Syncytialization alters the extracellular matrix and barrier function of placental trophoblasts.

The human placenta is of vital importance for proper nutrient and waste exchange, immune regulation, and overall fetal health and growth. Specifically, the extracellular matrix (ECM) of placental syncytiotrophoblasts, which extends outward from the placental chorionic villi into maternal blood, acts on a molecular level to regulate and maintain this barrier. Importantly, placental barrier dysfunction has been linked to diseases of pregnancy such as preeclampsia and intrauterine growth restriction. To help facilitate our understanding of the interface and develop therapeutics to repair or prevent dysfunction of the placental barrier, in vitro models of the placental ECM would be of great value. In this study, we aimed to characterize the ECM of an in vitro model of the placental barrier using syncytialized BeWo choriocarcinoma cells. Syncytialization caused a marked change in syndecans, integral proteoglycans of the ECM, which matched observations of in vivo placental ECM. Syndecan-1 expression increased greatly and predominated the other variants. Barrier function of the ECM, as measured by electric cell-substrate impedance sensing (ECIS), increased significantly during and after syncytialization, whereas the ability of THP-1 monocytes to adhere to syncytialized BeWos was greatly reduced compared with nonsyncytialized controls. Furthermore, ECIS measurements indicated that ECM degradation with matrix metalloproteinase-9 (MMP-9), but not heparanase, decreased barrier function. This decrease in ECIS-measured barrier function was not associated with any changes in THP-1 adherence to syncytialized BeWos treated with heparanase or MMP-9. Thus, syncytialization of BeWos provides a physiologically accurate placental ECM with a barrier function matching that seen in vivo.

NAA10 polyadenylation signal variants cause syndromic microphthalmia.

BACKGROUND: A single variant in NAA10 (c.471+2T>A), the gene encoding N-acetyltransferase 10, has been associated with Lenz microphthalmia syndrome. In this study, we aimed to identify causative variants in families with syndromic X-linked microphthalmia. METHODS: Three families, including 15 affected individuals with syndromic X-linked microphthalmia, underwent analyses including linkage analysis, exome sequencing and targeted gene sequencing. The consequences of two identified variants in NAA10 were evaluated using quantitative PCR and RNAseq. RESULTS: Genetic linkage analysis in family 1 supported a candidate region on Xq27-q28, which included NAA10. Exome sequencing identified a hemizygous NAA10 polyadenylation signal (PAS) variant, chrX:153,195,397T>C, c.*43A>G, which segregated with the disease. Targeted sequencing of affected males from families 2 and 3 identified distinct NAA10 PAS variants, chrX:g.153,195,401T>C, c.*39A>G and chrX:g.153,195,400T>C, c.*40A>G. All three variants were absent from gnomAD. Quantitative PCR and RNAseq showed reduced NAA10 mRNA levels and abnormal 3' UTRs in affected individuals. Targeted sequencing of NAA10 in 376 additional affected individuals failed to identify variants in the PAS. CONCLUSION: These data show that PAS variants are the most common variant type in NAA10-associated syndromic microphthalmia, suggesting reduced RNA is the molecular mechanism by which these alterations cause microphthalmia/anophthalmia. We reviewed recognised variants in PAS associated with Mendelian disorders and identified only 23 others, indicating that NAA10 harbours more than 10% of all known PAS variants. We hypothesise that PAS in other genes harbour unrecognised pathogenic variants associated with Mendelian disorders. The systematic interrogation of PAS could improve genetic testing yields.

Gsx transcription factors control neuronal versus glial specification in ventricular zone progenitors of the mouse lateral ganglionic eminence.

