Multiple receptors shape the estrogen response pathway and are critical considerations for the future of in vitro-based risk assessment efforts.

Current in life toxicity testing paradigms are being challenged as the future of risk assessment moves towards more comprehensive mode of action/adverse outcome pathway based approaches. In particular, endocrine disruption screening is now a global activity and key initiatives in the United States focus on the use of high throughput in vitro assays to prioritize compounds for further testing of estrogen, androgen or thyroid disruption. Of these pathways, much of the emphasis to date has been on high-throughput methods for estrogenic activity primarily using ligand binding and trans-activation assays. However, as the knowledge regarding estrogen receptor signaling pathways continues to evolve, it is clear that the assumption of a simple one-receptor pathway underlying current in vitro screening assays is out of date. To develop more accurate models for estrogen-initiated pathways useful for quantitative safety assessments, we must design assays that account for the key signaling processes driving cellular dose response based on up-to-date understanding of the biological network. In this review, we summarize the state of the science for the estrogen receptor signaling network, particularly with regard to proliferative effects, and highlight gaps in current high throughput approaches. From the sum of this literature, we propose a model for the estrogen-signaling pathway that should serve as a starting point for development of new in vitro methods fit for the purpose of predicting dose response for estrogenic chemicals in the human.

Modulating DNA methylation in activated CD8+ T cells inhibits regulatory T cell-induced binding of Foxp3 to the CD8+ T Cell IL-2 promoter.

We have previously demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) activated during the course of feline immunodeficiency virus (FIV) infection suppress CD8(+) CTL function in a TGF-ß-dependent fashion, inhibiting IFN-γ and IL-2 production and inducing G1 cell-cycle arrest. In this article, we describe the molecular events occurring at the IL-2 promoter leading to suppression of IL-2 production. These experiments demonstrate that Foxp3 induced by lentivirus-activated Tregs in the CD8(+) target cells binds to the IL-2 promoter, actively repressing IL-2 transcription. We further demonstrate that the chronic activation of CD8(+) T cells during FIV infection results in chromatin remodeling at the IL-2 promoter, specifically, demethylation of CpG residues. These DNA modifications occur during active transcription and translation of IL-2; however, these changes render the IL-2 promoter permissive to Foxp3-induced transcriptional repression. These data help explain, in part, the seemingly paradoxical observations that CD8(+) T cells displaying an activation phenotype exhibit altered antiviral function. Further, we demonstrate that blocking demethylation of CpG residues at the IL-2 promoter inhibits Foxp3 binding, suggesting a potential mechanism for rescue and/or reactivation of CD8(+) T cells. Using the FIV model for lentiviral persistence, these studies provide a framework for understanding how immune activation combined with Treg-mediated suppression may affect CD8(+) T cell IL-2 transcription, maturation, and antiviral function.

Infection with feline immunodeficiency virus directly activates CD4+ CD25+ T regulatory cells.

Lentivirus infection activates CD4(+) CD25(+) T regulatory (Treg) cells. Activation of Treg cells may be due to direct virus infection or chronic antigenic stimulation. Herein we demonstrate that in vitro feline immunodeficiency virus (FIV) infection, but not UV-inactivated virus, activates Treg cells as measured by immunosuppressive function and upregulation of GARP, FoxP3, and membrane-bound transforming growth factor ß (TGF-ß). These data demonstrate for the first time that AIDS lentiviruses infect and activate Treg cells, potentially contributing to immune dysfunction.

CD4+CD25+ T regulatory cells activated during feline immunodeficiency virus infection convert T helper cells into functional suppressors through a membrane-bound TGFß / GARP-mediated mechanism.

