Identification of immune cell markers associated with ulcerative colitis histological disease activity in colonic biopsies.

AIMS: Accurate determination of histological activity in ulcerative colitis (UC) is essential given its diagnostic and prognostic importance. Data on the relationship between histology and immune cell markers are limited. We aimed to evaluate the association between histological disease activity and immune cell marker concentration in colonic biopsies from patients with UC. METHODS: Sigmoid colon biopsies from 20 patients with UC were retrospectively assessed using the Robarts Histopathology Index (RHI). Targeted mass spectrometry determined the concentration of 18 immune cell markers (cluster of differentiation (CD) 4, CD8, CD19, CD20, CD40, CD56, CD68, CD103, forkhead box p3 (FOXP3), human leucocyte antigen, DR alpha chain (HLA-DRA), interleukin 10 (IL-10), IL-23 subunit alpha (IL-23A), IL-23 receptor (IL-23R), IL-2 receptor alpha chain (IL-2RA), Ki67, lymphocyte-activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1) and PD ligand 1 (PD-L1)). The association between RHI score and immune cell marker concentration was quantified using Spearman's rank correlation coefficient (ρ) and related 95% CIs. RESULTS: Fourteen of the 18 immune cell marker proteins were detected, with tissue concentration ranging from 0.003 to 11.53 fmol/µg. The overall RHI score was positively correlated with CD19, CD20, CD40, FOXP3, LAG-3, PD-1 and PD-L1 concentration (ρ=0.596-0.799) and negatively correlated with CD56 concentration (ρ=-0.460). There was no significant association between RHI score and CD4, CD8, CD68, CD103, HLA-DRA or Ki67 concentration. CONCLUSIONS: This study provides insight into the correlation between immune cell marker expression and histological disease activity and the possible molecular and immunological determinants underlying microscopic disease activity in UC.

Effect of storage time on peripheral blood mononuclear cell isolation from blood collected in vacutainer CPT™ tubes.

BACKGROUND: Clinical trials of novel therapies for the treatment of ulcerative colitis (UC) may benefit from immune cell profiling, however implementation of this methodology is limited in the multicenter trial setting by necessity of timely (within 6 to 8 h) isolation and processing of peripheral blood mononuclear cells (PBMC) from whole blood samples. Becton Dickinson Vacutainer CPT™ Cell Preparation Tubes (CPT™) limit required processing prior to shipping to a central lab to an initial centrifugation step within 24 h of sample collection. As shipping may delay final processing beyond 24 h, we analyzed cell viability and T cell composition in whole blood stored in CPT™ to determine if their use may accommodate processing delays typical for multicenter clinical trials. METHODS: Whole blood samples from 3 patients with UC were collected in CPT™ (15 tubes/patient) and PBMC were processed at various timepoints (24-96 h). Cell viability and T cell composition (26 types) were evaluated by flow cytometry. Variability between technical and biological replicates was evaluated in the context of cell-type abundance, delayed processing time, and data normalization. RESULTS: Total cell viability was <50% when processing was delayed to 48 h after collection and was further reduced at later processing timepoints. The effect of delayed processing on cell abundance varied widely across cell types, with CD4+, CD8+, naïve effector CD8+, and Tcm CD4 + T cells displaying the least variability in abundance with delayed processing. Normalization of cell counts to cell types other than total T cells corrected for the effect of delayed processing for several cell types, particularly Th17. CONCLUSIONS: Based on these data, processing of PBMC in CPT™ should ideally be performed within 48 h. Delayed processing of PBMC in CPT™ may be considered for cell types that are robust to these conditions. Normalization of cell abundance to different parental cell-types may reduce variability in quantitation and should be used in conjunction with the expected effect size to meet the experimental goals of a multicenter clinical trial.

Development and Validation of a Digital Analysis Method to Quantify CD3-immunostained T Lymphocytes in Whole Slide Images of Crohn's Disease Biopsies.

