Subunit Vaccine ESAT-6:c-di-AMP Delivered by Intranasal Route Elicits Immune Responses and Protects Against Mycobacterium tuberculosis Infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, remains the most common cause of death from a single infectious disease. More safe and effective vaccines are necessary for preventing the prevalence of TB. In this study, a subunit vaccine of ESAT-6 formulated with c-di-AMP (ESAT-6:c-di-AMP) promoted mucosal and systemic immune responses in spleen and lung. ESAT-6:c-di-AMP inhibited the differentiations of CD8+ T cells as well as macrophages, but promoted the differentiations of ILCs in lung. The co-stimulation also enhanced inflammatory cytokines production in MH-S cells. It was first revealed that ESAT-6 and c-di-AMP regulated autophagy of macrophages in different stages, which together resulted in the inhibition of Mtb growth in macrophages during early infection. After Mtb infection, the level of ESAT-6-specific immune responses induced by ESAT-6:c-di-AMP dropped sharply. Finally, inoculation of ESAT-6:c-di-AMP led to significant reduction of bacterial burdens in lungs and spleens of immunized mice. Our results demonstrated that subunit vaccine ESAT-6:c-di-AMP could elicit innate and adaptive immune responses which provided protection against Mtb challenge, and c-di-AMP as a mucosal adjuvant could enhance immunogenicity of antigen, especially for innate immunity, which might be used for new mucosal vaccine against TB.

Synthetic female gonadal hormones alter neurodevelopmental programming and behavior in F1 offspring.

The increased prevalence of neurodevelopmental disorders during the last half-century led us to investigate the potential for intergenerational detrimental neurodevelopmental effects of synthetic female gonadal hormones, typically used in contraceptive pills. We examined 3 separate cohorts of mice over the span of 2 years, a total of 150 female F0 mice and over 300 male and female rodents from their F1 progeny. We demonstrate that F1 male offsprings of female mice previously exposed to the synthetic estrogen 17α-ethinylestradiol (EE2) in combination with the synthetic progestin Norethindrone, exhibit neurodevelopmental and behavioral differences compared to control mice. Because the EE2 + Norethindrone administration resulted in gene expression changes in the exposed F0 mice ovaries persisting after the end of treatment, it is likely that the synthetic hormone treatment caused changes in the germline cells and that led to altered neurodevelopment in the offsprings. An altered gene expression pattern was discovered in the frontal cortex of male mice from the first offspring (F1.1) at infancy and an ADHD-like hyperactive locomotor behavior was exhibited in young male mice from the second offspring (F1.2) of female mice treated with contraceptive pill doses of EE2 + Norethindrone prior to pregnancy. The intergenerational neurodevelopmental effects of EE2 + Norethindrone treatment were sex specific, predominantly affecting males. Our observations in mice support the hypothesis that the use of synthetic contraceptive hormones is a potential environmental factor impacting the prevalence of human neurodevelopmental disorders. Additionally, our results indicate that contraceptive hormone drug safety assessments may need to be extended to F1 offspring.

Testicular Lmcd1 regulates phagocytosis by Sertoli cells through modulation of NFAT1/Txlna signaling pathway.

Increased oxidative stress is well known to cause testicular dysfunction in aging males, but the detailed relationships between aging, oxidative stress, and testicular function remain to be elucidated. LIM and cysteine-rich domains 1 (LMCD1) regulates fundamentally cellular process by interacting with transcription factors. A recent study has identified Lmcd1 as one of the most upregulated nuclear proteins associated with Sertoli cell (SC) differentiation, raising the possibility that testicular actions of LMCD1 are likely to take place. Herein, we reported that LMCD1 was exclusively expressed in the nuclei of SCs. This expression was regulated by TNF-α signaling produced by apoptotic germ cells (GCs) and was suppressed by oxidative stress in a STAT3-dependent manner. Ablation of endogenous LMCD1 expression caused lipid accumulation and senescence in GC co-incubated SCs. Using a previously validated in vivo siRNA approach, we showed that LMCD1 depletion significantly impaired male fertility by inducing oligozoospermia and asthenospermia. Mechanistically, LMCD1 upregulation was associated with the nuclear enrichment of the nuclear factor of activated T cells 1 (NFAT1), a core component of Ca2+ /calmodulin-dependent pathway. LMCD1 facilitated the dephosphorylation and nuclear translocation of NFAT1, which consequently expedited the transactivation of Txlna, a binding partner of the syntaxin family essential for testicular phagocytosis, and thus promoted the removal of apoptotic GCs by phagocytic SCs. Collectively, LMCD1 may operate as a novel pretranscriptional integrator linking SC phagocytosis, lipid homeostasis, and cell senescence.

Developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure of either parent enhances the risk of necrotizing enterocolitis in neonatal mice.

BACKGROUND: Necrotizing enterocolitis (NEC) is a rare, but potentially fatal intestinal inflammatory condition most often arising in premature infants. Infants provided formula are also at greater risk of developing this disease. Although the majority of formula-fed, preterm infants do not develop NEC, up to 30% of infants with the disease do not survive. Thus, identifying additional, currently unrecognized factors, which may predispose a specific infant to NEC development would be a significant clinical advancement. In this regard, we have previously reported that offspring of female or male mice with a history of developmental exposure to the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exhibit altered sensitivity to inflammatory challenges and are frequently born premature. Herein, we examined the possibility that, compared to unexposed mice (F1NONE ), developmental TCDD exposure of either parent (maternal, F1MTCDD , or paternal, F1PTCDD ) would enhance the risk of NEC in offspring (F2TCDD mice) in association with supplemental formula feeding. METHODS: Beginning on postnatal day 7, all neonates were randomized to maternal milk only or maternal milk with up to 20 supplemental formula feedings. All pups remained with the Dams and were additionally allowed to nurse ad libitum. RESULTS: Formula-fed F2NONE pups rarely developed NEC while this disease was common in formula-fed F2MTCDD and F2PTCDD mice. Unexpectedly, 50% of F2MTCDD pups that were not provided supplemental formula also developed NEC. CONCLUSIONS: Our studies provide evidence that a history of parental TCDD exposure enhances the risk of NEC in offspring and suggest exposure to environmental immunotoxicants such as TCDD may also contribute to this inflammatory disease in humans.

Fetal Membrane Organ-On-Chip: An Innovative Approach to Study Cellular Interactions.

OBJECTIVE: Fetal membranes, a vital component that helps maintain pregnancy and contribute to parturition signaling, are often studied in segments due to its structural complexity. Transwells are traditionally used to study cell interactions; however, their usefulness is limited. To overcome these difficulties, a fetal membrane-organ-on-chip (FM-OO-C) was created to study interactive properties of amnion epithelial cells (AECs) and decidual cells compared to transwell systems. METHODS: Primary AECs and decidual cells from term, nonlaboring fetal membranes were cultured in a 2-chamber (AEC/decidual cell) FM-OO-C device and sandwiched between a semipermeable membrane. Cells were treated with cigarette smoke extract (CSE) or dioxin, and membrane permeability and cellular senescence were measured after 48 hours. The same experiments were conducted in transwells for comparisons. RESULTS: Compared to transwell cultures, FM-OO-C model produced better membrane permeability readings regardless of the side of treatment or time point. Membrane permeabilization was higher in AECs directly treated with CSE (1.6 fold) compared to similar treatment on the decidual side (1.2 fold). In FM-OO-C, treatments forced changes between cellular layers. This was evident when CSE and dioxin-induced senescence on one side of the chamber produced similar changes on the opposite side. This effect was minimal in the transwell system. CONCLUSION: The controlled environment of an FM-OO-C allows for improved signal propagation between cells by minimizing noise and highlighting the small changes between treatments that cannot be seen in conventional transwell devices. Fetal membrane-organ-on-chip provides a better interaction between cell types that can be used to study fetal-maternal signaling during pregnancy in future studies.

