simian immunodeficiency virus (SIV) strains. Expression of CD4 and HIV/SIV coreceptors was examined by Western blotting and polymerase chain reaction.Mesangial cells were found to be susceptible to HIV-1 variant and mutants that infect brain-derived cells, but highly resistant to T-tropic (X4), M-tropic (R5) or dual-tropic (X4R5) HIV-1 strains. In addition, mesangial cells were also susceptible to HIV-2 and SIV strains that infect the brain-derived cells. Among HIV/SIV coreceptors we tested, the expression of GPR1 mRNA was detected in mesangial cells. Expression of CD4 mRNA and protein was also detected in them. Mesangial cells and GPR1-transduced CD4-positive cells showed similar susceptibilities to the HIV-1 variant and mutants and HIV-2 and SIV strains.CD4 and GPR1 mRNAs were detected in mesangial cells. Mesangial cells were susceptible to HIV/SIV strains that use GPR1 as a coreceptor. Our findings suggest that an orphan G protein-coupled receptor, GPR1, is a coreceptor expressed in mesangial cells. It remains to be investigated whether the interaction of mesangial cells with specific HIV-1 strains through GPR1 plays a role in the development of HIVAN.

New rationally designed i,i+7-hydrocarbon-stapled peptides that target both HIV-1 assembly and entry have been shown to have antiviral activity against HIV-1 subtypes circulating in Europe and North America. Here, we aimed to evaluate the antiviral activity of these peptides against HIV-1 subtypes predominantly circulating in China.The antiviral activity of three i,i+7-hydrocarbon-stapled peptides, NYAD-36, NYAD-67, and NYAD-66, against primary HIV-1 CRF07_BC and CRF01_AE isolates was evaluated in peripheral blood mononuclear cells (PBMCs). The activity against the CRF07_BC and CRF01_AE Env-pseudotyped viruses was analyzed in TZM-bl cells.We found that all the stapled peptides were effective in inhibiting infection by all the primary HIV-1 isolates tested, with 50% inhibitory concentration toward viral replication (IC50) in the low micromolar range. NYAD-36 and NYAD-67 showed better antiviral activity than NYAD-66 did. We further evaluated the sensitivity of CRF01_AE and CRF07_BC Env-pseudotyped viruses to these stapled peptides in a single-cycle virus infectivity assay. As observed with the primary isolates, the IC50s were in the low micromolar range, and NYAD-66 was less effective than NYAD-36 and NYAD-67.Hydrocarbon-stapled peptides appear to have broad antiviral activity against the predominant HIV-1 viruses in China. This finding may provide the impetus to the rational design of peptides for future antiviral therapy.

The goal of this study was to examine the role of E 2 in regulating innate immune protection by human uterine epithelial cells (UECs). Recognizing that UECs produce cytokines and chemokines to recruit and activate immune cells as well as viral and bacterial antimicrobials, we sought to examine the effect of E 2 on constitutive and Toll-like receptor (TLR) agonist (lipopolysaccharide (LPS) and poly (I:C))-induced immune responses. The secretion by polarized UECs in culture of interleukin (IL)-6, macrophage inhibitory factor (MIF), and secretory leukocyte protease inhibitor (SLPI) was examined as well as the mRNA expression of human β-defensin-2 (HBD2), tumor necrosis factor (TNF)-α, IL-8, and nuclear factor (NF)-kB. When incubated with E 2 for 24–48 h, we found that E 2 stimulated UEC secretion of SLPI (fourfold) and mRNA expression of HBD2 (fivefold). Moreover, when antibacterial activity in UEC secretions was measured using Staphylococcus aureus , E 2 increased the secretion of soluble factor(s) with antibacterial activity. In contrast, E 2 had no effect on constitutive secretion of proinflammatory cytokines and chemokines by UECs but completely inhibited LPS- and poly (I:C)-induced secretion of MIF, IL-6, and IL-8. Estradiol also reversed the stimulatory effects of IL-1β on mRNA expression of TNF-α, IL-8, and NF-kB by 85, 95, and 70%, respectively. As SLPI is known to inhibit NF-kB expression, these findings suggest that E 2 inhibition of proinflammatory cytokines may be mediated through SLPI regulation of NF-kB. Overall, these findings indicate that the production of cytokines, chemokines, and antimicrobials by UECs are differentially regulated by E 2 . Further, it suggests that with E 2 regulation, epithelial cells that line the uterine cavity have evolved immunologically to be sensitive to viral and bacterial infections as well as the constraints of procreation.