The T-lymphocyte-mediated inflammation in Crohn's disease can be assessed by quantifying CD3-positive T-lymphocyte counts in colonic sections. We developed and validated a process to reliably quantify immunohistochemical marker-positive cells in a high-throughput setting using whole slide images (WSIs) of CD3-immunostained colonic and ileal tissue sections. In regions of interest (ROIs) and/or whole tissue sections of 40 WSIs from 36 patients with Crohn's disease, CD3-positive cells were quantified by an expert gastrointestinal pathologist (gold standard) and by image analysis algorithms developed with software from 3 independent vendors. Semiautomated quantification of CD3-positive cell counts estimated in 1 ROI per section were accurate when compared with manual analysis (Pearson correlation coefficient, 0.877 to 0.925). Biological variability was acceptable in digitally determined CD3-positive cell measures between 2 to 5 ROIs annotated on the same tissue section (coefficient of variation <25%). Results from computer-aided analysis of CD3-positive T lymphocytes in a whole tissue section and the average of results from 2 to 5 ROIs per tissue section lacked reliability (overestimation or underestimation and systematic bias), suggesting that absolute quantification of CD3-positive T lymphocytes in a whole tissue section may be more accurate. Semiautomated image analysis in WSIs demonstrated reproducible CD3-positive cell measures across 3 independent algorithms. A computer-aided digital image analysis method was developed and validated to quantify CD3-positive T lymphocytes in colonic and ileal biopsy sections from patients with Crohn's disease. Results support consideration of this digital analysis method for use in future Crohn's disease clinical studies.

Pharmacological Interventions for the Prevention and Treatment of Immune Checkpoint Inhibitor-Associated Enterocolitis: A Systematic Review.

BACKGROUND: Patients treated with immune checkpoint inhibitors (ICIs) may develop ICI-associated enterocolitis, for which there is no approved treatment. AIMS: We aimed to systematically review the efficacy and safety of medical interventions for the prevention and treatment of ICI-associated enterocolitis. METHODS: MEDLINE, EMBASE, and the Cochrane Library were searched to identify randomized controlled trials (RCTs), cohort and case-control studies, and case series/reports, evaluating interventions (including corticosteroids, biologics, aminosalicylates, immunosuppressants, and fecal transplantation) for ICI-associated enterocolitis. Clinical, endoscopic, and histologic efficacy endpoints were evaluated. The Grading of Recommendations, Assessment, Development, and Evaluation criteria were used to assess overall quality of evidence. RESULTS: A total of 160 studies (n = 1514) were included (one RCT, 3 retrospective cohort studies, 156 case reports/case series). Very low quality evidence from one RCT suggests budesonide is not effective for prevention of ICI-associated enterocolitis in ipilimumab-treated patients (relative risk 0.93 [95% confidence interval 0.56, 1.56]). Very low quality evidence suggests that corticosteroids, infliximab, and vedolizumab may be effective for treatment of ICI-associated enterocolitis by inducing clinical response and remission. No validated indices for measuring disease activity were used. Biologic treatment was used in 42% (641/1528) of patients, as reported in 97 studies. ICIs were discontinued in 65% (457/702) of patients, as reported in 63 studies. CONCLUSIONS: Current treatment recommendations for ICI-associated enterocolitis are based on very low quality evidence, primarily from case reports and case series. Large-scale prospective cohort studies and RCTs are needed to develop prophylactic and therapeutic treatments to minimize interruption or discontinuation of oncological therapies.

A Clinical Prediction Model to Determine Probability of Response to Certolizumab Pegol for Crohn's Disease.