Recombinant BCG With Bacterial Signaling Molecule Cyclic di-AMP as Endogenous Adjuvant Induces Elevated Immune Responses After Mycobacterium tuberculosis Infection.

Bacillus Calmette-Guerin (BCG) is a live attenuated vaccine against tuberculosis (TB) and remains the most commonly used vaccine worldwide. However, BCG has varied protective efficiency in adults and has safety concerns in immunocompromised population. Thus, effective vaccines are necessary for preventing the prevalence of TB. Cyclic di-AMP (c-di-AMP) is a bacterial second messenger which regulates various cellular processes and host immune response. Previous work found that c-di-AMP regulates bacterial physiological function, pathogenicity and host type I IFN response. In this study, we constructed a recombinant BCG (rBCG) by overexpressing DisA, the diadenylate cyclase of Mycobacterium tuberculosis (Mtb), and observed the physiological changes of rBCG-DisA. The immunological characteristics of rBCG-DisA were investigated on humoral and cellar immune responses in a mice infection model. Our study demonstrated that overexpression of DisA in BCG does not affect the growth but reduces the length of BCG. rBCG-DisA-immunized mice show similar humoral and cellar immune responses in BCG-immunized mice. After Mtb infection, the splenic lymphocytes from both BCG and rBCG-DisA-immunized mice produced more IFN-γ, IL-2, and IL-10 than the un-immunized (UN) mice, while the cytokine levels of the rBCG-DisA group increased significantly than those of the BCG group. The transcription of IFN-ß, IL-1ß and autophagy related genes (Atgs) were up-regulated in macrophages after treated with c-di-AMP or bacterial infection. The productions of IL-6 were increased after Mtb challenge, especially in the rBCG-DisA-immunized mice. Strikingly, H3K4me3, the epigenetic marker of innate immune memory, was found in both two immunized groups, and the rBCG-DisA group showed stronger expression of H3K4me3 than that of BCG. In addition, the pathological changes of rBCG-DisA immunized mice were similar to that of BCG-immunized mice. The bacterial burdens in the lungs and spleens of BCG- and rBCG-DisA-immunized mice were significantly decreased, but there was no significant difference between the two immunized groups. Together, these results suggested that compared to BCG, rBCG-DisA vaccination, induces stronger immune responses but did not provided additional protection against Mtb infection in this study, which may be related to the innate immunity memory. Hence, c-di-AMP is a promising immunomodulator for a further developed BCG as a better vaccine.

Hemodynamic forces enhance decidualization via endothelial-derived prostaglandin E2 and prostacyclin in a microfluidic model of the human endometrium.