PROBLEM:Epithelial cells, as sentinels of immune protection in the endometrium, use innate immune mechanisms to protect against infection from pathogenic microbes. Our goal in this study was to assess the ability of human uterine epithelial cells to present antigen to cells of the adaptive immune system.METHOD OF STUDY:Highly purified preparations of uterine epithelial cells from 11 patients were assessed for their ability to present tetanus toxoid (TT) to autologous T cells. Leukocyte contamination in the epithelial cell preparations was numerically and functionally determined. Using confocal microscopy, epithelial cells were tested for the expression of CD40 and CD1d.RESULTS:Purified preparations of endometrial epithelial cells isolated from every patient presented TT recall antigen to autologous T cells. Leukocyte contamination of epithelial cell preparations was insignificant. Uterine epithelial cells express CD40 and CD1d.CONCLUSION:Antigen presentation is an additional aspect of uterine epithelial cell function in maintaining women's health.

The mucosal immune system in the female reproductive tract (FRT) has evolved to meet the unique requirements of dealing with sexually transmitted bacterial and viral pathogens, allogeneic spermatozoa, and the immunologically distinct fetus. Analysis of the FRT indicates that the key cells of the innate and adaptive immune systems are present and functionally responsive to antigens. Acting through Toll-like receptors in the Fallopian tubes, uterus, cervix, and in the vagina, epithelial cells, macrophages, natural killer cells, and neutrophils confer protection through the production of chemokines and cytokines, which recruit and activate immune cells, as well as bactericidal and virucidal agents, which confer protection at times when adaptive immunity is downregulated by sex hormones to meet the constraints of procreation. The overall goal of this paper is to define the innate immune system in the FRT and, where possible, to define the regulatory influences that occur during the menstrual cycle that contribute to protection from and susceptibility to potential pathogens. By understanding the nature of this protection and the ways in which innate and adaptive immunity interact, these studies provide the opportunity to contribute to the foundation of information essential for ensuring reproductive health.

PROBLEM:Uterine epithelial cells produce cytokines that stimulate leukocytes in response to a microbial insult. The goals of this study were to determine if uterine epithelial cells produce the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), and to see if toll-like receptor (TLR) agonists stimulate MIF secretion.METHODS OF STUDY:Human uterine epithelial cells were isolated and grown in cell culture inserts. Levels of MIF secretion were examined by ELISA and MIF messenger RNA (mRNA) expression was examined using real time RT-PCR.RESULTS:Uterine epithelial cells constitutively secrete MIF and exposure to the TLR3 agonist poly (I:C) resulted in enhanced apical secretion of MIF. MIF secretion appeared to be from pre-formed intracellular stores, since exposure of epithelial cells to poly (I:C) had little effect on the expression of MIF-mRNA.CONCLUSIONS:These results demonstrate that uterine epithelial cells constitutively produce MIF and stimulation with poly (I:C) results in enhanced MIF production. This suggests that MIF secretion by uterine epithelial cells may play a critical role in innate immune responses against viral pathogens mediated through TLR3.

The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist, poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human beta-defensin-1 and -2 (HBD1 and HBD2), IFN-beta, and the IFN-beta-stimulated genes myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase. The expression of TLR1-9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-alpha, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1beta. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-beta mRNA expression 70-fold and myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelia cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express beta-defensins, IFN-beta, and IFN-beta-stimulated genes that can have a direct inhibiting effect on viral replication.

In mature female rats, sex hormones regulate the reproductive (estrous) cycle to optimize mating and fertility. During the part of the estrous cycle when mating occurs, and when estrogen is the dominant sex hormone, the uterus is susceptible to infection with bacteria that can be deleterious for survival and fertility. The present study investigated whether sex hormones regulate innate immunity in the female reproductive tract by affecting the secretion of an anti-bacterial factor(s) in the rat uterus. Uterine fluids from intact rats at the proestrous stage of the estrous cycle significantly inhibited Staphylococcus aureus growth. When ovariectomized rats were treated with estradiol, anti-bacterial activity against both S. aureus and Escherichia coli increased in uterine secretions with hormone treatment. In contrast, rats injected with either progesterone and estradiol or progesterone alone displayed no bactericidal activity indicating that progesterone reversed the stimulatory effect of estradiol on anti-bacterial activity. In other studies, isolated uterine epithelial cells from intact animals were grown to confluence and high transepithelial resistance on cell inserts. Analysis of apical secretions indicated that a soluble factor(s) is released by polarized epithelial cells which inhibits bacterial growth. These results demonstrate that sex hormones influence the presence of a broad-spectrum bactericidal factor(s) in luminal secretions of the rat uterus. Further these studies suggest that epithelial cells which line the uterine lumen are a primary source of anti-bacterial activity.