The homeobox gene Gsx2 has previously been shown to inhibit oligodendroglial specification in dorsal lateral ganglionic eminence (dLGE) progenitors of the ventral telencephalon. The precocious specification of oligodendrocyte progenitor cells (OPCs) observed in Gsx2 mutants, however, is transient and begins to normalize by late stages of embryogenesis. Interestingly, this normalization correlates with the expansion of Gsx1, a close homolog of Gsx2, in a subset of progenitors in the Gsx2 mutant LGE. Here, we interrogated the mechanisms underlying oligodendroglial specification in Gsx2 mutants in relation to Gsx1. We found that Gsx1/2 double mutant embryos exhibit a more robust expansion of Olig2+ cells (i.e. OPCs) in the subventricular zone (SVZ) of the dLGE than Gsx2 mutants. Moreover, misexpression of Gsx1 throughout telencephalic VZ progenitors from E15 and onward resulted in a significant reduction of cortical OPCs. These results demonstrate redundant roles of Gsx1 and Gsx2 in suppressing early OPC specification in LGE VZ progenitors. However, Gsx1/2 mutants did not show a significant increase in adjacent cortical OPCs at later stages compared to Gsx2 mutants. This is likely due to reduced proliferation of OPCs within the SVZ of the Gsx1/2 double mutant LGE, suggesting a novel role for Gsx1 in expansion of migrating OPCs in the ventral telencephalon. We further investigated the glial specification mechanisms downstream of Gsx2 by generating Olig2/Gsx2 double mutants. Consistent with the known essential role for Olig2 in OPC specification, ectopic production of cortical OPCs observed in Gsx2 mutants disappeared in Olig2/Gsx2 double mutants. These mutants, however, maintained the expanded expression of gliogenic markers Zbtb20 and Bcan in the VZ of the LGE similarly to Gsx2 single mutants, suggesting that Gsx2 suppresses gliogenesis via Olig2-dependent and -independent mechanisms.

PARTIAL HOSPITALIZATION: AN INTERVENTION FOR YOUTH WITH POORLY CONTROLLED DIABETES MELLITUS.

Objective: To examine the efficacy of an integrated medical/psychiatric partial hospitalization program (PHP) to improve glycemic control in youth with both diabetes mellitus and mental health disorders. Methods: This retrospective chart review is of patients admitted to a PHP between 2005-2015 with concerns about diabetes mellitus care. Clinical characteristics, laboratory data, diabetic ketoacidosis hospitalizations, and outpatient clinic visit frequency were collected from the year prior to the year after PHP admission. Results: A total of 43 individuals met inclusion criteria: 22 (51%) were female, 40 (93%) had type 1 diabetes, the mean age was 15.2 ± 2.3 years, and the mean diabetes mellitus duration was 4.6 ± 3.6 years. Of those individuals, 35 of these patients had hemoglobin A1c (HbA1c) data available at baseline, 6 months, and 1 year after PHP. The average HbA1c before PHP admission was 11.3 ± 2.3% (100.5 ± 25 mmol/mol), and decreased to 9.2 ± 1.3% (76.7 ± 14.8 mmol/mol) within 6 months of PHP admission (P<.001). The average HbA1c 1 year after PHP was 10.7 ± 1.7 % (93.3 ± 19.1 mmol/mol). Overall, 24 patients (68%) had lower HbA1c, and 75% of those with improvement maintained an HbA1c reduction of ≥1% (≥10 mmol/mol) at 1 year compared to before PHP. Conclusion: Most patients demonstrated improved glycemic control within 6 months of PHP admission, and many of those maintained a ≥1% (≥10 mmol/mol) reduction in HbA1c at 1 year following PHP admission. This program may represent a promising intervention that could serve as a model for intensive outpatient management of youth with poorly controlled diabetes mellitus. Abbreviations: ADA = American Diabetes Association; DKA = diabetic ketoacidosis; EMR = electronic medical record; HbA1c = hemoglobin A1c; ICD-9 = International Classification of Diseases, 9th revision; PHP = partial hospitalization program.

The homeobox gene Gsx2 controls the timing of oligodendroglial fate specification in mouse lateral ganglionic eminence progenitors.

