Dietary Cholesterol Causes Inflammatory Imbalance and Exacerbates Morbidity in Mice Infected with Influenza A Virus.

Influenza is a common cause of pneumonia-induced hospitalization and death, but how host factors function to influence disease susceptibility or severity has not been fully elucidated. Cellular cholesterol levels may affect the pathogenesis of influenza infection, as cholesterol is crucial for viral entry and replication, as well as immune cell proliferation and function. However, there is still conflicting evidence on the extent to which dietary cholesterol influences cholesterol metabolism. In this study, we examined the effects of a high-cholesterol diet in modulating the immune response to influenza A virus (IAV) infection in mice. Mice were fed a standard or a high-cholesterol diet for 5 wk before inoculation with mouse-adapted human IAV (Puerto Rico/8/1934), and tissues were collected at days 0, 4, 8, and 16 postinfection. Cholesterol-fed mice exhibited dyslipidemia characterized by increased levels of total serum cholesterol prior to infection and decreased triglycerides postinfection. Cholesterol-fed mice also displayed increased morbidity compared with control-fed mice, which was neither a result of immunosuppression nor changes in viral load. Instead, transcriptomic analysis of the lungs revealed that dietary cholesterol caused upregulation of genes involved in viral-response pathways and leukocyte trafficking, which coincided with increased numbers of cytokine-producing CD4+ and CD8+ T cells and infiltrating dendritic cells. Morbidity as determined by percent weight loss was highly correlated with numbers of cytokine-producing CD4+ and CD8+ T cells as well as granulocytes. Taken together, dietary cholesterol promoted IAV morbidity via exaggerated cellular immune responses that were independent of viral load.

PRESCIENT: platform for the rapid evaluation of antibody success using integrated microfluidics enabled technology.

Identifying antibodies (Abs) that neutralize infectious agents is the first step for developing therapeutics, vaccines, and diagnostic tools for these infectious agents. However, current approaches for identifying neutralizing Abs (nAbs) typically rely on dilution-based assays that are costly, inefficient, and only survey a small subset of the entire repertoire. There are also intrinsic biases in many steps of conventional nAb identification processes. More importantly, conventional assays rely on simple Ab-antigen binding assays, which may not result in identifying the most potent nAbs, as the strongest binder may not be the most potent nAb. Droplet microfluidic systems have the capability to overcome such limitations by conducting complex multi-step assays with high reliability, resolution, and throughput in a pico-liter volume water-in-oil emulsion droplet format. Here, we describe the development of PRESCIENT (Platform for the Rapid Evaluation of antibody SucCess using Integrated microfluidics ENabled Technology), a droplet microfluidic system that can enable high-throughput single-cell resolution identification of nAb repertoires elicited in response to viral infection. We demonstrate PRESCIENT's ability to identify Abs that neutralize a model viral agent, Murine coronavirus (murine hepatitis virus), which causes high mortality rates in experimentally infected mice. In-droplet infection of host cells by the virus was first demonstrated, followed by demonstration of in-droplet neutralization by nAbs produced from a single Ab-producing hybridoma cell. Finally, fluorescence intensity analyses of two populations of hybridoma cell lines (nAb-producing and non-nAb-producing hybridoma cell lines) successfully discriminated between the two populations. The presented strategy and platform have the potential to identify and investigate neutralizing activities against a broad range of potential infectious agents for which nAbs have yet to be discovered, significantly advancing the nAb identification process as well as reinvigorating the field of Ab discovery, characterization, and development.

Fetal Alcohol Exposure Alters Blood Flow and Neurological Responses to Transient Cerebral Ischemia in Adult Mice.

BACKGROUND: Prenatal alcohol exposure (PAE) can result in physical and neurocognitive deficits that are collectively termed "fetal alcohol spectrum disorders" (FASD). Although FASD is associated with lifelong intellectual disability, the mechanisms mediating the emergence of secondary mental health and physical disabilities are poorly understood. Based on our previous data showing that maternal ethanol (EtOH) exposure in mice resulted in an immediate reduction in cranially directed fetal blood flow, we hypothesized that such exposure would also result in persistent alterations in cranially directed blood flow in the prenatally alcohol-exposed (PAE) adult. We also hypothesized that PAE adults exposed to an acute cerebrovascular insult would exhibit more brain damage and neurobehavioral impairment compared to non-PAE adult controls. METHODS: Pregnant C57BL/6 mice were exposed to EtOH, 3 g/kg, or water by intragastric gavage. Blood flow in carotid, renal, and femoral arteries was assessed by ultrasound imaging in PAE and control adults at 3, 6, and 12 months of age. To mimic ischemic stroke in young adult populations, 3-month-old PAE and control animals were subject to transient middle cerebral artery occlusion (MCAo) and subsequently assessed for behavioral recovery, stroke infarct volume, and brain cytokine profiles. RESULTS: PAE resulted in a significant age-related decrease in blood acceleration in adult mice, specifically in the carotid artery. A unilateral transient MCAo resulted in equivalent cortico-striatal damage in both PAE and control adults. However, PAE adult mice exhibited significantly decreased poststroke behavioral recovery compared to controls. CONCLUSIONS: Our data collectively show that PAE adult mice exhibit a persistent, long-term loss of cranially directed blood flow, and decreased capacity to compensate for brain trauma due to acute-onset adult diseases like ischemic stroke.