BACKGROUND: We and others have previously reported that cell membrane-bound TGFß (mTGFß) on activated T regulatory (Treg) cells mediates suppressor function. Current findings suggest that a novel protein known as Glycoprotein A Repetitions Predominant (GARP) anchors mTGFß to the Treg cell surface and facilitates suppressor activity. Recently, we have described that GARP+TGFß+ Treg cells expand during the course of FIV infection. Because Treg cells are anergic and generally exhibit poor proliferative ability, we asked how Treg homeostasis is maintained during the course of feline immunodeficiency virus (FIV) infection. RESULTS: Here, we report that Treg cells from FIV+ cats express GARP and mTGFß and convert T helper (Th) cells into phenotypic and functional Treg cells. Th to Treg conversion was abrogated by anti-TGFß or anti-GARP treatment of Treg cells or by anti-TGFßRII treatment of Th cells, suggesting that Treg cell recruitment from the Th pool is mediated by TGFß/TGFßRII signaling and that cell-surface GARP plays a major role in this process. CONCLUSIONS: These findings suggest Th to Treg conversion may initiate a cascade of events that contributes to the maintenance of virus reservoirs, progressive Th cell immunosuppression, and the development of immunodeficiency, all of which are central to the pathogenesis of AIDS lentivirus infections.

T-cell factor/ß-catenin activity is suppressed in two different models of autosomal dominant polycystic kidney disease.

During murine kidney development, canonical WNT signaling is highly active in tubules until about embryonic days E16-E18. At this time, ß-catenin transcriptional activity is progressively restricted to the nephrogenic zone. The cilial protein genes PKD1 and PKD2 are known to be mutated in autosomal dominant polycystic kidney disease (ADPKD), and previous studies proposed that these mutations could lead to a failure to suppress canonical WNT signaling activity. Several in vitro studies have found a link between cilial signaling and ß-catenin regulation, suggesting that aberrant activity might contribute to the cystic phenotype. To study this, we crossed T-cell factor (TCF)/ß-catenin-lacZ reporter mice with mice having Pkd1 or Pkd2 mutations and found that there was no ß-galactosidase staining in cells lining the renal cysts. Thus, suppression of canonical WNT activity, defined by the TCF/ß-catenin-lacZ reporter, is normal in these two different models of polycystic kidney disease. Hence, excessive ß-catenin transcriptional activity may not contribute to cystogenesis in these models of ADPKD.

Optimization and qualification of an assay that demonstrates that a FimH vaccine induces functional antibody responses in women with histories of urinary tract infections.

Recurrent urinary tract infections (rUTI) are a serious disease associated with morbidities and mortality. Resistance to the standard of care antibiotics is now widespread because of the continued use of antibiotics among people who suffer from rUTI. We are therefore developing a vaccine to prevent recurrences among patients with rUTI. The antigen of the vaccine is FimH, a bacterial adhesin protein, and the vaccine is adjuvanted with a TLR-4 agonist. In a Phase 1 clinical study evaluating the vaccine, immunized individuals produced FimH-binding antibodies. Here we describe the optimization, qualification, and use of an assay to assess the functionality of these anti-FimH antibodies. The suitability of the assay for its intended purpose was demonstrated by selectivity, specificity, sensitivity, and intra-assay and inter-assay precision. The acceptance criteria were achieved for all parameters including intra-assay precision with ≤10% relative standard deviations and inter-assay precision with ≤25% relative standard deviations. The results presented herein suggest this functional assay will be important for supporting the vaccine's efficacy in future human studies. Furthermore and of great significance, these results prove that vaccine-induced functional antibodies can be elicited in rUTI patients against an essential virulence factor, FimH.

Editor's Highlight: Development of an In vitro Assay Measuring Uterine-Specific Estrogenic Responses for Use in Chemical Safety Assessment.