BACKGROUND:Pro-inflammatory chemokines that attract and cytokines that activate immune cells contribute to normal physiological homeostasis in the female reproductive tract, and are needed to deal effectively with potential pathogenic microbes. Mucosal epithelial cells are capable of producing these factors that communicate with cells of the innate and adaptive immune systems.METHODS:Epithelial cells from Fallopian tube, endometrium and endocervix were isolated and grown to high transepithelial resistance in cell inserts from seven patients who had hysterectomies. Interleukin (IL)-8, IL-6, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha) and macrophage inflammatory peptide-1beta (MIP-1beta) were assessed by Luminex bead analysis or enzyme-linked immunosorbent assay (ELISA) in epithelial cell conditioned media from the apical and basolateral compartments.RESULTS:With the exception of MCP-1, the seven chemokines/cytokines constitutively produced by the polarized epithelial cells were preferentially secreted apically. A concentration pattern was found in all cases, with IL-8 and IL-6 produced in the greatest quantity.CONCLUSIONS:The concentrations of IL-8, IL-6, G-CSF and MCP-1 are similar to the levels found in reproductive tract fluids of patients with infection. The constitutive secretion and compartmentalization of large quantities of bioactive chemokines and cytokines provide additional evidence for the role of epithelial cells as gatekeepers of innate immune protection in the female reproductive tract.

OBJECTIVE:To determine whether primary human uterine epithelial cells in culture are able to influence monocyte chemotaxis and to establish whether the causal agent of chemotaxis is monocyte chemotactic protein (MCP)-1. DESIGN:Tissue culture study. SETTING:University medical center. PATIENT(S):Women aged 23 to 53 years who were undergoing hysterectomy (n=7). INTERVENTION(S):Primary human endometrial epithelial cells were acquired from surgical specimens and grown to confluence and high transepithelial resistance. Conditioned media from epithelial cultures were analyzed for the presence of MCP-1 and for capacity to affect monocyte chemotaxis using the THP-1 monocyte line. Antibody neutralization of conditioned media was used to establish the role of MCP-1 in chemotaxis. MAIN OUTCOME MEASURE(S):Assay of conditioned media for MCP-1, quantitative measurement of monocyte chemotaxis to conditioned media, and inhibition of chemotaxis by antibody neutralization of MCP-1. RESULT(S):Primary endometrial epithelial cells in monolayer culture secrete MCP-1 to both the apical and basolateral compartments. Monocyte chemotactic protein-1 was identified as the primary agent of monocyte chemotaxis by antibody neutralization. CONCLUSION(S):These findings suggest that biologically active MCP-1 is secreted into both the uterine lumen as well as the underlying stroma and that it mediates the presence of monocytes, macrophages, and other immune cells in the uterine endometrium.

Neutrophils occur in tissues of the female reproductive tract (FRT) under non-infected conditions. These cells generally enter tissues under the influence of chemoattractants called chemokines. Primary epithelial cells (EC) from FRT were a potent source of chemokines, IL-8 being the chief neutrophil chemoattractant secreted. Blocking with neutralizing anti-IL-8 showed that IL-8 did not account for all of the chemoattraction observed. A mixture of 25ng/mL rIL-8 and 1ng/mL rGM-CSF mediated 2.7-fold more chemotaxis than that expected if the two agents were additive. We then found that GM–CSF was produced by EC in amounts that synergised strongly with IL-8 to enhance chemotaxis. Treatment of uterine EC conditioned medium with saturating doses of anti-IL-8 plus anti-GM–CSF antibodies produced an 84% inhibition of chemotaxis, These findings demonstrate that the majority of neutrophil chemoattractant activity produced by FRT EC results from the synergistic effects of IL-8 and GM–CSF.

Uterine epithelial cells express toll-like receptors (TLR) that mediate innate immune responses. A critical regulator of innate immune responses is MIF. This study was performed to measure TLR mRNA expression by the uterine epithelial cell lines ECC-1 and RL95-2 and the effect of TLR agonists on MIF secretion by uterine epithelial cells. These studies demonstrate that ECC-1 and RL95-2 uterine epithelial cells express TLR3 and TLR5 mRNA, while only ECC-1 cells express TLR2 mRNA. Furthermore, only the TLR3 agonist poly (I:C) induced the secretion of MIF by ECC-1 and RL95-2 cells. These results indicate that epithelial cells lining the uterine cavity are sensitive to viral dsRNA and may be crucial to defending the female reproductive tract against viral pathogens.