Identification of optimal conditions for human placental explant culture and extracellular vesicle release.

Extracellular vesicles (EVs) can mediate intercellular communication, including signaling between the placenta and maternal tissues. Human placental explant culture is a versatile in vitro model system to investigate placental function. We performed systematic studies in different tissue culture media types and oxygen tensions to identify a defined serum-free culture condition that supports high trophoblast viability and metabolism, as well as the release of similar populations of EVs, compared to traditional undefined conditions that contain media additives potentially contaminated with exogenous EVs. We also determined the time frame in which trophoblast viability and functionality remain optimal. Multiplex vesicle flow cytometry with classical EV and placenta-specific markers revealed three separate populations of explant-derived EVs: small CD63+ EVs; large PLAP+ EVs; and CD63-/PLAP- EVs. These culture and analytical approaches will enable in vitro modeling of short-term effects of environmental perturbations associated with pregnancy complications on placental function and EV release.

An Atypical Presentation of Kawasaki Disease and Potential Markers for Diagnosis.

Cervical lymphadenopathy is seldom the initial symptom of Kawasaki disease (KD), making diagnosis difficult in early node-first Kawasaki disease (NFKD). Early treatment is important to prevent cardiovascular sequelae. This report discusses a case of a 4-year-old African American female with NFKD and retropharyngeal phlegmon who was initially treated with antibiotics for cervical lymphadenitis. She later developed classic symptoms of KD, including mucositis, conjunctivitis, palmar erythema, and truncal rash. KD was then suspected and treated appropriately, with the patient experiencing rapid clinical improvement. Early misdiagnosis of NFKD is not uncommon, but certain indices, such as patient age, elevated absolute neutrophil count, or elevated liver enzymes, may be helpful in increasing clinical suspicion. NFKD and retropharyngeal phlegmon remain a rare presentation of an already known condition. The case presented here emphasizes the need for KD to be a differential diagnosis in cases of cervical lymphadenitis and retropharyngeal abscess refractory to antibiotic treatment.

The effects of progesterone on immune cellular function at the maternal-fetal interface and in maternal circulation.

Progesterone is a sex steroid hormone that plays a critical role in the establishment and maintenance of pregnancy. This hormone drives numerous maternal physiological adaptations to ensure the continuation of pregnancy and to facilitate fetal growth, including broad and potent modulation of the maternal immune system to promote maternal-fetal tolerance. In this brief review, we provide an overview of the immunomodulatory functions of progesterone in the decidua, placenta, myometrium, and maternal circulation during pregnancy. Specifically, we summarize current evidence of the regulated functions of innate and adaptive immune cells induced by progesterone and its downstream effector molecules in these compartments, including observations in human pregnancy and in animal models. Our review highlights the gaps in knowledge of interactions between progesterone and maternal cellular immunity that may direct future research.

Defining a role for Interferon Epsilon in normal and complicated pregnancies.

Interferon epsilon (IFNe) is a recently described cytokine that is constitutively expressed in the female reproductive tract. However, the role of this hormonally regulated cytokine during human pregnancy is poorly understood. Moreover, whether IFNe participates in host immune response against bacteria-driven intra-amniotic infection or cervical human papillomavirus infection during pregnancy is unknown. Herein, using a unique set of human samples derived from multiple study cohorts, we aimed to uncover the role of IFNe in normal and complicated pregnancies. We showed that IFNe is expressed in the myometrium, cervix, and chorioamniotic membranes, and may therefore represent a constitutive element of host defense mechanisms in these tissues during pregnancy. The expression of IFNe in the myometrium and cervix appeared greater in late gestation than in mid-pregnancy, but did not seem to be impacted by labor. Notably, concentrations of IFNe in amniotic fluid, but not cervical fluid, were increased in a subset of women undergoing spontaneous preterm labor with intra-amniotic infection, indicating that IFNe could participate in anti-microbial responses in the amniotic cavity. However, stimulation with Ureaplasma parvum and/or lipopolysaccharide did not enhance IFNE expression by amnion epithelial or cervical cells in vitro, implicating alternative sources of this cytokine during intra-amniotic or cervical infection, respectively. Collectively, our results represent the first characterization of IFNe expression by human reproductive and gestational tissues during normal pregnancy and suggest a role for this cytokine in intra-amniotic infection leading to preterm birth.

Clarithromycin prevents preterm birth and neonatal mortality by dampening alarmin-induced maternal-fetal inflammation in mice.

BACKGROUND: One of every four preterm neonates is born to a woman with sterile intra-amniotic inflammation (inflammatory process induced by alarmins); yet, this clinical condition still lacks treatment. Herein, we utilized an established murine model of sterile intra-amniotic inflammation induced by the alarmin high-mobility group box-1 (HMGB1) to evaluate whether treatment with clarithromycin prevents preterm birth and adverse neonatal outcomes by dampening maternal and fetal inflammatory responses. METHODS: Pregnant mice were intra-amniotically injected with HMGB1 under ultrasound guidance and treated with clarithromycin or vehicle control, and pregnancy and neonatal outcomes were recorded (n = 15 dams each). Additionally, amniotic fluid, placenta, uterine decidua, cervix, and fetal tissues were collected prior to preterm birth for determination of the inflammatory status (n = 7-8 dams each). RESULTS: Clarithromycin extended the gestational length, reduced the rate of preterm birth, and improved neonatal mortality induced by HMGB1. Clarithromycin prevented preterm birth by interfering with the common cascade of parturition as evidenced by dysregulated expression of contractility-associated proteins and inflammatory mediators in the intra-uterine tissues. Notably, clarithromycin improved neonatal survival by dampening inflammation in the placenta as well as in the fetal lung, intestine, liver, and spleen. CONCLUSIONS: Clarithromycin prevents preterm birth and improves neonatal survival in an animal model of sterile intra-amniotic inflammation, demonstrating the potential utility of this macrolide for treating women with this clinical condition, which currently lacks a therapeutic intervention.

Feedback Control of Snf1 Protein and Its Phosphorylation Is Necessary for Adaptation to Environmental Stress.

Snf1, a member of the AMP-activated protein kinase family, plays a critical role in metabolic energy control in yeast cells. Snf1 activity is activated by phosphorylation of Thr-210 on the activation loop of its catalytic subunit; following activation, Snf1 regulates stress-responsive transcription factors. Here, we report that the level of Snf1 protein is dramatically decreased in a UBP8- and UBP10-deleted yeast mutant (ubp8Δ ubp10Δ), and this is independent of transcriptional regulation and proteasome-mediated degradation. Surprisingly, most Snf1-mediated functions, including glucose limitation regulation, utilization of alternative carbon sources, stress responses, and aging, are unaffected in this strain. Snf1 phosphorylation in ubp8Δ ubp10Δ cells is hyperactivated upon stress, which may compensate for the loss of the Snf1 protein and protect cells against stress and aging. Furthermore, artificial elevation of Snf1 phosphorylation (accomplished through deletion of REG1, which encodes a protein that regulates Snf1 dephosphorylation) restored Snf1 protein levels and the regulation of Snf1 activity in ubp8Δ ubp10Δ cells. Our results reveal the existence of a feedback loop that controls Snf1 protein level and its phosphorylation, which is masked by Ubp8 and Ubp10 through an unknown mechanism. We propose that this dynamic modulation of Snf1 phosphorylation and its protein level may be important for adaptation to environmental stress.