STUDY QUESTION: Does the uterine vasculature play a localized role in promoting stromal cell decidualization in the human endometrium? SUMMARY ANSWER: Our study demonstrated that hemodynamic forces induced secretion of specific endothelial cell-derived prostanoids that enhanced endometrial perivascular decidualization via a paracrine mechanism. WHAT IS KNOWN ALREADY: Differentiation of stromal cell fibroblasts into the specialized decidua of the placenta is a progesterone-dependent process; however, histologically, it has long been noted that the first morphological signs of decidualization appear in the perivascular stroma. These observations suggest that the human endometrial vasculature plays an active role in promoting stromal differentiation. STUDY DESIGN, SIZE, DURATION: Primary human endometrial stromal cells were co-cultured for 14 days with primary uterine microvascular endothelial cells within a microfluidic Organ-on-Chip model of the endometrium. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cultures were maintained with estradiol and a progestin, with or without continuous laminar perfusion to mimic hemodynamic forces derived from the blood flow. Some cultures additionally received exogenous agonist-mediated challenges. Decidualization in the microfluidic model was assessed morphologically and biochemically. ELISA was used to examine the culture effluent for expression of decidualization markers and prostaglandins. Immunofluorescence was used to monitor cyclooxygenase-2 expression in association with decidualization. MAIN RESULTS AND THE ROLE OF CHANCE: A significantly enhanced stromal decidualization response was observed in the co-cultures when the endothelial cells were stimulated with hemodynamic forces (e.g. laminar shear stress) derived from controlled microfluidic perfusion (<0.001). Furthermore, the enhanced progestin-driven stromal differentiation was mediated via cyclooxygenase-2 and the paracrine action of prostaglandin E2 and prostacyclin. Altogether, these translational findings indicate that the vascular endothelium plays a key physiologic role during the early events of perivascular decidualization in the human endometrium. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This report is largely an in vitro study. Although we were able to experimentally mimic hemodynamic forces in our microfluidic model, we have not yet determined the contribution of additional cell types to the decidualization process or determined the precise physiological rates of shear stress that the microvasculature of the endometrium undergoes in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Identification of specific endothelial-derived prostaglandins and their role during endometrial reproductive processes may have clinical utility as therapeutic targets for reproductive disorders such as infertility, endometriosis, adenomyosis, pre-eclampsia and poor pregnancy outcomes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Veterans Affairs (I01 BX002853), the Bill and Melinda Gates Foundation Grand Challenges Exploration (OPP1159411), the Environmental Toxicology Training Grant (NIH T32 ES007028) and the Environmental Protection Agency STAR Center Grant (83573601). CONFLICT OF INTEREST: The authors report no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Fetal Membrane Organ-On-Chip: An Innovative Approach to Study Cellular Interactions.

OBJECTIVE:: Fetal membranes, a vital component that helps maintain pregnancy and contribute to parturition signaling, are often studied in segments due to its structural complexity. Transwells are traditionally used to study cell interactions; however, their usefulness is limited. To overcome these difficulties, a fetal membrane-organ-on-chip (FM-OO-C) was created to study interactive properties of amnion epithelial cells (AECs) and decidual cells compared to transwell systems. METHODS:: Primary AECs and decidual cells from term, nonlaboring fetal membranes were cultured in a 2-chamber (AEC/decidual cell) FM-OO-C device and sandwiched between a semipermeable membrane. Cells were treated with cigarette smoke extract (CSE) or dioxin, and membrane permeability and cellular senescence were measured after 48 hours. The same experiments were conducted in transwells for comparisons. RESULTS:: Compared to transwell cultures, FM-OO-C model produced better membrane permeability readings regardless of the side of treatment or time point. Membrane permeabilization was higher in AECs directly treated with CSE (1.6 fold) compared to similar treatment on the decidual side (1.2 fold). In FM-OO-C, treatments forced changes between cellular layers. This was evident when CSE and dioxin-induced senescence on one side of the chamber produced similar changes on the opposite side. This effect was minimal in the transwell system. CONCLUSION:: The controlled environment of an FM-OO-C allows for improved signal propagation between cells by minimizing noise and highlighting the small changes between treatments that cannot be seen in conventional transwell devices. Fetal membrane-organ-on-chip provides a better interaction between cell types that can be used to study fetal-maternal signaling during pregnancy in future studies.

Paternal Environmental Toxicant Exposure and Risk of Adverse Pregnancy Outcomes.

PURPOSE OF REVIEW: Current clinical efforts to predict and prevent preterm birth are primarily focused on the mother and have made minimal progress in improving outcomes. However, recent data indicate that paternal factors can also influence timing of birth. Herein, we will review recent human and murine data examining the contribution of the father to pregnancy outcomes with an emphasis on environmental exposures that can negatively impact fertility and the timing of birth. RECENT FINDINGS: Human epidemiology studies now clearly indicate that a variety of paternal factors (age, race, weight, smoking status) can influence sperm quality, birth timing and, in some studies, offspring health. Utilizing a mouse model, our data have 57demonstrated that developmental exposure to the environmental toxicant TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is associated with a transgenerational reduction in sperm number and quality and an increased risk of preterm birth in an unexposed partner. SUMMARY: Toxicant exposure history can clearly influence sperm quality in men and mice. Murine data further indicate that exposures which negatively affect sperm quality also impair placental function, potentially leading to preterm birth and other adverse outcomes. Of particular concern, these changes have been linked to epigenetic alterations within the male germ cell which can then be transmitted across multiple generations. Since it is not possible to prevent an ancestral toxicant exposure in a human population, identifying lifestyle modifications that can be implemented during the preconception period to improve sperm quality should be explored for the therapeutic potential to reduce the incidence of PTB and its sequelae.