The homeobox gene Gsx2 has previously been shown to be required for the specification of distinct neuronal subtypes derived from lateral ganglionic eminence (LGE) progenitors at specific embryonic time points. However, its role in the subsequent generation of oligodendrocytes from these progenitors remains unclear. We have utilized conditional gain-of-function and loss-of-function approaches in order to elucidate the role of Gsx2 in the switch between neurogenesis and oligodendrogenesis within the embryonic ventral telencephalon. In the absence of Gsx2 expression, an increase in oligodendrocyte precursor cells (OPCs) with a concomitant decrease in neurogenesis is observed in the subventricular zone of the LGE at mid-stages of embryogenesis (i.e. E12.5-15.5), which subsequently leads to an increased number of Gsx2-derived OPCs within the adjacent mantle regions of the cortex before birth at E18.5. Moreover, using Olig2(cre) to conditionally inactivate Gsx2 throughout the ventral telencephalon with the exception of the dorsal (d)LGE, we found that the increase in cortical OPCs in Gsx2 germline mutants are derived from dLGE progenitors. We also show that Ascl1 is required for the expansion of these dLGE-derived OPCs in the cortex of Gsx2 mutants. Complementing these results, gain-of-function experiments in which Gsx2 was expressed throughout most of the late-stage embryonic telencephalon (i.e. E15.5-18.5) result in a significant decrease in the number of cortical OPCs. These results support the notion that high levels of Gsx2 suppress OPC specification in dLGE progenitors and that its downregulation is required for the transition from neurogenesis to oligodendrogenesis.

Synergistic defects of different molecules in the cytotoxic pathway lead to clinical familial hemophagocytic lymphohistiocytosis.

Several molecules (LYST, AP3, RAB27A, STX11, STXBP2, MUNC13-4, and PRF1) have been associated with the function of cytotoxic lymphocytes. Biallelic defects in all of these molecules have been associated with familial hemophagocytic lymphohistiocytosis (FHL). We retrospectively reviewed the genetic and immunology test results from 2701 patients with a clinically suspected diagnosis of hemophagocytic lymphohistiocytosis and found 28 patients with single heterozygous mutations in 2 FHL-associated genes. Of these patients, 21 had mutations within PRF1 and a degranulation gene, and 7 were found to have mutations within 2 genes involved in the degranulation pathway. In patients with combination defects involving 2 genes in the degranulation pathway, CD107a degranulation was decreased, comparable to patients with biallelic mutations in one of the genes in the degranulation pathway. This suggests a potential digenic mode of inheritance of FHL as a result of a synergistic function effect within genes involved in cytotoxic lymphocyte degranulation.

Reliability, Validity, and Classification Accuracy of the DSM-5 Diagnostic Criteria for Gambling Disorder and Comparison to DSM-IV.

The DSM-5 was published in 2013 and it included two substantive revisions for gambling disorder (GD). These changes are the reduction in the threshold from five to four criteria and elimination of the illegal activities criterion. The purpose of this study was to twofold. First, to assess the reliability, validity and classification accuracy of the DSM-5 diagnostic criteria for GD. Second, to compare the DSM-5-DSM-IV on reliability, validity, and classification accuracy, including an examination of the effect of the elimination of the illegal acts criterion on diagnostic accuracy. To compare DSM-5 and DSM-IV, eight datasets from three different countries (Canada, USA, and Spain; total N = 3247) were used. All datasets were based on similar research methods. Participants were recruited from outpatient gambling treatment services to represent the group with a GD and from the community to represent the group without a GD. All participants were administered a standardized measure of diagnostic criteria. The DSM-5 yielded satisfactory reliability, validity and classification accuracy. In comparing the DSM-5 to the DSM-IV, most comparisons of reliability, validity and classification accuracy showed more similarities than differences. There was evidence of modest improvements in classification accuracy for DSM-5 over DSM-IV, particularly in reduction of false negative errors. This reduction in false negative errors was largely a function of lowering the cut score from five to four and this revision is an improvement over DSM-IV. From a statistical standpoint, eliminating the illegal acts criterion did not make a significant impact on diagnostic accuracy. From a clinical standpoint, illegal acts can still be addressed in the context of the DSM-5 criterion of lying to others.

Functional Neurological Disorder in Pediatrics: Diagnostic Considerations.

Functional neurological disorder (FND) is a common diagnosis of varied neuropsychiatric symptoms presenting to pediatric healthcare settings, including primary, urgent and subspecialty care. A key diagnostic shift appearing in the DSM-V is that FND is no longer a diagnosis of exclusion; rather, a rule-in diagnosis based on suggestive elements of symptom presentation. This article reviews diagnostic criteria, clarifying features, risks, and prognostic factors. This is the first in a series of six articles on FND and will introduce an FND case that will be examined in each subsequent article in the context of their more specific subject matter.

