Joseph C. Chen

Trachoma and LGV biovars of Chlamydia trachomatis share the same glycosaminoglycan‐dependent mechanism for infection of eukaryotic cells

A sulphated glycosaminoglycan‐dependent mechanism of microbial infection for mammailan cells was characterized for the Chlamydia trachomatis trachoma and lymphogranuloma venereum (LGV) biovars. We demonstrated that the trachoma and LGV biovars compete for the same receptor(s) on host cells and that their infectivity was inhibited by heparin or heparan sulphate. Using a specific heparan suiphate lyase (heparitinase) to treat organisms, the Infectivity of both biovars was abolished. Furthermore, exogenous heparan sulphate rescued chlamydial infectivity following treatment with heparitinase and the restored infectivity was neutralized by an anti‐heparan sulphate monoclonal antibody. These data suggest that heparan sulphate‐like‐mediated Interactions between C. trachomatis and eukaryotic cells are essential for infectivity.

Relaxin (RLX) and estrogen affect estrogen receptor α, vascular endothelial growth factor, and RLX receptor expression in the neonatal porcine uterus and cervix

The porcine female reproductive tract undergoes estrogen receptor (ER) alpha-dependent development after birth (postnatal day=PND 0), the course of which can determine adult uterine function. Uterotrophic effects of relaxin (RLX) in the porcine neonate are age specific and may involve ER activation. Here, objectives were to determine effects of RLX and estrogen administered from birth on uterine and cervical growth and expression of ERalpha, vascular endothelial growth factor (VEGF), and the RLX receptor (RXFP1). On PND 0, gilts were treated with the antiestrogen ICI 182 780 (ICI) or vehicle alone and, 2 h later, were given estradiol-17beta (E) or porcine RLX for 2 days. Neither RLX nor E affected uterine wet weight or protein content on PND 2. However, RLX, but not E, increased cervical wet weight and protein content when compared with controls. Pretreatment with ICI did not inhibit RLX-stimulated cervical growth. Uterine and cervical ERalpha increased in response to RLX, but not E. Both RLX and E increased VEGF in the uterus and cervix on PND 2. Pretreatment with ICI increased VEGF in both tissues and increased RLX-induced cervical VEGF. In the uterus E, but not RLX, increased RXFP1 mRNA. In the cervix, E increased RXFP1 gene expression whereas RLX decreased it. Results indicate that the neonatal uterus and cervix are sensitive to E and RLX and that growth responses to RLX in these tissues differ by PND 2. Effects of RLX on uterine and cervical ERalpha and VEGF expression may be important for neonatal reproductive tract development.

Human Endometrial Fibroblasts Derived from Mesenchymal Progenitors Inherit Progesterone Resistance and Acquire an Inflammatory Phenotype in the Endometrial Niche in Endometriosis

ABSTRACT Human endometrium undergoes cyclic regeneration involving stem/progenitor cells, but the role of resident endometrial mesenchymal stem cells (eMSC) as progenitors of endometrial stromal fibroblasts (eSF) has not been definitively demonstrated. In endometriosis, eSF display progesterone (P4) resistance with impaired decidualization in vivo and in vitro. To investigate eMSC as precursors of eSF and whether endometriosis P4 resistance is inherited from eMSC, we analyzed transcriptomes of eutopic endometrium eMSC and eSF isolated by fluorescence-activated cell sorting (FACS) from endometriosis (eMSCendo, eSFendo) and controls (eMSCcontrol, eSFcontrol) and their derived primary cultures. Differentially expressed lineage-associated genes (LG) of FACS-isolated eMSC and eSF were largely conserved in endometriosis. In culture, eSFcontrol maintained in vitro expression of a subset of eSF LG and decidualized in vitro with P4. The eMSCcontrol cultures differentiated in vitro to eSF lineage, down-regulating eMSC LG and up-regulating eSF LG, showing minimal transcriptome differences versus eSFcontrol cultures and decidualizing in vitro. Cultured eSFendo displayed less in vitro LG stability and did not decidualize in vitro. In vitro, eMSCendo differentiated to eSF lineage but showed more differentially expressed genes versus eSFendo cultures, and did not decidualize in vitro, demonstrating P4 resistance inherited from eMSCendo. Compared to controls, cultures from tissue-derived eSFendo uniquely had a pro-inflammatory phenotype not present in eMSCendo differentiated to eSF in vitro, suggesting divergent niche effects for in vivo versus in vitro lineage differentiation. These findings substantiate eMSC as progenitors of eSF and reveal eSF in endometriosis as having P4 resistance inherited from eMSC and a pro-inflammatory phenotype acquired within the endometrial niche.

Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts

STUDY QUESTIONnHow does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)?nnnSUMMARY ANSWERnExposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death.nnnWHAT IS KNOWN ALREADYnStudies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductive tract (FRT), which may participate in promoting reproductive success.nnnSTUDY DESIGN, SIZE, DURATIONnThis is a cross sectional study involving control samples versus treatment. SP (pooled from twenty donors) was first tested for dose- and time-dependent cytotoxic effects on eEC and eSF (n = 4). As exposure of eEC or eSF to 1% SP for 6 h proved to be non-toxic, a second set of eEC/eSF samples (n = 4) was treated under these conditions for transcriptome, protein and functional analysis. With a third set of samples (n = 3), we further compared the transcriptional response of the cells to SP versus fresh semen.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSneEC and eSF were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. RNA was isolated and processed for microarray studies to analyze global transcriptomic changes. Secreted factors in conditioned media from SP-treated cells were analyzed by Luminex and for the ability to stimulate migration of CD14+ monocytes and CD4+ T cells.nnnMAIN RESULTS AND THE ROLE OF CHANCEnPathway identifications were determined using the Z-scoring system in Ingenuity Pathways Analysis (Z scores ≥|1.5|). SP induced transcriptomic changes (P < 0.05) associated with promoting leukocyte and endothelial cell recruitment, and proliferation of eEC and eSF. Cell viability pathways were induced, while those associated with cell death were suppressed (P < 0.05). SP and fresh semen induced similar sets of pathways, suggesting that SP can model the signaling effects of semen in the endometrium. SP also induced secretion of pro-inflammatory and pro-chemotactic cytokines, as well as pro-angiogenic and proliferative growth factors (P < 0.05) in both eEC and eSF. Finally, functional assays revealed that conditioned media from SP-treated eEC and eSF significantly increased (P < 0.05) chemotaxis of CD14+ monocytes and CD4+ T cells.nnnLIMITATIONS, REASONS FOR CAUTIONnThis study is limited to in vitro analyses of the effects of SP on endometrial cells. In addition, the measured response to SP was conducted in the absence of the ovarian hormones estradiol and progesterone, as well as epithelial-stromal paracrine signaling. While this study focused on establishing the baseline cellular response of endometrial cells to SP, future work should assess how hormone signaling in the presence of appropriate paracrine interactions affects SP-induced genes in these cells.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe results of this study support previous findings that SP and semen contain bioactive factors capable of eliciting chemotactic responses in the uterus, which can lead to recruitment of leukocytes to the endometrium. Future directions will explore if similar changes in gene expression do indeed occur after coitus in vivo, and how the signaling cascades initiated by SP in the endometrium can affect reproductive success, female reproductive health and susceptibility to sexually transmitted diseases. The gene list provided by the transcriptome analysis reported here should prove a valuable resource for understanding the response of the upper FRT to SP exposure.nnnSTUDY FUNDING/COMPETING INTEREST(S)nThis project was supported by NIH AI083050-04 (W.C.G./L.C.G.); NIH U54HD 055764 (L.C.G.); NIH 1F32HD074423-02 (J.C.C.); DOD W81XWH-11-1-0562 (W.C.G.); NIH 5K12-DK083021-04, NIH 1K99AI104262-01A1, The UCSF Hellman Award (N.R.R.). The authors have nothing to disclose.

Coculturing human endometrial epithelial cells and stromal fibroblasts alters cell-specific gene expression and cytokine production

OBJECTIVEnTo determine the effects of coculturing endometrial epithelial cells (eEC) with paired endometrial stromal fibroblasts (eSF) on cell-specific gene expression and cytokine secretion patterns.nnnDESIGNnIn vitro study.nnnSETTINGnUniversity research laboratory.nnnPATIENT(S)nEndometrial biopsies were obtained from premenopausal women.nnnINTERVENTION(S)nPolarized eEC and subject-paired eSF were cultured for 12.5 hours alone (monoculture) or combined in a two-chamber coculture system without cell-cell contact. Cells and conditioned media were analyzed for global gene expression and cytokine secretion, respectively. Purified, endometrial tissue-derived eEC and eSF isolated by fluorescent activated cell sorting (FACS) were used as noncultured controls.nnnMAIN OUTCOME MEASURE(S)nCell-specific global gene expression profiling and analysis of secreted cytokines in eEC/eSF cocultures and respective monocultures.nnnRESULT(S)nTransepithelial resistance, diffusible tracer exclusion, expression of tight junction proteins, and apical/basolateral vectorial secretion confirmed eEC structural and functional polarization. Distinct transcriptomes of eEC and eSF were consistent with their respective lineages and their endometrial origin. Coculture of eEC with eSF resulted in altered cell-specific gene expression and cytokine secretion.nnnCONCLUSION(S)nThis coculture model provides evidence that interactions between endometrial functionally polarized epithelium and stromal fibroblasts affect cell-specific gene expression and cytokine secretion underscoring their relevance when modeling endometrium in vitro.