BACKGROUND: Certolizumab pegol (CZP) is effective for moderately to severely active Crohn's disease (CD). Higher plasma concentrations are associated with better outcomes and increased drug clearance is the driver of subtherapeutic CZP concentrations. OBJECTIVE: We aimed to develop a prediction model incorporating predicted CZP clearance and patient variables to allow estimation of the probability for remission prior to initiating therapy. METHODS: A population pharmacokinetic model estimated baseline CZP clearance in patients with CD from nine phase II and III trials. Multivariable prediction models were developed and validated using the PRECiSE 1 and PRECiSE 2 datasets to identify candidate predictors for a composite remission outcome (Crohn's Disease Activity Index ≤ 150 and fecal calprotectin concentration ≤ 250 µg/g) at Weeks 6 or 26. An online clinical decision support tool (CDST) was developed. RESULTS: Baseline predicted CZP clearance ≥ 0.5 L/day was associated with subtherapeutic Week 6 CZP plasma concentrations. Baseline weight (odds ratio [OR] 1.04; 95% confidence interval [CI] 1.02-1.07), calculated CZP clearance (OR 0.92; 95% CI 0.87-0.96), hematocrit (OR 2.55; 95% CI 1.43-4.54), and fecal calprotectin (OR 0.66; 95% CI 0.54-0.80) were associated with Week 6 remission (p ≤ 0.0015 for all predictors). Baseline weight (OR 1.04; 95% CI 1.02-1.07), calculated CZP clearance (OR 0.93; 95% CI 0.88-0.97), and Patient-Reported Outcome-2 (PRO2) (OR 0.93; 95% CI 0.87-0.99) were associated with Week 26 remission (p ≤ 0.033 for all predictors). CONCLUSIONS: Patients who are predicted to have accelerated baseline CZP clearance are at risk of subtherapeutic CZP concentrations. Patient-level probabilities for a composite remission outcome can be predicted for patients with CD by entering commonly available patient- and disease-related factors into an online CDST ( https://premedibd.com ) incorporating predicted CZP clearance.

Polybrominated Diphenyl Ethers in Human Follicular Fluid Dysregulate Mural and Cumulus Granulosa Cell Gene Expression.

Polybrominated diphenyl ethers (PBDEs), a major class of flame retardants incorporated into numerous consumer products, leach out into dust resulting in widespread exposure. There is evidence from in vitro and in vivo animal studies that PBDEs affect ovarian granulosa cell function and follicular development, yet human studies of their association with female infertility are inconclusive. Here, we tested the hypothesis that exposure to the PBDEs in follicular fluid is associated with dysregulation of gene expression in the mural and cumulus granulosa cells collected from women undergoing in vitro fertilization by intracytoplasmic sperm injection. The median concentration of the ∑ 10PBDEs detected in the follicular fluid samples (n = 37) was 15.04 pg/g wet weight. RNA microarray analyses revealed that many genes were differentially expressed in mural and cumulus granulosa cells. Highest vs lowest quartile exposure to the Σ 10PBDEs or to 2 predominant PBDE congeners, BDE-47 or BDE-153, was associated with significant effects on gene expression in both cell types. Mural granulosa cells were generally more sensitive to PBDE exposure compared to cumulus cells. Overall, gene expression changes associated with BDE-47 exposure were similar to those for ∑ 10PBDEs but distinct from those associated with BDE-153 exposure. Interestingly, exposure to BDE-47 and ∑ 10PBDEs activated the expression of genes in pathways that are important in innate immunity and inflammation. To the best of our knowledge, this is the first demonstration that exposure to these environmental chemicals is associated with the dysregulation of pathways that play an essential role in ovulation.

In Utero and Lactational Exposure to Flame Retardants Disrupts Rat Ovarian Follicular Development and Advances Puberty.

Brominated flame retardants (BFRs), including polybrominated diphenyl ethers and hexabromocyclododecane, leach out from consumer products into the environment. Exposure to BFRs has been associated with effects on endocrine homeostasis. To test the hypothesis that in utero and lactational exposure to BFRs may affect the reproductive system of female offspring, adult female Sprague Dawley rats were fed diets formulated to deliver nominal doses (0, 0.06, 20, or 60 mg/kg/day) of a BFR dietary mixture mimicking the relative congener levels in house dust from prior to mating until weaning. Vaginal opening and the day of first estrus occurred at a significantly earlier age among offspring from the 20 mg/kg/day BFR group, indicating that the onset of puberty was advanced. Histological analysis of ovaries from postnatal day 46 offspring revealed an increase in the incidence of abnormal follicles. A toxicogenomic analysis of ovarian gene expression identified upstream regulators, including HIF1A, CREB1, EGF, the ß-estradiol, and PPARA pathways, predicted to be downregulated in the 20 or 60 mg/kg/day group and to contribute to the gene expression patterns observed. Thus, perinatal exposure to BFRs dysregulated ovarian folliculogenesis and signaling pathways that are fundamental for ovarian function in the adult.