The Rehabilitation Therapist's Role in the Treatment of Functional Neurological Disorder in Children and Adolescents.

Functional Neurological Disorder (FND) is a common condition encountered by rehabilitation therapists (physical therapists-PT, occupational therapists-OT, and speech and language pathologists-SLP) in pediatric practice. In an effort to bridge the knowledge and experience gap, PT, OT and SLP experts have published consensus recommendations for the rehab therapist's treatment of FND1-3 with additional research emphasizing a psychologically informed4,5 and wellness approach.6 This article highlights the unique role of each rehab discipline in treating FND, reviews functional diagnoses specific to PT, OT and SLP, and introduces a unique stepwise or ladder approach to treatment that can be utilized in the inpatient or outpatient setting.

Considerations in Prescribing and De-Prescribing in Pediatric Functional Neurological Disorders.

Functional neurological disorder (FND) is common among children and adolescents, results in significant impairments in quality of life, and places a substantial burden on healthcare systems. Despite this, there is minimal literature to guide prescribing for this population. The purpose of this article is to provide common sense prescribing recommendations for providers who treat pediatric FND. A narrative review was conducted by searching PubMed using keywords related to FND and pharmacology. The narrative synthesis was guided by the objective of providing evidence for generally accepted practices and highlighting contributions and gaps in the literature. There is a dearth of evidence, and unique challenges exist in prescribing for pediatric patients with FND. Efforts should be made to limit prescribing and to discontinue, or de-prescribe, medications that may contribute to polypharmacy or overmedicalization of functional symptoms. Pediatric patients with FND require a thoughtful, multidisciplinary approach.

Functional Neurological Disorder in Adults.

Functional neurological disorder (FND) is a heterogenous group of disorders with limitations in one or more neurologic systems without a structural or anatomic cause. Elements of diagnosis, evaluation and management are similar between children and adults though diagnostic considerations can be more complex with suspected FND in adults. This article will focus on issues specific and relevant to adults.

Substance use disorders among armed forces veterans with gambling disorder: Insights from an inpatient treatment sample.

Armed forces veterans are especially prone to experiencing a variety of addictive disorders, including gambling disorder. Even so, gambling disorder in veterans remains understudied generally, and there remain significant gaps in the research literature with regards to how gambling disorder relates to the experience and expression of comorbid substance use disorders. The present work examines the prevalence, presentation, and clinical associations of substance use disorders in U.S. Armed Forces veterans receiving inpatient treatment for gambling disorder. Participants (N = 664) were veterans from all branches of the U.S. Armed Forces receiving inpatient treatment for gambling disorder through the Department of Veteran Affairs Healthcare System. Clinical data from the time of intake was analyzed. A substantial portion of veterans (36.1%) met current criteria for an SUD, with another 16.5% reporting a history of SUD. Alcohol use disorder was the most commonly reported SUD (76.1% of those with a current SUD), with polysubstance use disorders and stimulant use disorders each occurring in at least 25% of those with SUDs. SUD status was related to greater levels of impulsivity, but there was no evidence that SUD status was related to gambling symptom severity, gambling preferences, or further psychiatric comorbidities.

Comorbid psychiatric diagnoses and gaming preferences in US armed forces veterans receiving inpatient treatment for gambling disorder.