A toxicity pathway approach was taken to develop an in vitro assay using human uterine epithelial adenocarcinoma (Ishikawa) cells as a replacement for measuring an in vivo uterotrophic response to estrogens. The Ishikawa cell was determined to be fit for the purpose of recapitulating in vivo uterine response by verifying fidelity of the biological pathway components and the dose-response predictions to women of child-bearing age. Expression of the suite of estrogen receptors that control uterine proliferation (ERα66, ERα46, ERα36, ERß, G-protein coupled estrogen receptor (GPER)) were confirmed across passages and treatment conditions. Phenotypic responses to ethinyl estradiol (EE) from transcriptional activation of ER-mediated genes, to ALP enzyme induction and cellular proliferation occurred at concentrations consistent with estrogenic activity in adult women (low picomolar). To confirm utility of this model to predict concentration-response for uterine proliferation with xenobiotics, we tested the concentration-response for compounds with known uterine estrogenic activity in humans and compared the results to assays from the ToxCast and Tox21 suite of estrogen assays. The Ishikawa proliferation assay was consistent with in vivo responses and was a more sensitive measure of uterine response. Because this assay was constructed by first mapping the key molecular events for cellular response, and then ensuring that the assay incorporated these events, the resulting cellular assay should be a reliable tool for identifying estrogenic compounds and may provide improved quantitation of chemical concentration response for in vitro-based safety assessments.

Human intravenous immunoglobulin (hIVIG) inhibits anti-CD32 antibody binding to canine DH82 cells and canine monocytes in vitro.

The IgG receptors CD16 and CD32 (Fc(γ)RIII and Fc(γ)RII) link the humoral immune response to effector cell immune responses by binding immune complexes. Human intravenous immunoglobulin (hIVIG) consisting of immunoglobulin from pooled donors is reported to block Fc(γ)Rs and has been used to treat a variety of canine autoimmune disorders. Fc(γ)Rs have been poorly described for canine monocytes; therefore, the objectives of this study were to: (1) identify canine monocyte/macrophage Fc(γ)R (CD16 and CD32) expression and (2) demonstrate in vitro hIVIG binding to these receptors. The canine monocyte/macrophage-like cell line (DH82) and monocytes isolated from peripheral blood of healthy dogs were evaluated by flow cytometry (FACS) for CD16 and CD32 expression using commercially available anti-CD16 and anti-CD32 antibodies directed against the human isoforms. The mean percentage of cells expressing CD16 was 55% of DH82 cells and 13% of blood monocytes and the mean percentage of cells expressing CD32 was 85% of DH82 cells and 73% of blood monocytes. Immunoprecipitation of canine DH82 cells lysate using the same anti-CD16 or anti-CD32 antibodies suggested that these anti-human antibodies recognize the canine homologues. To demonstrate Fc(γ)R blockade, cells were incubated with increasing concentrations of hIVIG and then incubated with anti-CD16 or anti-CD32 antibodies. The percentage of CD32 expression decreased in a concentration dependent fashion in DH82 cells and blood monocytes after incubation with increasing concentrations of IVIG, suggesting that hIVIG was binding to CD32 and inhibiting anti-CD32 antibody binding. The same results were not demonstrated with anti-CD16 antibody. We believe this is the first report to demonstrate Fc(γ) receptors CD16 and CD32 expression on canine monocytes and in vitro CD32 binding by human IgG, which may represent one of the immunomodulatory mechanisms of hIVIG.

Feline glycoprotein A repetitions predominant anchors transforming growth factor beta on the surface of activated CD4(+)CD25(+) regulatory T cells and mediates AIDS lentivirus-induced T cell immunodeficiency.

Using the feline immunodeficiency virus (FIV) model for AIDS-lentivirus infection, our laboratory has previously demonstrated that T regulatory (Treg) cell-mediated immune T and B cell dysfunction contributes to lentivirus persistence and chronic disease through membrane bound transforming growth factor beta (mTGFb). Studying Treg cells in the context of infection has been problematic as no inducible marker for activated Treg cells had been identified. However, recent reports in human Treg studies have described a novel protein, glycoprotein A repetitions predominant (GARP), as a unique marker of activated human Treg cells that anchors mTGFb. Herein we extend these studies to the feline Treg system, identifying feline GARP and demonstrating that human and feline GARP proteins are homologous in structure, expression pattern, and ability to form a complex with TGFb. We further demonstrate that GARP and TGFb form a complex on the surface of activated Treg cells and that these GARP(+)TGFb(+) Treg cells are highly efficient suppressor cells. Analysis of expression of this Treg activation marker in the FIV-AIDS model reveals an up-regulation of GARP expressing Treg cells during chronic FIV infection. We demonstrate that the GARP(+) Treg cells from FIV-infected cats suppress T helper cells in vivo and that blocking GARP or TGFb eliminates this suppression. These data suggest that GARP is expressed in complex with TGFb on the surface of activated Treg cells and plays an important role in TGFb(+) Treg-mediated T cell immune suppression during lentivirus infection.