Rodent Models of Experimental Endometriosis: Identifying Mechanisms of Disease and Therapeutic Targets.

BACKGROUND: Although it has been more than a century since endometriosis was initially described in the literature, understanding the etiology and natural history of the disease has been challenging. However, the broad utility of murine and rat models of experimental endometriosis has enabled the elucidation of a number of potentially targetable processes which may otherwise promote this disease. OBJECTIVE: To review a variety of studies utilizing rodent models of endometriosis to illustrate their utility in examining mechanisms associated with development and progression of this disease. RESULTS: Use of rodent models of endometriosis has provided a much broader understanding of the risk factors for the initial development of endometriosis, the cellular pathology of the disease and the identification of potential therapeutic targets. CONCLUSION: Although there are limitations with any animal model, the variety of experimental endometriosis models that have been developed has enabled investigation into numerous aspects of this disease. Thanks to these models, our under-standing of the early processes of disease development, the role of steroid responsiveness, inflammatory processes and the peritoneal environment has been advanced. More recent models have begun to shed light on how epigenetic alterations con-tribute to the molecular basis of this disease as well as the multiple comorbidities which plague many patients. Continued de-velopments of animal models which aid in unraveling the mechanisms of endometriosis development provide the best oppor-tunity to identify therapeutic strategies to prevent or regress this enigmatic disease.

Paternal developmental toxicant exposure is associated with epigenetic modulation of sperm and placental Pgr and Igf2 in a mouse model.

Preterm birth (PTB), parturition prior to 37 weeks' gestation, is the leading cause of neonatal mortality. The causes of spontaneous PTB are poorly understood; however, recent studies suggest that this condition may arise as a consequence of the parental fetal environment. Specifically, we previously demonstrated that developmental exposure of male mice (F1 animals) to the environmental endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was associated with reduced sperm quantity/quality in adulthood and control female partners frequently delivered preterm. Reproductive defects persisted in the F2 and F3 descendants, and spontaneous PTB was common. Reproductive changes in the F3 males, the first generation without direct TCDD exposure, suggest the occurrence of epigenetic alterations in the sperm, which have the potential to impact placental development. Herein, we conducted an epigenetic microarray analysis of control and F1 male-derived placentae, which identified 2171 differentially methylated regions, including the progesterone receptor (Pgr) and insulin-like growth factor (Igf2). To assess if Pgr and Igf2 DNA methylation changes were present in sperm and persist in future generations, we assessed methylation and expression of these genes in F1/F3 sperm and F3-derived placentae. Although alterations in methylation and gene expression were observed, in most tissues, only Pgr reached statistical significance. Despite the modest gene expression changes in Igf2, offspring of F1 and F3 males consistently exhibited IUGR. Taken together, our data indicate that paternal developmental TCDD exposure is associated with transgenerational placental dysfunction, suggesting epigenetic modifications within the sperm have occurred. An evaluation of additional genes and alternative epigenetic mechanisms is warranted.

Structural basis for neutralization of Japanese encephalitis virus by two potent therapeutic antibodies.

