P2X7 receptor blockade prevents preterm birth and perinatal brain injury in a mouse model of intrauterine inflammation.

The P2X7 is an adenosine triphosphate (ATP)-gated ion channel involved in several facets of immune activation and neuronal function through its importance in interleukin (IL)-1ß secretion. We hypothesized that blockade of P2X7 would prevent perinatal brain injury associated with exposure to intrauterine (IU) inflammation. Dams received 45 mg/kg of Brilliant Blue G (BBG), a specific P2X7 receptor (P2X7R) antagonist, on gestation day 17 (E17) prior to administration of lipopolysaccharide (LPS) or phosphate-buffered saline (PBS). Furthermore, we utilized embryo transfer experiments to delineate whether the P2X7 was the key mediator of IU inflammation-associated brain injury on maternal or fetal sides. In these experiments, P2X7-/- dams were embryo-transferred wild type embryos and wild type dams were embryo-transferred P2X7-/- embryos. In the mouse model of intrauterine inflammation, pharmacologic blockade of P2X7R reduced preterm birth rate, improved offspring performance on neuromotor tests as well as the dendritic arborization and density of cortical neurons. Embryo transfer experiments demonstrated the importance of maternal P2X7R in IU inflammation-mediated effects on offspring. Both genetic and pharmacologic blockade of IL-1ß signaling, by targeting maternal P2X7R, ameliorated perinatal brain injury following exposure to IU inflammation. Specific targeting of maternal P2X7R may provide a clinically useful tool to prevent both preterm birth and prematurity-associated perinatal brain injury, and further studies are urgently needed.

In utero localized diffusion MRI of the embryonic mouse brain microstructure and injury.

PURPOSE: To develop an in vivo diffusion magnetic resonance imaging (dMRI) technique to study embryonic mouse brain structure and injury. MATERIALS AND METHODS: Pregnant CD-1 mice were examined on embryonic day 17 on an 11.7T scanner. Spatially selective excitation pulses were used to achieve localized imaging of individual mouse brains, in combination with a 3D fast imaging sequence to acquire dMRI at 0.16-0.2 mm isotropic resolution. Subject motions were corrected by navigator echoes and image registration. Further acceleration was achieved by simultaneous imaging of two embryos in an interleaved fashion. We applied this technique to detect embryonic brain injury in a mouse model of intrauterine inflammation. RESULTS: With the localized imaging technique, we achieved in utero high-resolution T2 -weighted and dMRI of the embryonic mouse brain for the first time. Early embryonic brain structures were delineated from diffusion tensor images, and major white matter tracts were reconstructed in 3D. Comparison with ex vivo data showed significant changes in the apparent diffusion coefficient (ADC), but mostly unchanged fractional anisotropy. In the inflammation-affected embryonic brains, ADC in the cortical regions was reduced at 6 hours after the injury, potentially caused by cellular edema. CONCLUSION: The feasibility of in utero dMRI of embryonic mouse brains was demonstrated. The technique is important for noninvasive monitoring of embryonic mouse brain microstructure and injury.

Murine model: maternal administration of stem cells for prevention of prematurity.

OBJECTIVE: Using a mouse model of intrauterine inflammation, we have demonstrated that exposure to inflammation induces preterm birth and perinatal brain injury. Mesenchymal stem cells (MSCs) have been shown to exhibit immunomodulatory effects in many inflammatory conditions. We hypothesized that treatment with human adipose tissue-derived MSCs may decrease the rate of preterm birth and perinatal brain injury through changes in antiinflammatory and regulatory milieu. STUDY DESIGN: A mouse model of intrauterine inflammation was used with the following groups: (1) control; (2) intrauterine inflammation (lipopolysaccharide); and (3) intrauterine lipopolysaccharide+intraperitoneal (MSCs). Preterm birth was investigated. Luminex multiplex enzyme-linked immunosorbent assays were performed for protein levels of cytokines in maternal and fetal compartments. Immunofluorescent staining was used to identify and localize MSCs and to examine microglial morphologic condition and neurotoxicity in perinatal brain. Behavioral testing was performed at postnatal day 5. RESULTS: Pretreatment with MSCs significantly decreased the rate of preterm birth by 21% compared with the lipopolysaccharide group (P<.01). Pretreatment was associated with increased interleukin-10 in maternal serum, increased interleukin-4 in placenta, decreased interleukin-6 in fetal brain (P<.05), decreased microglial activation (P<.05), and decreased fetal neurotoxicity (P<.05). These findings were associated with improved neurobehavioral testing at postnatal day 5 (P<.05). Injected MSCs were localized to placenta. CONCLUSION: Maternally administered MSCs appear to modulate maternal and fetal immune response to intrauterine inflammation in the model and decrease preterm birth, perinatal brain injury, and motor deficits in offspring mice.