Armed Forces Veterans are uniquely vulnerable to problem gambling and gambling disorder. Even so, research regarding the full clinical profile of veterans with gambling problems lags. Gambling activities vary widely from each other, but most gambling activities can be understood as either strategic (i.e., involving some measure of skill and decision-making as a part of the gambling practice) or non-strategic (i.e., gambling activities that are entirely based on chance). Prior works have found that gamblers that prefer strategic gambling activities and those that prefer nonstrategic gambling activities often differ from each other in key ways, with the two preferences being linked to varying motivations for gambling, varying cognitions about gambling, and the course of gambling disorder. The present work sought to examine how preferences for strategic vs. nonstrategic gambling might be related to psychiatric comorbidities among U.S. Armed Forces Veterans receiving inpatient treatment for Gambling Disorder. Data from U.S. Armed Forces Veterans (N = 401) receiving residential treatment for GD between the years of 2010-2016 were analyzed. Results demonstrated that gamblers that preferred strategic gambling, as opposed to non-strategic gambling, were more likely to be younger, more likely to be men, less likely to have a nicotine use disorder, and less likely to have PTSD. Such findings suggest that gamblers with PTSD are likely to prefer nonstrategic games and may imply a unique vulnerability to gambling problems related to non-strategic gambling among armed forces veterans.

A randomized control trial of integrated care for families managing infant colic.

This article presents a randomized clinical trial examining the effectiveness of a unique model of integrated care for the treatment of infant colic. Families seeking help for infant colic were randomized to either the family-centered treatment (TX; n = 31) or standard pediatric care (SC; n = 31). All parents completed 3 days of Infant Behavior Diaries (Barr et al., 1998) and the Colic Symptom Checklist (Lester, 1997), Beck Depression Inventory (Beck & Steer, 1984), and Parenting Stress Index 3rd ed.-SF (Abidin, 1995). TX families were seen three times by a pediatrician and a mental health clinician within 1, 2, and 6 weeks of baseline data. TX families received individualized treatment plans addressing problem areas of sleep, feeding, routine, and family mental health. SC families were seen only by their own healthcare provider. All families were visited at home by a research assistant to retrieve data at 2, 6, and 10 weeks after baseline. Family-based treatment accelerated the rate of reduction of infant crying faster than did standard pediatric care. Infants in the TX group had more hours of sleep at 2 weeks posttreatment and spent less time feeding at 2, 6, and 10 weeks posttreatment than did SC infants. Results indicate that individualized family-based treatment reduces infant colic more rapidly than does standard pediatric care.

Preventing the degradation of mps1 at centrosomes is sufficient to cause centrosome reduplication in human cells.

Supernumerary centrosomes promote the assembly of abnormal mitotic spindles in many human tumors. In human cells, overexpression of the cyclin-dependent kinase (Cdk)2 partner cyclin A during a prolonged S phase produces extra centrosomes, called centrosome reduplication. Cdk2 activity protects the Mps1 protein kinase from proteasome-mediated degradation, and we demonstrate here that Mps1 mediates cyclin A-dependent centrosome reduplication. Overexpression of cyclin A or a brief proteasome inhibition increases the centrosomal levels of Mps1, whereas depletion of Cdk2 leads to the proteasome-dependent loss of Mps1 from centrosomes only. When a Cdk2 phosphorylation site within Mps1 (T468) is mutated to alanine, Mps1 cannot accumulate at centrosomes or participate in centrosome duplication. In contrast, phosphomimetic mutations at T468 or deletion of the region surrounding T468 prevent the proteasome-dependent removal of Mps1 from centrosomes in the absence of Cdk2 activity. Moreover, cyclin A-dependent centrosome reduplication requires Mps1, and these stabilizing Mps1 mutations cause centrosome reduplication, bypassing cyclin A. Together, our data demonstrate that the region surrounding T468 contains a motif that regulates the accumulation of Mps1 at centrosomes. We suggest that phosphorylation of T468 attenuates the degradation of Mps1 at centrosomes and that preventing this degradation is necessary and sufficient to cause centrosome reduplication in human cells.

The impact of communication barriers on diagnostic confidence and ancillary testing in the emergency department.