Dysregulation of microRNA expression and function contributes to the etiology of fetal alcohol spectrum disorders.

MicroRNAs (miRNAs) are members of a large class of non-protein-coding RNA (ncRNA) molecules that represent a significant, but until recently unappreciated, layer of cellular regulation. Assessment of the generation and function of miRNAs suggests that these ncRNAs are vulnerable to interference from genetic, epigenetic, and environmental factors. A small but rapidly expanding body of studies using a variety of animal- and cell culture-based experimental models also has shown that miRNAs are important targets of alcohol during fetal development and that their dysregulation likely plays a significant role in the etiology of fetal alcohol spectrum disorders (FASD). Accordingly, an analysis of the regulation and function of these miRNAs may yield important clues to the management of FASD.

CD24 expression identifies teratogen-sensitive fetal neural stem cell subpopulations: evidence from developmental ethanol exposure and orthotopic cell transfer models.

BACKGROUND: Ethanol is a potent teratogen. Its adverse neural effects are partly mediated by disrupting fetal neurogenesis. The teratogenic process is poorly understood, and vulnerable neurogenic stages have not been identified. Identifying these is a prerequisite for therapeutic interventions to mitigate effects of teratogen exposures. METHODS: We used flow cytometry and qRT-PCR to screen fetal mouse-derived neurosphere cultures for ethanol-sensitive neural stem cell (NSC) subpopulations, to study NSC renewal and differentiation. The identity of vulnerable NSC populations was validated in vivo, using a maternal ethanol exposure model. Finally, the effect of ethanol exposure on the ability of vulnerable NSC subpopulations to integrate into the fetal neurogenic environment was assessed following ultrasound guided, adoptive transfer. RESULTS: Ethanol decreased NSC mRNAs for c-kit, Musashi-1and GFAP. The CD24(+) NSC population, specifically the CD24(+)CD15(+) double-positive subpopulation, was selectively decreased by ethanol. Maternal ethanol exposure also resulted in decreased fetal forebrain CD24 expression. Ethanol pre-exposed CD24(+) cells exhibited increased proliferation, and deficits in cell-autonomous and cue-directed neuronal differentiation, and following orthotopic transplantation into naïve fetuses, were unable to integrate into neurogenic niches. CD24(depleted) cells retained neurosphere regeneration capacity, but following ethanol exposure, generated increased numbers of CD24(+) cells relative to controls. CONCLUSIONS: Neuronal lineage committed CD24(+) cells exhibit specific vulnerability, and ethanol exposure persistently impairs this population's cell-autonomous differentiation capacity. CD24(+) cells may additionally serve as quorum sensors within neurogenic niches; their loss, leading to compensatory NSC activation, perhaps depleting renewal capacity. These data collectively advance a mechanistic hypothesis for teratogenesis leading to microencephaly.

Identification of cell-specific patterns of reference gene stability in quantitative reverse-transcriptase polymerase chain reaction studies of embryonic, placental and neural stem models of prenatal ethanol exposure.

Identification of the transcriptional networks disrupted by prenatal ethanol exposure remains a core requirement to better understanding the molecular mechanisms of alcohol-induced teratogenesis. In this regard, quantitative reverse-transcriptase polymerase chain reaction (qPCR) has emerged as an essential technique in our efforts to characterize alterations in gene expression brought on by exposure to alcohol. However, many publications continue to report the utilization of inappropriate methods of qPCR normalization, and for many in vitro models, no consistent set of empirically tested normalization controls have been identified. In the present study, we sought to identify a group of candidate reference genes for use within studies of alcohol exposed embryonic, placental, and neurosphere stem cells under both conditions maintaining stemness as well as throughout in vitro differentiation. To this end, we surveyed the recent literature and compiled a short list of fourteen candidate genes commonly used as normalization controls in qPCR studies of gene expression. This list included: Actb, B2m, Gapdh, Gusb, H2afz, Hk2, Hmbs, Hprt, Mrpl1, Pgk1, Ppia, Sdha, Tbp, and Ywhaz. From these studies, we find no single candidate gene was consistently refractory to the influence of alcohol nor completely stable throughout in vitro differentiation. Accordingly, we propose normalizing qPCR measurements to the geometric mean C(T) values obtained for three independent reference mRNAs as a reliable method to accurately interpret qPCR data and assess alterations in gene expression within alcohol treated cultures. Highlighting the importance of careful and empirical reference gene selection, the commonly used reference gene Actb was often amongst the least stable candidate genes tested. In fact, it would not serve as a valid normalization control in many cases. Data presented here will aid in the design of future experiments using stem cells to study the transcriptional processes driving differentiation, and model the developmental impact of teratogens.

Ethanol exposure during pregnancy persistently attenuates cranially directed blood flow in the developing fetus: evidence from ultrasound imaging in a murine second trimester equivalent model.