KLF4 modulates expression of IL-6 in dendritic cells via both promoter activation and epigenetic modification.

The initiation and maintenance of the immune response require a coordinated regulation of signal transduction pathways. Identifying the mechanisms by which these pathways are controlled and modulated is a significant goal of immunology. In the present report, we show a novel role for the zinc finger transcription factor Kruppel-like factor 4 (KLF4) in the modulation of the inflammatory immune response via its regulation of IL-6. We analyzed the role of KLF4 in the production of IL-6 by dendritic cells. Our data indicate that KLF4 can act in a dual function manner. It acts as a transcription factor in that it can bind to and activate the IL-6 promoter at specific binding sites. KLF4 also has a role in the chromatin remodeling of the IL-6 promoter in that cells deficient in KLF4 exhibited a relative hypoacetylation. These results indicate a molecular role for KLF4 in modulating the intensity of the inflammatory response and help to explain its pleiotropic role in different settings.

Mouse model of intrauterine inflammation: sex-specific differences in long-term neurologic and immune sequelae.

Preterm infants, especially those that are exposed to prenatal intrauterine infection or inflammation, are at a major risk for adverse neurological outcomes, including cognitive, motor and behavioral disabilities. We have previously shown in a mouse model that there is an acute fetal brain insult associated with intrauterine inflammation. The objectives of this study were: (1) to elucidate long-term (into adolescence and adulthood) neurological outcomes by assessing neurobehavioral development, MRI, immunohistochemistry and flow cytometry of cells of immune origin and (2) to determine whether there are any sex-specific differences in brain development associated with intrauterine inflammation. Our results have shown that prenatal exposure appeared to lead to changes in MRI and behavior patterns throughout the neonatal period and during adulthood. Furthermore, we observed chronic brain inflammation in the offspring, with persistence of microglial activation and increased numbers of macrophages in the brain, ultimately resulting in neuronal loss. Moreover, our study highlights the sex-specific differences in long-term sequelae. This study, while extending the growing literature of adverse neurologic outcomes following exposure to inflammation during early development, presents novel findings in the context of intrauterine inflammation.

Functional blockade of the voltage-gated potassium channel Kv1.3 mediates reversion of T effector to central memory lymphocytes through SMAD3/p21cip1 signaling.

The maintenance of T cell memory is critical for the development of rapid recall responses to pathogens, but may also have the undesired side effect of clonal expansion of T effector memory (T(EM)) cells in chronic autoimmune diseases. The mechanisms by which lineage differentiation of T cells is controlled have been investigated, but are not completely understood. Our previous work demonstrated a role of the voltage-gated potassium channel Kv1.3 in effector T cell function in autoimmune disease. In the present study, we have identified a mechanism by which Kv1.3 regulates the conversion of T central memory cells (T(CM)) into T(EM). Using a lentiviral-dominant negative approach, we show that loss of function of Kv1.3 mediates reversion of T(EM) into T(CM), via a delay in cell cycle progression at the G2/M stage. The inhibition of Kv1.3 signaling caused an up-regulation of SMAD3 phosphorylation and induction of nuclear p21(cip1) with resulting suppression of Cdk1 and cyclin B1. These data highlight a novel role for Kv1.3 in T cell differentiation and memory responses, and provide further support for the therapeutic potential of Kv1.3 specific channel blockers in T(EM)-mediated autoimmune diseases.

Primary retinal pathology in multiple sclerosis as detected by optical coherence tomography.