Japanese encephalitis virus (JEV), closely related to dengue, Zika, yellow fever and West Nile viruses, remains neglected and not well characterized 1 . JEV is the leading causative agent of encephalitis, and is responsible for thousands of deaths each year in Asia. Humoral immunity is essential for protecting against flavivirus infections and passive immunization has been demonstrated to be effective in curing disease2,3. Here, we demonstrate that JEV-specific monoclonal antibodies, 2F2 and 2H4, block attachment of the virus to its receptor and also prevent fusion of the virus. Neutralization of JEV by these antibodies is exceptionally potent and confers clear therapeutic benefit in mouse models. A single 20 µg dose of these antibodies resulted in 100% survival and complete clearance of JEV from the brains of mice. The 4.7 Å and 4.6 Å resolution cryo-electron microscopy structures of JEV-2F2-Fab and JEV-2H4-Fab complexes, together with the crystal structure of 2H4 Fab and our recent near-atomic structure of JEV 4 , unveil the nature and location of epitopes targeted by the antibodies. Both 2F2 and 2H4 Fabs bind quaternary epitopes that span across three adjacent envelope proteins. Our results provide a structural and molecular basis for the application of 2F2 and 2H4 to treat JEV infection.

Sex-Dependent Influence of Developmental Toxicant Exposure on Group B Streptococcus-Mediated Preterm Birth in a Murine Model.

Infectious agents are a significant risk factor for preterm birth (PTB); however, the simple presence of bacteria is not sufficient to induce PTB in most women. Human and animal data suggest that environmental toxicant exposures may act in concert with other risk factors to promote PTB. Supporting this "second hit" hypothesis, we previously demonstrated exposure of fetal mice (F1 animals) to the environmental endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to an increased risk of spontaneous and infection-mediated PTB in adult animals. Surprisingly, adult F1males also confer an enhanced risk of PTB to their control partners. Herein, we used a recently established model of ascending group B Streptococcus (GBS) infection to explore the impact of a maternal versus paternal developmental TCDD exposure on infection-mediated PTB in adulthood. Group B Streptococcus is an important contributor to PTB in women and can have serious adverse effects on their infants. Our studies revealed that although gestation length was reduced in control mating pairs exposed to low-dose GBS, dams were able to clear the infection and bacterial transmission to pups was minimal. In contrast, exposure of pregnant F1females to the same GBS inoculum resulted in 100% maternal and fetal mortality. Maternal health and gestation length were not impacted in control females mated to F1males and exposed to GBS; however, neonatal survival was reduced compared to controls. Our data revealed a sex-dependent impact of parental TCDD exposure on placental expression of Toll-like receptor 2 and glycogen production, which may be responsible for the differential impact on fetal and maternal outcomes in response to GBS infection.

Heat shock protein 90ß in the Vero cell membrane binds Japanese encephalitis virus.

The pathogenesis of Japanese encephalitis virus (JEV) is complex and unclearly defined, and in particular, the effects of the JEV receptor (JEVR) on diverse susceptible cells are elusive. In contrast to previous studies investigating JEVR in rodent or mosquito cells, in this study, we used primate Vero cells instead. We noted that few novel proteins co­immunoprecipitated with JEV, and discovered that one of these was heat shock protein 90ß (HSP90ß), which was probed by mass spectrometry with the highest score of 60.3 after questing the monkey and human protein databases. The specific HSP90ß­JEV binding was confirmed by western blot analysis under non­reducing conditions, and this was significantly inhibited by an anti­human HSP90ß monoclonal antibody in a dose­dependent manner, as shown by immunofluorescence assay and flow cytometry. In addition, the results of confocal laser scanning microscopic examination demonstrated that the HSP90ß­JEV binding occurred on the Vero cell surface. Finally, JEV progeny yields determined by plaque assay were also markedly decreased in siRNA­treated Vero cells, particularly at 24 and 36 h post­infection. Thus, our data indicate that HSP90ß is a binding receptor for JEV in Vero cells.

Therapeutically Targeting the Inflammasome Product in a Chimeric Model of Endometriosis-Related Surgical Adhesions.