BACKGROUND: Ethanol (EtOH) consumption during pregnancy can lead to fetal growth retardation, mental retardation, and neurodevelopmental delay. The fetal brain initiates neurogenesis and vasculogenesis during the second trimester, and depends on maternal-fetal circulation for nutrition and growth signals. We used high-resolution in vivo ultrasound imaging to test the hypothesis that EtOH interferes with fetal brain-directed blood flow during this critical developmental period. METHODS: Pregnant mice were lightly anesthetized on gestational day 12 with an isoflurane/oxygen mixture. We assessed the effect of single and repeated binge-like maternal EtOH exposures at 3 g/kg, administered by intragastric gavage or intraperitoneal injection, on maternal circulation and fetal umbilical, aortic, internal carotid, and middle cerebral arterial circulation. RESULTS: Binge maternal EtOH exposure, regardless of exposure route, significantly reduced fetal arterial blood acceleration and velocity time integral (VTI), from umbilical to cerebral arteries, without a change in fetal heart rate and resistivity indices. Importantly a single maternal binge EtOH exposure induced persistent suppression of fetal arterial VTI for at least 24 hours. Repeated binge episodes resulted in a continuing and persistent suppression of fetal VTI. Qualitative assessments showed that maternal EtOH exposure induced oscillatory, nondirectional blood flow in fetal cerebral arteries. Maternal cardiac and other physiological parameters remained unaltered. CONCLUSIONS: These data show that binge-type maternal EtOH exposure results in rapid and persistent loss of blood flow from the umbilical artery to the fetal brain, potentially compromising nutrition and the maternal/fetal endocrine environment during a critical period for neuron formation and angiogenesis in the maturing brain.

Hydralazine modifies Aß fibril formation and prevents modification by lipids in vitro.

Lipid oxidative damage and amyloid ß (Aß) misfolding contribute to Alzheimer's disease (AD) pathology. Thus, the prevention of oxidative damage and Aß misfolding are attractive targets for drug discovery. At present, no AD drugs approved by the Food and Drug Administration (FDA) prevent or halt disease progression. Hydralazine, a smooth muscle relaxant, is a potential drug candidate for AD drug therapy as it reduces Aß production and prevents oxidative damage via its antioxidant hydrazide group. We evaluated the efficacy of hydralazine, and related hydrazides, in reducing (1) Aß misfolding and (2) Aß protein modification by the reactive lipid 4-hydroxy-2-nonenal (HNE) using transmission electron microscopy and Western blotting. While hydralazine did not prevent Aß aggregation as measured using the protease protection assay, there were more oligomeric species observed by electron microscopy. Hydralazine prevented lipid modification of Aß, and Aß was used as a proxy for classes of proteins which either misfold or are modified by HNE. All of the other hydrazides prevented lipid modification of Aß and also did not prevent Aß aggregation. Surprisingly, a few of the compounds, carbazochrome and niclosamide, appeared to augment Aß formation. Thus, hydrazides reduced lipid oxidative damage, and hydralazine additionally reduced Aß misfolding. While hydralazine would require specific chemical modifications for use as an AD therapeutic itself (to improve blood brain barrier permeability, reduce vasoactive side effects, and optimization for amyloid inhibition), this study suggests its potential merit for further AD drug development.

Ethanol induces cell-cycle activity and reduces stem cell diversity to alter both regenerative capacity and differentiation potential of cerebral cortical neuroepithelial precursors.

BACKGROUND: The fetal cortical neuroepithelium is a mosaic of distinct progenitor populations that elaborate diverse cellular fates. Ethanol induces apoptosis and interferes with the survival of differentiating neurons. However, we know little about ethanol's effects on neuronal progenitors. We therefore exposed neurosphere cultures from fetal rat cerebral cortex, to varying ethanol concentrations, to examine the impact of ethanol on stem cell fate. RESULTS: Ethanol promoted cell cycle progression, increased neurosphere number and increased diversity in neurosphere size, without inducing apoptosis. Unlike controls, dissociated cortical progenitors exposed to ethanol exhibited morphological evidence for asymmetric cell division, and cells derived from ethanol pre-treated neurospheres exhibited decreased proliferation capacity. Ethanol significantly reduced the numbers of cells expressing the stem cell markers CD117, CD133, Sca-1 and ABCG2, without decreasing nestin expression. Furthermore, ethanol-induced neurosphere proliferation was not accompanied by a commensurate increase in telomerase activity. Finally, cells derived from ethanol-pretreated neurospheres exhibited decreased differentiation in response to retinoic acid. CONCLUSION: The reduction in stem cell number along with a transient ethanol-driven increase in cell proliferation, suggests that ethanol promotes stem to blast cell maturation, ultimately depleting the reserve proliferation capacity of neuroepithelial cells. However, the lack of a concomitant change in telomerase activity suggests that neuroepithelial maturation is accompanied by an increased potential for genomic instability. Finally, the cellular phenotype that emerges from ethanol pre-treated, stem cell depleted neurospheres is refractory to additional differentiation stimuli, suggesting that ethanol exposure ablates or delays subsequent neuronal differentiation.