Clinical Pharmacology of Janus Kinase Inhibitors in Inflammatory Bowel Disease.

Inflammatory bowel disease, including ulcerative colitis and Crohn's disease, are chronic inflammatory disorders of the gastrointestinal tract which are characterised, in part, by an imbalance in the production of several pro- and anti-inflammatory cytokines. Although various agents are effective for inducing and maintaining remission, approximately 20% of patients are treatment-refractory and require surgery. Parenterally administered monoclonal antibody-based biologics are associated with adverse effects resulting in treatment discontinuation and/or immunogenicity, leading to loss of response to therapy. Approximately 50% of patients who initially respond to treatment with tumour necrosis factor antagonists lose response to therapy within the 1st year of treatment. Incidence of immunogenicity tends to decrease over time, but once present can persist for years, even after treatment discontinuation. Nonimmunogenic oral small molecule therapies, including Janus kinase inhibitors, are currently being developed and have demonstrated efficacy in early phase clinical trials, which has already led to regulatory approval of tofacitinib for the treatment of patients with moderate-to-severe ulcerative colitis. Differentiation of T cells into T helper cells, which are mediators of the inflammatory response in inflammatory bowel disease, is mediated by the Janus kinase signal transducer and activator of the transcription signalling pathway. Absorption and distribution of Janus kinase inhibitors occurs at the site of action in the gastrointestinal tract, and newer compounds are being developed with limited systemic absorption, potentially reducing the risk of adverse effects. The current review describes the clinical pharmacology of approved Janus kinase inhibitors, as well as those in clinical development for the treatment of inflammatory bowel disease.

Factors Influencing Drug Disposition of Monoclonal Antibodies in Inflammatory Bowel Disease: Implications for Personalized Medicine.

Monoclonal antibody (mAb) therapies have revolutionized the treatment of several chronic inflammatory diseases, including the inflammatory bowel diseases (IBD), Crohn's disease, and ulcerative colitis. While efficacious, responses to these therapies vary considerably from patient to patient, due in part to inter- and intra-individual variability in pharmacokinetics (PK) and drug exposure. The concept of personalized medicine to monitor drug exposure and to adjust dosing in individual patients is consequently gaining acceptance as a powerful tool to optimize mAb therapy for improved outcomes in IBD. This review provides a brief overview of the different mAbs currently approved or in development for the treatment of IBD, including their presumed mechanisms of action and PK properties. Specifically described are (1) the factors known to affect mAb PK and drug exposure in patients with IBD, (2) the value of population PK/pharmacodynamic (PD) modeling to identify and understand the influence of these factors on drug exposure and effect, and (3) the clinical evidence for the potential of therapeutic drug monitoring (TDM) to improve IBD outcomes in response to mAb-based therapy. Incorporation of PK/PD parameters into clinical decision support tools has the potential to guide therapeutic decision making and aid implementation of personalized medicine strategies in patients with IBD.

Gestational and Early Postnatal Exposure to an Environmentally Relevant Mixture of Brominated Flame Retardants: General Toxicity and Skeletal Variations.