Optical coherence tomography studies in multiple sclerosis have primarily focused on evaluation of the retinal nerve fibre layer. The aetiology of retinal changes in multiple sclerosis is thought to be secondary to optic nerve demyelination. The objective of this study was to use optical coherence tomography to determine if a subset of patients with multiple sclerosis exhibit primary retinal neuronopathy, in the absence of retrograde degeneration of the retinal nerve fibre layer and to ascertain if such patients may have any distinguishing clinical characteristics. We identified 50 patients with multiple sclerosis with predominantly macular thinning (normal retinal nerve fibre-layer thickness with average macular thickness < 5th percentile), a previously undescribed optical coherence tomography defined phenotype in multiple sclerosis, and compared them with 48 patients with multiple sclerosis with normal optical coherence tomography findings, 48 patients with multiple sclerosis with abnormal optical coherence tomography findings (typical for multiple sclerosis) and 86 healthy controls. Utilizing a novel retinal segmentation protocol, we found that those with predominant macular thinning had significant thinning of both the inner and outer nuclear layers, when compared with other patients with multiple sclerosis (P < 0.001 for both), with relative sparing of the ganglion cell layer. Inner and outer nuclear layer thicknesses in patients with non-macular thinning predominant multiple sclerosis were not different from healthy controls. Segmentation analyses thereby demonstrated extensive deeper disruption of retinal architecture in this subtype than may be expected due to retrograde degeneration from either typical clinical or sub-clinical optic neuropathy. Functional corroboration of retinal dysfunction was provided through multi-focal electroretinography in a subset of such patients. These findings support the possibility of primary retinal pathology in a subset of patients with multiple sclerosis. Multiple sclerosis-severity scores were also significantly increased in patients with the macular thinning predominant phenotype, compared with those without this phenotype (n = 96, P=0.006). We have identified a unique subset of patients with multiple sclerosis in whom there appears to be disproportionate thinning of the inner and outer nuclear layers, which may be occurring as a primary process independent of optic nerve pathology. In vivo analyses of retinal layers in multiple sclerosis have not been previously performed, and structural demonstration of pathology in the deeper retinal layers, such as the outer nuclear layer, has not been previously described in multiple sclerosis. Patients with inner and outer nuclear layer pathology have more rapid disability progression and thus retinal neuronal pathology may be a harbinger of a more aggressive form of multiple sclerosis.

Maternal CD8+ T-cell depletion alleviates intrauterine inflammation-induced perinatal brain injury.

We investigated the mechanisms by which CD8+ T-cell trafficking in placenta contributes to perinatal brain injury by studying effects of maternal CD8+ T-cell depletion (DEP) in a mouse model of intrauterine inflammation (IUI). Maternal CD8+ T cells were depleted with anti-CD8+ antibodies. IUI was induced with lipopolysaccharide (LPS). DEP was confirmed using flow cytometry. Preterm birth rate was evaluated. Offspring neurologic sequelae were assessed by Nissl staining, immune arrays, confirmatory individual TaqMan® gene assays, and neurobehavioral tests. DEP did not significantly prevent LPS-induced preterm birth but improved neurobehavioral performance (P < .001) and increased cortical neuronal density (P < .05) in LPS-exposed pups compared to controls. These changes were associated with decreased CCL3 and CXCL10 and increased CCL5 in DEP LPS-exposed mice. We demonstrate that DEP reduces perinatal brain injury following IUI. This supports a role for maternal CD8+ T-cell trafficking in placenta in mediating perinatal brain injury separate from preterm birth mechanisms.

Umbilical Cord Blood NOS1 as a Potential Biomarker of Neonatal Encephalopathy.

BACKGROUND: There are no definitive markers to aid in diagnosis of neonatal encephalopathy (NE). The purpose of our study was (1) to identify and evaluate the utility of neuronal nitric oxide synthase (NOS1) in umbilical cord blood as a NE biomarker and (2) to identify the source of NOS1 in umbilical cord blood. METHODS: This was a nested case-control study of neonates >35 weeks of gestation. ELISA for NOS1 in umbilical cord blood was performed. Sources of NOS1 in umbilical cord were investigated by immunohistochemistry, western blot, ELISA, and quantitative PCR. Furthermore, umbilical cords of full-term neonates were subjected to 1% hypoxia ex vivo. RESULTS: NOS1 was present in umbilical cord blood and increased in NE cases compared with controls. NOS1 was expressed in endothelial cells of the umbilical cord vein, but not in artery or blood cells. In ex vivo experiments, hypoxia was associated with increased levels of NOS1 in venous endothelial cells of the umbilical cord as well as in ex vivo culture medium. CONCLUSION: This is the first study to investigate an early marker of NE. NOS1 is elevated with hypoxia, and further studies are needed to investigate it as a valuable tool for early diagnosis of neonatal brain injury.

Maternal dendrimer-based therapy for inflammation-induced preterm birth and perinatal brain injury.