Development of adhesions commonly occurs in association with surgery for endometriosis. Even in the absence of surgery, women with endometriosis appear to be at an enhanced risk of developing adhesions. In the current study, we utilized a chimeric mouse model of experimental endometriosis in order to examine the role of inflammasome activation in the development of postsurgical adhesions. Mice were randomized to receive peritoneal injections of human endometrial tissue fragments or endometrial tissue conditioned media (CM) from women with or without endometriosis 16 hours after ovariectomy and placement of an estradiol-releasing silastic capsule. A subset of mice receiving CM was also treated with interleukin (IL) 1 receptor antagonist (IL-1ra). Our studies demonstrate that peritoneal injection of endometrial tissue fragments near the time of surgery resulted in extensive adhesive disease regardless of tissue origin. However, adhesion scores were significantly higher in mice receiving CM from tissues acquired from patients with endometriosis compared to control tissue CM ( P = .0001). Cytokine bead array analysis of endometrial CM revealed enhanced expression of IL-1ß from patients with endometriosis compared to controls ( P < .01). Finally, the ability of human tissue CM to promote adhesive disease was dramatically reduced in mice cotreated with IL-1ra ( P < .0001). Our data implicate enhanced expression of IL-1ß in women with endometriosis as a potential causal factor in their increased susceptibility of developing postsurgical adhesions. Thus, targeting inflammasome activation may be an effective strategy for the prevention of surgical adhesions in patients with endometriosis.

Comparison of lens oxidative damage induced by vitrectomy and/or hyperoxia in rabbits.

AIM: To compare of lens oxidative damage induced by vitrectomy and/or hyperoxia in rabbit. METHODS: Sixteen New Zealand rabbits (2.4-2.5 kg) were randomly divided into two groups (Group A, n=12; Group B, n=4). In Group A, the right eyes were treated with vitrectomy and systemic hyperoxia (oxygen concentration: 80%-85%, 1 ATA, 4h/d) (Group A-right), and the left eyes were treated with hyperoxia without vitrectomy surgery (Group A-left). Four rabbits in group B (eight eyes) were untreated as the controls. Lens transparency was monitored with a slit lamp and recorded before and after vitrectomy. After hyperoxic treatment for 6mo, the eyeballs were removed and the lens cortices (containing the capsules) and nuclei were separated for further morphological and biochemical evaluation. RESULTS: Six months after treatments, there were no significant morphological changes in the lenses in any experimental group when observed with a slit lamp. However, the levels of water-soluble proteins and ascorbate, and the activities of catalase and Na+-K+-ATPase were significantly reduced, whereas the levels of malondialdehyde and transforming growth factor ß2 (TGF-ß2) were significantly elevated, in both the cortices and nuclei of eyes treated with vitrectomy and hyperoxia. The increase in protein-glutathione mixed disulfides and the reduction in water-soluble proteins were more obvious in the lens nuclei. The levels of ascorbate in the vitreous fluid were also reduced after vitrectomy, whereas TGF-ß2 increased after vitrectomy and hyperoxia. Systemic hyperoxia exposure increased these effects. CONCLUSION: Removal of the intact vitreous gel with vitrectomy and exposing the lens to increased oxygen from the retina induce lens oxidation and aggregation. Thus, an intact vitreous gel structure may protect the lens from oxidative insult and maintain lens transparency.

Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium.

The endometrium is the inner lining of the uterus. Following specific cyclic hormonal stimulation, endometrial stromal fibroblasts (stroma) and vascular endothelial cells exhibit morphological and biochemical changes to support embryo implantation and regulate vascular function, respectively. Herein, we integrated a resin-based porous membrane in a dual chamber microfluidic device in polydimethylsiloxane that allows long term in vitro co-culture of human endometrial stromal and endothelial cells. This transparent, 2-µm porous membrane separates the two chambers, allows for the diffusion of small molecules and enables high resolution bright field and fluorescent imaging. Within our primary human co-culture model of stromal and endothelial cells, we simulated the temporal hormone changes occurring during an idealized 28-day menstrual cycle. We observed the successful differentiation of stroma into functional decidual cells, determined by morphology as well as biochemically as measured by increased production of prolactin. By controlling the microfluidic properties of the device, we additionally found that shear stress forces promoted cytoskeleton alignment and tight junction formation in the endothelial layer. Finally, we demonstrated that the endometrial perivascular stroma model was sustainable for up to 4 weeks, remained sensitive to steroids and is suitable for quantitative biochemical analysis. Future utilization of this device will allow the direct evaluation of paracrine and endocrine crosstalk between these two cell types as well as studies of immunological events associated with normal vs. disease-related endometrial microenvironments.