Brominated flame retardants (BFRs) are stable environmental contaminants known to exert endocrine-disrupting effects. Developmental exposure to polybrominated diphenyl ethers (PBDEs) is correlated with impaired thyroid hormone signaling, as well as estrogenic and anti-androgenic effects. As previous studies have focused on a single congener or technical mixture, the purpose of the current study was to examine the effects of gestational and early postnatal exposure to an environmentally relevant mixture of BFRs designed to reflect house dust levels of PBDEs and hexabromocyclododecane on postnatal developmental outcomes. Pregnant Sprague-Dawley rats were exposed to the PBDE mixture from preconception to weaning (PND 21) through the diet containing 0, 0.75, 250, and 750 mg mixture/kg diet. BFR exposure induced transient reductions in body weight at PND 35 in male and from PND 30-45 in female offspring (250 and 750 mg/kg). Liver weights (PND 21) and xenobiotic metabolizing enzyme activities (PND 21 and 46) were increased in both male and female offspring exposed to 250 and 750 mg/kg diets. Furthermore, serum T4 levels were reduced at PND 21 in both,male and female offspring (250 and 750 mg/kg). At PND 21, Serum alkaline phosphatase (ALP) was decreased in males exposed to 750 mg/kg dietat, and females exposed to 250 and 750 mg/kg diets. At PND 46 ALP was significantly elevated in males (250 and 750 mg/kg). Variations in the cervical vertebrae and phalanges were observed in pups at PND 4 (250 and 750 mg/kg). Therefore, BFR exposure during gestation through to weaning alters developmental programming in the offspring. The persistence of BFRs in the environment remains a cause for concern with regards to developmental toxicity.

Polyamine-Mediated Effects of Prolactin Dictate Emergence from Mink Obligate Embryonic Diapause.

Embryonic diapause is an evolutionary strategy to ensure that offspring are born when maternal and environmental conditions are optimal for survival. In many species of carnivores, obligate embryonic diapause occurs in every gestation. Reciprocal embryo transplant studies indicate that embryo arrest during diapause is conferred by uterine conditions and is due to a lack of specific factors necessary for continued development. In previous studies, global gene expression analysis revealed reduced uterine expression during diapause of a cluster of genes in the mink that regulate the abundance of polyamines, including ornithine decarboxylase 1 (ODC1). In addition, in vivo inhibition of the conversion of ornithine to the polyamine, putrescine, induced a reversible arrest in mink embryonic development and an arrest in trophoblast cell proliferation in vitro. Previous studies have implicated prolactin as the principal endocrine signal to terminate diapause. In this study, uterine expression of both the progesterone and estrogen receptors remained low at reactivation whilst the prolactin receptor was expressed at all times. Treatment of mink uterine epithelial cells with varying doses of prolactin indicated that this hormone induces ODC1 expression in the uterus via pSTAT1 and mTOR, thereby regulating uterine polyamine levels. In addition, we performed global gene expression analysis on mink embryos to further explore dynamic changes during diapause and found 94 genes upregulated at reactivation from diapause. Three polyamine-related genes, including ODC1, were also upregulated at reactivation from diapause. To establish whether polyamines mitigate escape from embryonic diapause, we collected mink embryos in diapause and incubated them in vitro with putrescine. Increase in embryo volume, the first indication of emergence from diapause, was observed within the first 5 days of culture in all viable embryos treated with putrescine, and the duration of embryo survival was increased threefold. Concomitant increases were also observed in both the total number of cells and the proportion of dividing cells in putrescine-treated embryos whilst control embryos remained in the diapause state. In further studies, inhibition of polyamine synthesis abrogated proliferation in cells derived from the inner cell mass of the mink embryo, while putrescine induced dose-dependent increases in cell division. We conclude that supplementation of embryos in diapause with putrescine results in their escape from developmental dormancy. These results provide strong evidence that obligate diapause in vivo is caused by the paucity of polyamines necessary for activation of the embryo after prolactin-induced termination of diapause.

A Mixture Reflecting Polybrominated Diphenyl Ether (PBDE) Profiles Detected in Human Follicular Fluid Significantly Affects Steroidogenesis and Induces Oxidative Stress in a Female Human Granulosa Cell Line.