CD8+ clonality is associated with prolonged acute plasma viremia and altered mRNA cytokine profiles during the course of feline immunodeficiency virus infection.

Acute lentiviral infection is characterized by early CD8(+) cytotoxic T cell (CTL) activity and a subsequent decline in plasma viremia. However, CD8(+) lymphocytes fail to eliminate the virus and a progressive T cell immune dysfunction develops during the course of chronic lentiviral infection. To further define this CD8(+) immune dysfunction we utilized PARR (PCR for antigen receptor rearrangements), a technique which measures clonally expanded lymphocyte populations by comparison of highly conserved T cell receptor (TCR) regions to identify the prevalence of clonal CD8(+) T cells following FIV infection. We then compared phenotype, mRNA profiles, CD8(+) proliferation and plasma viremia during acute and chronic infection for PARR positive (PARR(+)) and PARR negative (PARR(-)) Feline Immunodeficiency Virus (FIV) infected cats. We demonstrated that approximately forty percent of the FIV(+) cats examined exhibit CD8(+) clonality compared to none of the FIV(-) control cats. There were no phenotypic differences between PARR(+) and PARR(-) CD8(+) lymphocytes from FIV(+) cats but retrospective analysis of plasma viremia over the course of infection revealed a delayed peak in plasma viremia and a decline in lymphocyte counts were observed in the PARR(+) group during acute infection. CD8(+) lymphocytes isolated from chronically infected PARR(-) cats exhibited significantly higher mRNA expression of IFN-γ and IL-2 following mitogenic stimulation when compared to PARR(+) CD8(+) lymphocytes. These data suggest that clonal CD8(+) expansion may be related to impaired control of acute viremia and less effective CD8(+) anti-viral function. Using PARR to assess changes in CD8(+) clonality during the progression from acute to chronic FIV infection may help to better characterize the factors which contribute to CD8(+) anergy and lentiviral persistence.

Administration of Fozivudine tidoxil as a single-agent therapeutic during acute feline immunodeficiency virus infection does not alter chronic infection.

Initiating combination antiretroviral therapy (ART) during acute HIV infection has been correlated with decreased viral set point and improved lymphocyte function. However, the long term effects of single-agent therapy administered only during the acute stage of infection (interrupted treatment) remain largely uncharacterized. In this study we provide longitudinal data using the feline immunodeficiency virus (FIV) model for HIV infection. Infected cats were treated with a prophylactic single-agent therapy, Fozivudine tidoxil (FZD), for six weeks, starting one day before infection. The initial acute infection study, reported elsewhere, demonstrated a decrease in plasma- and cell-associated viremia at two weeks post-infection (PI) in FZD-treated cats as compared to placebo-treated cats. We hypothesized that this early alteration in plasma- and cell-associated viremia would alter the virus set point and ultimately affect the outcome of chronic infection. Here we provide data at one, two and three years PI for plasma- and/or cell-associated viremia, total lymphocyte counts and CD4:CD8 ratios. There was no difference in viremia or cell counts between treated and nontreated groups at all time points tested. Contrary to our hypothesis, these results suggest that treatment with a single agent anti-retroviral drug during acute lentivirus infection does not significantly alter viral load and immune function during the chronic, asymptomatic stage of infection.