Exposure to the environmental endocrine disruptor TCDD and human reproductive dysfunction: Translating lessons from murine models.

Humans and other animals are exposed to a wide array of man-made toxicants, many of which act as endocrine disruptors that exhibit differential effects across the lifespan. In humans, while the impact of adult exposure is known for some compounds, the potential consequences of developmental exposure to endocrine disrupting chemicals (EDCs) is more difficult to ascertain. Animal studies have revealed that exposure to EDCs prior to puberty can lead to adult reproductive disease and dysfunction. Specifically, in adult female mice with an early life exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we demonstrated a transgenerational occurrence of several reproductive diseases that have been linked to endometriosis in women. Herein, we review the evidence for TCDD-associated development of adult reproductive disease as well as known epigenetic alterations associated with TCDD and/or endometriosis. We will also introduce new "Organ-on-Chip" models which, combined with our established murine model, are expected to further enhance our ability to examine alterations in gene-environment interactions that lead to heritable disease.

LSm1 binds to the Dengue virus RNA 3' UTR and is a positive regulator of Dengue virus replication.

Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans. The DENV positive strand RNA genome contains 5' and 3' untranslated regions (UTRs) that have been shown to be required for virus replication and interaction with host cell proteins. In the present study LSm1 was identified as a host cellular protein involved in DENV RNA replication. By using two independent methodologies, we demonstrated a critical interaction between LSm1 and the 3' UTR of DENV. Furthermore, the confocal immunofluorescence analysis showed that the interaction between LSm1 and viral RNA is located in P-body around nucleoli in the cytoplasm. LSm1 knockdown by siRNA specifically reduced the levels of viral RNA in DENV-infected cells and infectious DENV particles in the supernatant. These results provide evidence that LSm1 binding to the DENV RNA 3' UTR positively regulates DENV RNA replication.

Cross-species transcriptomic approach reveals genes in hamster implantation sites.

The mouse model has greatly contributed to understanding molecular mechanisms involved in the regulation of progesterone (P4) plus estrogen (E)-dependent blastocyst implantation process. However, little is known about contributory molecular mechanisms of the P4-only-dependent blastocyst implantation process that occurs in species such as hamsters, guineapigs, rabbits, pigs, rhesus monkeys, and perhaps humans. We used the hamster as a model of P4-only-dependent blastocyst implantation and carried out cross-species microarray (CSM) analyses to reveal differentially expressed genes at the blastocyst implantation site (BIS), in order to advance the understanding of molecular mechanisms of implantation. Upregulation of 112 genes and downregulation of 77 genes at the BIS were identified using a mouse microarray platform, while use of the human microarray revealed 62 up- and 38 down-regulated genes at the BIS. Excitingly, a sizable number of genes (30 up- and 11 down-regulated genes) were identified as a shared pool by both CSMs. Real-time RT-PCR and in situ hybridization validated the expression patterns of several up- and down-regulated genes identified by both CSMs at the hamster and mouse BIS to demonstrate the merit of CSM findings across species, in addition to revealing genes specific to hamsters. Functional annotation analysis found that genes involved in the spliceosome, proteasome, and ubiquination pathways are enriched at the hamster BIS, while genes associated with tight junction, SAPK/JNK signaling, and PPARα/RXRα signalings are repressed at the BIS. Overall, this study provides a pool of genes and evidence of their participation in up- and down-regulated cellular functions/pathways at the hamster BIS.