Brominated flame retardants are incorporated into consumer products to prevent flame propagation. These compounds leach into the domestic environment, resulting in chronic exposure. Pregnancy failure is associated with high levels of polybrominated diphenyl ethers (PBDEs), a major class of brominated flame retardants, in human follicular fluid, raising serious questions regarding their impact on female fertility. Our goal was to elucidate the effects of a mixture of PBDEs, similar to the profile found in human follicular fluid, on an immortalized human granulosa cell line, the KGN cell line. We showed that cell viability was altered and oxidative stress was induced as reflected by increased reactive oxygen species formation at 100 µM of the PBDE mixture. Transcriptomic analysis revealed that PBDE treatments of 1, 5, and 20 µM altered the expression of several genes involved in the reactive oxygen species signaling pathway. Significant dose-dependent reductions in progesterone and estradiol levels in the culture medium were measured after PBDE treatment; in parallel, the expression of genes involved in estradiol metabolism, namely CYP1A1, was up-regulated by 5 and 20 µM of the PBDE mixture. Treatment with 20 µM PBDE also increased the expression and secretion of the proinflammatory factor, IL-6, into the KGN cell culture medium. Our results demonstrate that PBDEs can alter human granulosa cell functions by inducing oxidative stress and disrupting steroidogenesis. These results indicate that PBDEs may be detrimental to ovarian functions and thus may adversely affect female reproductive health after chronic exposure.

Exposure of Female Rats to an Environmentally Relevant Mixture of Brominated Flame Retardants Targets the Ovary, Affecting Folliculogenesis and Steroidogenesis.

Brominated flame retardants (BFRs) are incorporated into various consumer products to prevent flame propagation. These compounds leach into the domestic environment, resulting in chronic exposure and contamination. Pregnancy failure is associated with high levels of BFRs in human follicular fluid, raising serious questions regarding their impact on female reproductive health. The goal of this study is to elucidate the effects of an environmentally relevant BFR mixture on female rat ovarian functions (i.e., folliculogenesis and steroidogenesis). A BFR dietary mixture formulated to mimic the relative BFR congener levels in North American house dust was administered to adult female Sprague-Dawley rats from 2 to 3 wk before mating until Gestational Day 20; these diets were designed to deliver nominal doses of 0, 0.06, 20, or 60 mg/kg/day of the BFR mixture. Exposure to BFRs triggered an approximately 50% increase in the numbers of preantral and antral follicles and an enlargement of the antral follicles in the ovaries of the dams. A significant reduction in the expression of catalase, an antioxidant enzyme, and downregulation of the expression of insulin-like factor 3 (Insl3) and 17alpha-hydroxylase (Cyp17a1) were observed in the ovary. In addition, BFR exposure affected steroidogenesis; we observed a significant decrease in circulating 17-hydroxypregnenolone and an increase in testosterone concentrations in BFR-exposed dams. Thus, BFRs target ovarian function in the rat, adversely affecting both folliculogenesis and steroidogenesis.

Exposure to an environmentally relevant mixture of brominated flame retardants affects fetal development in Sprague-Dawley rats.

Brominated flame retardants are incorporated into a wide variety of consumer products and are known to enter into the surrounding environment, leading to human exposure. There is accumulating evidence that these compounds have adverse effects on reproduction and development in humans and animal models. Animal studies have generally characterized the outcome of exposure to a single technical mixture or congener. Here, we determined the impact of exposure of rats prior to mating and during gestation to a mixture representative of congener levels found in North American household dust. Adult female Sprague-Dawley rats were fed a diet containing 0, 0.75, 250 or 750mg/kg of a mixture of flame retardants (polybrominated diphenyl ethers, hexabromocyclododecane) from two weeks prior to mating to gestation day 20. This formulation delivered nominal doses of 0, 0.06, 20 and 60mg/kg body weight/day. The lowest dose approximates high human exposures based on house dust levels and the dust ingestion rates of toddlers. Litter size and resorption sites were counted and fetal development evaluated. No effects on maternal health, litter size, fetal viability, weights, crown rump lengths or sex ratios were detected. The proportion of litters with fetuses with anomalies of the digits (soft tissue syndactyly or malposition of the distal phalanges) was increased significantly in the low (0.06mg/kg/day) dose group. Skeletal analysis revealed a decreased ossification of the sixth sternebra at all exposure levels. Thus, exposure to an environmentally relevant mixture of brominated flame retardants results in developmental abnormalities in the absence of apparent maternal toxicity. The relevance of these findings for predicting human risk is yet to be determined.

A new role for muscle segment homeobox genes in mammalian embryonic diapause.