BACKGROUND: Communication barriers (CBs) compromise the diagnostic power of the medical interview and may result in increased reliance on diagnostic tests or incorrect test ordering. The prevalence and degree to which these barriers affect diagnosis, testing, and treatment are unknown. STUDY OBJECTIVES: To quantify and characterize CBs encountered in the Emergency Department (ED), and assess the effect of CBs on initial diagnosis and perceived reliance on ancillary testing. METHODS: This was a prospective survey completed by emergency physicians after initial adult patient encounters. CB severity, diagnostic confidence, and reliance on ancillary testing were quantified on a 100-mm Visual Analog Scale (VAS) from least (0) to most (100). RESULTS: Data were collected on 417 ED patient encounters. CBs were reported in 46%; with a mean severity of 50 mm on a 100-mm VAS with endpoints of "perfect communication and "no communication." Language was the most commonly reported form of CB (28%). More than one CB was identified in 6%. The 100-mm VAS rating of diagnostic confidence was lower in patients with perceived CBs (64 mm) vs. those without CBs (80 mm), p < 0.001. VAS ratings of physician reliance on ancillary testing was higher in patients with perceived CBs (50 mm) vs. patients without a perceived CB (38 mm), p < 0.001. CONCLUSIONS: Communication barriers in our ED setting were common, and resulted in lower diagnostic confidence and increased perception that ancillary tests are needed to narrow the diagnosis.

Unfractionated heparin displaces sFlt-1 from the placental extracellular matrix.

Soluble vascular endothelial growth factor receptor-1 (sFlt-1) is an anti-angiogenic protein which is secreted by numerous cell types and acts as a decoy receptor for the angiogenic protein vascular endothelial growth factor (VEGF). Despite its physiologic importance in maintaining angiogenic balance, excess sFlt-1 levels are associated with the pathogenesis of many diseases, especially those with angiogenic imbalance, endothelial dysfunction, and hypertension. Although sFlt-1 is a soluble protein, it contains a binding site for the extracellular matrix component heparan sulfate. This allows cells to retain and localize sFlt-1 in order to prevent excessive VEGF signaling. During pregnancy, placental syncytiotrophoblasts develop a large extracellular matrix which contains significant amounts of heparan sulfate. Consequently, the placenta becomes a potential storage site for large amounts of sFlt-1 bound to extracellular heparan sulfate. Additionally, it should be noted that sFlt-1 can bind to the anticoagulant unfractionated heparin due to its molecular mimicry to heparan sulfate. However, it remains unknown whether unfractionated heparin can compete with heparan sulfate for binding of localized sFlt-1. In this study, we hypothesized that administration of unfractionated heparin would displace and solubilize placental extracellular matrix(ECM)-bound sFlt-1. If unfractionated heparin can displace this large reservoir of sFlt-1 in the placenta and mobilized it into the maternal circulation, we should be able to observe its effects on maternal angiogenic balance and blood pressure. To test this hypothesis, we utilized in vitro, ex vivo, and in vivo methods. Using the BeWo placental trophoblast cell line, we observed increased sFlt-1 in the media of cells treated with unfractionated heparin compared to controls. The increase in media sFlt-1 was found in conjunction with decreased localized cellular Flt (sFlt-1 and Flt-1) as measured by total cell fluorescence. Similar results were observed using ex vivo placental villous explants treated with unfractionated heparin. Real-time quantitative PCR of the explants showed no change in sFlt-1 or heparanase-1 mRNA expression, eliminating increased production and enzymatic cleavage of heparan sulfate as causes for sFlt-1 media increase. Timed-pregnant rats given a continuous infusion of unfractionated heparin exhibited an increased mean arterial pressure as well as decreased bioavailable VEGF compared to vehicle-treated animals. These data demonstrate that chronic unfractionated heparin treatment is able to displace matrix-bound sFlt-1 into the maternal circulation to such a degree that mean arterial pressure is significantly affected. Here we have shown that the placental ECM is a storage site for large quantities of sFlt-1, and that it should be carefully considered in future studies concerning angiogenic balance in pregnancy.

Potential Perinatally Acquired Extended Spectrum ß-Lactamase Escherichia coli Urinary Tract Infection in an Infant.