Preterm birth is a major risk factor for adverse neurological outcomes in ex-preterm children, including motor, cognitive, and behavioral disabilities. N-acetyl-L-cysteine therapy has been used in clinical studies; however, it requires doses that cause significant side effects. In this study, we explore the effect of low dose N-acetyl-L-cysteine therapy, delivered using a targeted, systemic, maternal, dendrimer nanoparticle (DNAC), in a mouse model of intrauterine inflammation. Our results demonstrated that intraperitoneal maternal DNAC administration significantly reduced the preterm birth rate and altered placental immune profile with decreased CD8+ T-cell infiltration. Furthermore, we demonstrated that DNAC improved neurobehavioral outcomes and reduced fetal neuroinflammation and long-term microglial activation in offspring. Our study is the first to provide evidence for the role of CD8+ T-cell in the maternal-fetal interface during inflammation and further support the efficacy of DNAC in preventing preterm birth and prematurity-related outcomes.

Three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer.

Early detection remains the most promising approach to improve long-term survival of patients with ovarian cancer. In a five-center case-control study, serum proteomic expressions were analyzed on 153 patients with invasive epithelial ovarian cancer, 42 with other ovarian cancers, 166 with benign pelvic masses, and 142 healthy women. Data from patients with early stage ovarian cancer and healthy women at two centers were analyzed independently and the results cross-validated to discover potential biomarkers. The results were validated using the samples from two of the remaining centers. After protein identification, biomarkers for which an immunoassay was available were tested on samples from the fifth center, which included 41 healthy women, 41 patients with ovarian cancer, and 20 each with breast, colon, and prostate cancers. Three biomarkers were identified as follows: (a) apolipoprotein A1 (down-regulated in cancer); (b) a truncated form of transthyretin (down-regulated); and (c) a cleavage fragment of inter-alpha-trypsin inhibitor heavy chain H4 (up-regulated). In independent validation to detect early stage invasive epithelial ovarian cancer from healthy controls, the sensitivity of a multivariate model combining the three biomarkers and CA125 [74% (95% CI, 52-90%)] was higher than that of CA125 alone [65% (95% CI, 43-84%)] at a matched specificity of 97% (95% CI, 89-100%). When compared at a fixed sensitivity of 83% (95% CI, 61-95%), the specificity of the model [94% (95% CI, 85-98%)] was significantly better than that of CA125 alone [52% (95% CI, 39-65%)]. These biomarkers demonstrated the potential to improve the detection of early stage ovarian cancer.

Peri-Implantation Hormonal Milieu: Elucidating Mechanisms of Adverse Neurodevelopmental Outcomes.

While live births resulting from assisted reproductive technology (ART) exceed 1% of total births annually, the effect of ART on fetal development is not well understood. Data have demonstrated that IVF leads to alterations in DNA methylation and gene expression in the placenta that may have long-term effects on health and disease. Studies have linked adverse neurodevelopmental outcomes to ART, although human studies are inconclusive. In order to isolate the peri-implantation environment and its effects on brain development, we utilized a mouse model with and without superovulation and examined the effect of adult behavior as well as adult cortical neuronal density. Adult offspring of superovulated dams showed increased anxiety-like behavior compared to offspring of naturally mated dams (P < .05). There was no difference in memory and learning tests between the 2 groups. The adult brains from offspring of superovulated recipients had fewer neurons per field compared to naturally mated control offspring (P < .05). In order to examine potential pathways leading to these changes, we measured messenger RNA and microRNA (miRNA) expression in fetal brains at E18.5. Microarray analysis found that miRNAs miR-122, miR-144, and miR-211, involved in regulation of neuronal migration and differentiation, were downregulated in brains of offspring exposed to a superovulated environment(P < .05). There was also altered expression of genes involved in neuronal development. These results suggest that the peri-implantation environment can affect neurodevelopment and can lead to behavioral changes in adulthood. Human studies with long-term follow-up of children from ART are necessary to further investigate the influence of ART on the offspring.

Interleukin-1 receptor blockade in perinatal brain injury.

Interleukin-1 (IL-1) is a potent inflammatory cytokine that can be produced by a variety of cell types throughout the body. While IL-1 is a central mediator of inflammation and response to infection, the role of IL-1 signaling in adult and pediatric brain injury is becoming increasingly clear. Although the mechanisms of IL-1 expression are largely understood, the downstream effects and contributions to excitotoxicity and oxidative stress are poorly defined. Here, we present a review of mechanisms of IL-1 signaling with a focus on the role of IL-1 in perinatal brain injury. We highlight research models of perinatal brain injury and the use of interleukin-1 receptor antagonist (IL-1RA) as an agent of therapeutic potential in preventing perinatal brain injury due to exposure to inflammation.