Mammalian embryonic diapause is a phenomenon defined by the temporary arrest in blastocyst growth and metabolic activity within the uterus which synchronously becomes quiescent to blastocyst activation and implantation. This reproductive strategy temporally uncouples conception from parturition until environmental or maternal conditions are favourable for the survival of the mother and newborn. The underlying molecular mechanism by which the uterus and embryo temporarily achieve quiescence, maintain blastocyst survival and then resume blastocyst activation with subsequent implantation remains unknown. Here, we show that uterine expression of Msx1 or Msx2, members of an ancient, highly conserved homeobox gene family, persists in three unrelated mammalian species during diapause, followed by rapid downregulation with blastocyst activation and implantation. Mice with uterine inactivation of Msx1 and Msx2 fail to achieve diapause and reactivation. Remarkably, the North American mink and Australian tammar wallaby share similar expression patterns of MSX1 or MSX2 as in mice-it persists during diapause and is rapidly downregulated upon blastocyst activation and implantation. Evidence from mouse studies suggests that the effects of Msx genes in diapause are mediated through Wnt5a, a known transcriptional target of uterine Msx. These studies provide strong evidence that the Msx gene family constitutes a common conserved molecular mediator in the uterus during embryonic diapause to improve female reproductive fitness.

Pharmacological targeting of mammalian target of rapamycin inhibits ovarian granulosa cell tumor growth.

Few targeted therapies have been developed for ovarian granulosa cell tumor (GCT), even though it represents 5% of all malignant ovarian tumors in women. As misregulation of PI3K/AKT signaling has been implicated in GCT development, we hypothesized that the AKT signaling effector mammalian target of rapamycin (mTOR) may play a role in the pathogenesis of GCT and could represent a therapeutic target. Analyses of human GCT samples showed an increase in protein levels of mTOR and its downstream effectors RPS6KB1, RPS6, eIF4B and PPARG relative to normal granulosa cells, suggestive of an increase in mTOR pathway activity and increased translational activity and/or protein stability. We next sought to evaluate mTOR as a GCT therapeutic target using the Pten (tm1Hwu/tmiHwu);Ctnnb1 (tm1Mmt/+);Amhr2 (tm3(cre)Bhr/+) (PCA) mouse model, in which mTOR, RPS6KB1, eIF4B and PPARG are upregulated in tumor cells in a manner similar to human GCT. Treatment of PCA mice with the mTOR-specific inhibitor everolimus reduced tumor growth rate (1.5-fold; P < 0.05) and also reduced total tumor burden (4.7-fold; P < 0.05) and increased survival rate (78 versus 44% in the vehicle group) in a PCA surgical model of GCT peritoneal carcinomatosis. Everolimus decreased tumor cell proliferation and tumor cell volume relative to controls (P < 0.05), whereas apoptosis was unaffected. Phosphorylation of RPS6KB1 and RPS6 were decreased (P < 0.05) by everolimus, but RPS6KB1, RPS6, eIF4B and PPARG expressions were not affected. These results suggest that mTOR is a valid and clinically useful pharmacological target for the treatment of GCT, although its inhibition does not reverse all consequences of aberrant PI3K/AKT signaling in the PCA model.

Polyamines on the reproductive landscape.

The polyamines are ubiquitous polycationic compounds. Over the past 40 yr, investigation has shown that some of these, namely spermine, spermidine, and putrescine, are essential to male and female reproductive processes and to embryo/fetal development. Indeed, their absence is characterized by infertility and arrest in embryogenesis. Mammals synthesize polyamines de novo from amino acids or import these compounds from the diet. Information collected recently has shown that polyamines are essential regulators of cell growth and gene expression, and they have been implicated in both mitosis and meiosis. In male reproduction, polyamine expression correlates with stages of spermatogenesis, and polyamines appear to function in promoting sperm motility. There is evidence for polyamine involvement in ovarian follicle development and ovulation in female mammals, and polyamine synthesis is required for steroidogenesis in the ovary. Studies of the embryo indicate a polyamine requirement that can be met from maternal sources before implantation, whereas elimination of polyamine synthesis abrogates embryo development at gastrulation. Polyamines play roles in embryo implantation, in decidualization, and in placental formation and function, and polyamine privation during gestation results in intrauterine growth retardation. Emerging information implicates dietary arginine and dietary polyamines as nutritional regulators of fertility. The mechanisms by which polyamines regulate these multiple and diverse processes are not yet well explored; thus, there is fertile ground for further productive investigation.