Extended-spectrum ß-lactamases (ESBL) are produced mainly by members of the Enterobacteriaceae family and confer resistance to most ß-lactam antibiotics. Because of limited treatment options, ESBL infections are typically more challenging to treat resulting in poor outcomes, increased complications, and mortality. Because ESBL-producing organisms are primarily seen in critically ill patients, along with those patients having prolonged hospital stays, extensive courses of antimicrobials, and/or use of invasive medical devices (i.e., urinary catheters, central venous lines, or endotracheal tubes), guidelines regarding the management of ESBL-producing organisms in the pediatric population are scant. A review of current recommended treatment options for infections caused by ESBL-producing organisms centers on the use of carbapenems, with some supportive literature regarding the utility/effectiveness of other non-ß-lactam therapy. We present a case report of an 8-month-old female diagnosed with a urinary tract infection due to ESBL-producing Escherichia coli successfully treated with sulfamethoxazole/trimethoprim. Multidrug resistant infections in pediatric patients without risk factors remains an important field of study because these unique infections may pose a problem when choosing an effective empiric antimicrobial therapy.

Differential regulation of sFlt-1 splicing by U2AF65 and JMJD6 in placental-derived and endothelial cells.

Despite years of study, the gestational disorder preeclampsia (PE) remains poorly understood. One proposed mechanism of PE development is increased soluble VEGF receptor-1 (sFlt-1), ultimately causing angiogenic imbalance and endothelial dysfunction. The soluble protein is an alternative splice variant of FLT1, which also encodes for the full-length receptor Flt-1. The mechanism of the alternative splicing, and the reason for its inappropriate increase in preeclampsia, is not well understood. U2 auxiliary factor 65 (U2AF65) and jumonji C domain-containing protein 6 (JMJD6) have been implicated in the splicing of sFlt-1. Using siRNA knockdown and plasmid overexpression in immortalized placental trophoblasts (BeWo) and primary endothelial cells (HUVECs), we examined the role these proteins play in production of sFlt-1. Our results showed that U2AF65 has little, if any, effect on sFlt-1 splicing, and JMJD6 may enhance sFlt-1 splicing, but is not necessary for splicing to occur. Utilizing a hypoxic environment to mimic conditions of the preeclamptic placenta, as well as examining placentae in the reduced uterine perfusion pressure (RUPP) model of PE, which exhibits increased circulating sFlt-1, we found increased expression of JMJD6 in both hypoxic cells and placental tissue. Additionally, we observed a potential role for U2AF65 and JMJD6 to regulate the extracellular matrix enzyme heparanase, which may be involved in the release of sFlt-1 protein from the extracellular matrix. It will be important to study the role of these proteins in different tissues in the future, as changes in expression had differential effects on sFlt-1 splicing in the different cell types studied here.

Biopolymer-Delivered, Maternally Sequestered NF-κB (Nuclear Factor-κB) Inhibitory Peptide for Treatment of Preeclampsia.

Preeclampsia is a hypertensive disorder of pregnancy that causes significant acute and long-term risk to the mother and the baby. The multifaceted maternal syndrome is driven by overproduction of circulating anti-angiogenic factors, widespread inflammation, and endothelial dysfunction. Nuclear factor-κB (NF-κB) is a transcription factor that plays a central role in the inflammatory response. Its activity is increased in the preeclamptic placenta, and it promotes the systemic endothelial dysfunction present in preeclampsia. There is an acute need for new therapeutics targeted to the causative pathways of preeclampsia. Our group has developed a drug delivery system based on the bioengineered protein ELP (elastin-like polypeptide) that is capable of stabilizing therapeutics in the maternal circulation and preventing their placental transfer. Here we used the ELP carrier system to deliver a peptide known to inhibit the NF-κB pathway. This polypeptide, containing a cell-penetrating peptide and an NF-κB inhibitory peptide derived from the p50 nuclear localization sequence (abbreviated SynB1-ELP-p50i), blocked NF-κB activation and prevented TNF-α (tumor necrosis factor alpha)-induced endothelin production in vitro. Fusion of the p50i peptide to the SynB1-ELP carrier slowed its plasma clearance and prevented its placental transfer in pregnant rats, resulting in increased deposition in the maternal kidney, liver, and placenta relative to the free peptide. When administered in a rat model of placental ischemia, SynB1-ELP-p50i partially ameliorated placental ischemia-induced hypertension and reduced placental TNF-α levels with no signs of toxicity. These data support the continued development of ELP-delivered NF-κB inhibitors as maternally sequestered anti-inflammatory agents for preeclampsia therapy.