Progestin-Containing Contraceptives Alter Expression of Host Defense-Related Genes of the Endometrium and Cervix

Epidemiological studies indicate that progestin-containing contraceptives increase susceptibility to HIV, although the underlying mechanisms involving the upper female reproductive tract are undefined. To determine the effects of depot medroxyprogesterone acetate (DMPA) and the levonorgestrel intrauterine system (LNG-IUS) on gene expression and physiology of human endometrial and cervical transformation zone (TZ), microarray analyses were performed on whole tissue biopsies. In endometrium, activated pathways included leukocyte chemotaxis, attachment, and inflammation in DMPA and LNG-IUS users, and individual genes included pattern recognition receptors, complement components, and other immune mediators. In cervical TZ, progestin treatment altered expression of tissue remodeling and viability but not immune function genes. Together, these results indicate that progestins influence expression of immune-related genes in endometrium relevant to local recruitment of HIV target cells with potential to increase susceptibility and underscore the importance of the upper reproductive tract when assessing the safety of contraceptive products.

Unexpected Inflammatory Effects of Intravaginal Gels (Universal Placebo Gel and Nonoxynol-9) on the Upper Female Reproductive Tract: A Randomized Crossover Study

Intravaginal anti-HIV microbicides could provide women with a self-controlled means for HIV prevention, but results from clinical trials have been largely disappointing. We postulated that unrecognized effects of intravaginal gels on the upper female reproductive tract might contribute to the lower-than-expected efficacy of HIV microbicides. Our objective was to study the effects of intravaginal gels on the immune microenvironment of the cervix and uterus. In this randomized crossover study, 27 healthy female volunteers used a nightly application of intravaginal nonoxynol-9 (N9) gel as a “failed” microbicide or the universal placebo gel (UPG) as a “safe” gel (intervention cycles), or nothing (control cycle) from the end of menses to the mid-luteal phase. At a specific time-point following ovulation, all participants underwent sample collection for measurements of T-cell phenotypes, gene expression, and cytokine/chemokine protein concentrations from 3 anatomic sites above the vagina: the cervical transformation zone, the endocervix and the endometrium. We used hierarchical statistical models to estimate mean (95% CI) intervention effects, for N9 and UPG relative to control. Exposure to N9 gel and UPG generated a common “harm signal” that included transcriptional up-regulation of inflammatory genes chemokine (C-C motif) ligand 20 (macrophage inflammatory factor-3alpha) and interleukin 8 in the cervix, decreased protein concentrations of secretory leukocyte protease inhibitor, and transcriptional up-regulation of inflammatory mediators glycodelin-A and osteopontin in the endometrium. These results need to be replicated with a larger sample, but underscore the need to consider the effects of microbicide agents and gel excipients on the upper female reproductive tract in studies of vaginal microbicides.

Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells

Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells–by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV–without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy.

Infection of human uterine fibroblasts by Zika virus in vitro: implications for viral transmission in women

Zika virus (ZIKV) is a single-stranded RNA arbovirus belonging to the Flavivirus genus. Similar to other zoonotic arboviruses, ZIKV is transmitted by the Aedes mosquito. It causes Zika fever, a usually non-fatal condition with symptoms including headache, fever, malaise, maculopapular rash, joint pains, and conjunctivitis. Recently, it was revealed that ZIKV may have additional pathogenic effects on pregnant women and their fetuses. Reports from South America suggest that ZIKV infection during pregnancy causes fetal microcephaly, which has negative impacts up to and including death. In addition to mosquito-borne transmission, it has been reported that ZIKV may be transmitted through heterosexual intercourse, with concomitant implications for global women’s health. This is the first example of a mosquito-borne arbovirus that may also utilize a sexual route of transmission. This knowledge has initiated a massive effort by the translational science community to develop more effective detection methods and treatment strategies. Currently there are no approved vaccines or drugs for preventing or treating ZIKV infection or ZIKV-induced pathologies. Recent diagnostics development efforts have focused on ELISA and highly sensitive quantitative reverse transcription PCR

Stability of gels formed following coagulation of Limulus amebocyte lysate: lack of covalent crosslinking of coagulin.

Incubation of lysates prepared from amebocytes of the horseshoe crab (Limulus polyphemus) with bacterial endotoxin results in coagulation and formation of a solid gel. Although Limulus gels remained solid indefinitely, if undisturbed, they were easily disrupted by mechanical agitation. Chemical solubility studies of gelled lysates demonstrated rapid solubilization of gels in monochloroacetic acid, a property of clots that have not been covalently stabilized; but in contrast demonstrated resistance to solubilization by urea, a property of stabilized clots. Analysis of solubilized proteins by polyacrylamide gel electrophoresis in SDS demonstrated coagulin, the designation for the activated form of coagulogen (the clottable protein) that forms a gel, only in samples derived from clotted lysate that had been previously incubated with monochloroacetic acid, but not in samples following incubation with urea, confirming the results of the chemical solubility studies. Enzymatic assays for transpeptidase (Factor XIII-like) activity in either native or gelled Limulus lysates were negative. Furthermore, analysis for covalently crosslinked peptides in gelled coagulin confirmed the absence of intermolecular gamma-glutamyl-epsilon-lysyl bonds. Therefore, the stable gels formed following coagulation of Limulus lysate by bacterial endotoxin are not covalently crosslinked.