Polyamines are implicated in the emergence of the embryo from obligate diapause.

Embryonic diapause is a poorly understood phenomenon of reversible arrest of embryo development prior to implantation. In many carnivores, such as the mink (Neovison vison), obligate diapause characterizes each gestation. Embryo reactivation is controlled by the uterus by mechanisms that remain elusive. Because polyamines are essential regulators of cell proliferation and growth, it was hypothesized that they trigger embryo reactivation. To test this, mated mink females were treated with α-difluoromethylornithine, an inhibitor of ornithine decarboxylase 1, the rate-limiting enzyme in polyamine biosynthesis, or saline as a control during the first 5 d of reactivation. This treatment induced polyamine deprivation with the consequence of rearrest in embryo cell proliferation. A mink trophoblast cell line in vitro subjected to α-difluoromethylornithine treatment likewise displayed an arrest in cell proliferation, morphological changes, and intracellular translocation of ornithine decarboxylase 1 protein. The arrest in embryo development deferred implantation for a period consistent with the length of treatment. Successful implantation and parturition ensued. We conclude that polyamine deprivation brought about a reversible rearrest of embryo development, which returned the mink embryo to diapause and induced a second delay in embryo implantation. The results are the first demonstration of a factor essential to reactivation of embryos in obligate diapause.

Uterine signaling at the emergence of the embryo from obligate diapause.

Embryonic diapause is the reversible arrest of embryo development prior to implantation under a regime of uterine control that is not well understood. Our objective was to explore uterine modifications associated with the emergence of embryonic diapause in the mink, a species in which embryonic diapause characterizes every gestation. We investigated the uterine transcriptome at reactivation using the suppressive subtractive hybridization technique. A library of 123 differentially expressed genes between uteri with blastocysts in diapause and reactivated blastocysts was generated. Among those genes, 41.5% encode for potential secreted products that are implicated in regulation of cell proliferation (14%), homeostasis (14%), protein folding (11%), electron transport chain (8%), and innate immune response (8%), therefore suggesting that these biological processes are implicated in blastocyst reactivation. Two genes, the high-mobility group nucleosome binding domain 1 (HMGN1), a chromatin remodeling factor, and the secreted protein acidic and cystein-rich (SPARC), which is implicated in extracellular cell-cell interactions, were submitted to more detailed analysis of expression patterns in the mink uterus at blastocyst reactivation. Expression of both HMGN1 and SPARC was increased significantly in the uterus at embryo reactivation compared with diapause, principally in the endometrial epithelium and subepithelial stroma. These results provide new insight into uterine signaling at the emergence of the blastocyst from diapause and highlight the factors HMGN1 and SPARC as potential inductors of uterine environment modifications underlying uterine signaling during emergence of the embryo from embryonic diapause.

Differential gene expression in the uterus and blastocyst during the reactivation of embryo development in a model of delayed implantation.

Delayed implantation, a reversible arrest in embryo development while the embryo is at the blastocyst stage, provides an interesting window for observation of the preparation for implantation on both the embryonic and maternal sides. The American mink (Mustela vison) is a species in which delayed implantation is a normal aspect of embryo development as it consistently occurs at each breeding season. We used a transcriptome-wide approach to screen global gene expression and to identify new key genes expressed in the uterus and in the endometrium at the resumption of the embryo development, after delayed implantation. By using the suppressive subtractive hybridization (SSH) technique, two libraries of differentially expressed cDNAs, one at the uterine level and a second one at the blastocyst level, were successfully generated. Candidate genes from those two libraries were selected and their differentially expressed pattern of expression between reactivation and delayed implantation was investigated by real-time PCR and immunolocalization.