CD24 Expression Dampens the Basal Antiviral State in Human Neuroblastoma Cells and Enhances Permissivity to Zika Virus Infection.

Zika virus (ZIKV) exhibits distinct selectivity for infection of various cells and tissues, but how host cellular factors modulate varying permissivity remains largely unknown. Previous studies showed that the neuroblastoma cell line SK-N-AS (expressing low levels of cellular protein CD24) was highly restricted for ZIKV infection, and that this restriction was relieved by ectopic expression of CD24. We tested the hypothesis that CD24 expression allowed ZIKV replication by suppression of the antiviral response. SK-N-AS cells expressing an empty vector (termed CD24-low cells) showed elevated basal levels of phosphorylated STAT1, IRF-1, IKKE, and NFκB. In response to exogenously added type I interferon (IFN-I), CD24-low cells had higher-level induction of antiviral genes and activity against two IFN-I-sensitive viruses (VSV and PIV5-P/V) compared to SK-N-AS cells with ectopic CD24 expression (termed CD24-high cells). Media-transfer experiments showed that the inherent antiviral state of CD24-low cells was not dependent on a secreted factor such as IFN-I. Transcriptomics analysis revealed that CD24 expression decreased expression of genes involved in intracellular antiviral pathways, including IFN-I, NFκB, and Ras. Our findings that CD24 expression in neuroblastoma cells represses intracellular antiviral pathways support the proposal that CD24 may represent a novel biomarker in cancer cells for susceptibility to oncolytic viruses.

Dipyridamole Inhibits Lipogenic Gene Expression by Retaining SCAP-SREBP in the Endoplasmic Reticulum.

Sterol regulatory element-binding proteins (SREBPs) are master transcriptional regulators of the mevalonate pathway and lipid metabolism and represent an attractive therapeutic target for lipid metabolic disorders. SREBPs are maintained in the endoplasmic reticulum (ER) in a tripartite complex with SREBP cleavage-activating protein (SCAP) and insulin-induced gene protein (INSIG). When new lipid synthesis is required, the SCAP-SREBP complex dissociates from INSIG and undergoes ER-to-Golgi transport where the N-terminal transcription factor domain is released by proteolysis. The mature transcription factor translocates to the nucleus and stimulates expression of the SREBP gene program. Previous studies showed that dipyridamole, a clinically prescribed phosphodiesterase (PDE) inhibitor, potentiated statin-induced tumor growth inhibition. Dipyridamole limited nuclear accumulation of SREBP, but the mechanism was not well resolved. In this study, we show that dipyridamole selectively blocks ER-to-Golgi movement of the SCAP-SREBP complex and that this is independent of its PDE inhibitory activity.

SREBP-1a-stimulated lipid synthesis is required for macrophage phagocytosis downstream of TLR4-directed mTORC1.

There is a growing appreciation for a fundamental connection between lipid metabolism and the immune response. Macrophage phagocytosis is a signature innate immune response to pathogen exposure, and cytoplasmic membrane expansion is required to engulf the phagocytic target. The sterol regulatory element binding proteins (SREBPs) are key transcriptional regulatory proteins that sense the intracellular lipid environment and modulate expression of key genes of fatty acid and cholesterol metabolism to maintain lipid homeostasis. In this study, we show that TLR4-dependent stimulation of macrophage phagocytosis requires mTORC1-directed SREBP-1a-dependent lipid synthesis. We also show that the phagocytic defect in macrophages from SREBP-1a-deficient mice results from decreased interaction between membrane lipid rafts and the actin cytoskeleton, presumably due to reduced accumulation of newly synthesized fatty acyl chains within major membrane phospholipids. We show that mTORC1-deficient macrophages also have a phagocytic block downstream from TLR4 signaling, and, interestingly, the reduced level of phagocytosis in both SREBP-1a- and mTORC1-deficient macrophages can be restored by ectopic SREBP-1a expression. Taken together, these observations indicate SREBP-1a is a major downstream effector of TLR4-mTORC1 directed interactions between membrane lipid rafts and the actin cytoskeleton that are required for pathogen-stimulated phagocytosis in macrophages.

Zika virus as an oncolytic treatment of human neuroblastoma cells requires CD24.

Neuroblastoma is the second most common childhood tumor. Survival is poor even with intensive therapy. In a search for therapies to neuroblastoma, we assessed the oncolytic potential of Zika virus. Zika virus is an emerging mosquito-borne pathogen unique among flaviviruses because of its association with congenital defects. Recent studies have shown that neuronal progenitor cells are likely the human target of Zika virus. Neuroblastoma has been shown to be responsive to infection. In this study, we show that neuroblastoma cells are widely permissive to Zika infection, revealing extensive cytopathic effects (CPE) and producing high titers of virus. However, a single cell line appeared poorly responsive to infection, producing undetectable levels of non-structural protein 1 (NS1), limited CPE, and low virus titers. A comparison of these poorly permissive cells to highly permissive neuroblastoma cells revealed a dramatic loss in the expression of the cell surface glycoprotein CD24 in poorly permissive cells. Complementation of CD24 expression in these cells led to the production of detectable levels of NS1 expression after infection with Zika, as well as dramatic increases in viral titers and CPE. Complementary studies using the Zika virus index strain and a north African isolate confirmed these phenotypes. These results suggest a possible role for CD24 in host cell specificity by Zika virus and offer a potential therapeutic target for its treatment. In addition, Zika viral therapy can serve as an adjunctive treatment for neuroblastoma by targeting tumor cells that can lead to recurrent disease and treatment failure.

Skeletal muscle overexpression of nicotinamide phosphoribosyl transferase in mice coupled with voluntary exercise augments exercise endurance.

OBJECTIVE: Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway that produces nicotinamide adenine dinucleotide (NAD+), an essential co-substrate regulating a myriad of signaling pathways. We produced a mouse that overexpressed NAMPT in skeletal muscle (NamptTg) and hypothesized that NamptTg mice would have increased oxidative capacity, endurance performance, and mitochondrial gene expression, and would be rescued from metabolic abnormalities that developed with high fat diet (HFD) feeding. METHODS: Insulin sensitivity (hyperinsulinemic-euglycemic clamp) was assessed in NamptTg and WT mice fed very high fat diet (VHFD, 60% by kcal) or chow diet (CD). The aerobic capacity (VO2max) and endurance performance of NamptTg and WT mice before and after 7 weeks of voluntary exercise training (running wheel in home cage) or sedentary conditions (no running wheel) were measured. Skeletal muscle mitochondrial gene expression was also measured in exercised and sedentary mice and in mice fed HFD (45% by kcal) or low fat diet (LFD, 10% by kcal). RESULTS: NAMPT enzyme activity in skeletal muscle was 7-fold higher in NamptTg mice versus WT mice. There was a concomitant 1.6-fold elevation of skeletal muscle NAD+. NamptTg mice fed VHFD were partially protected against body weight gain, but not against insulin resistance. Notably, voluntary exercise training elicited a 3-fold higher exercise endurance in NamptTg versus WT mice. Mitochondrial gene expression was higher in NamptTg mice compared to WT mice, especially when fed HFD. Mitochondrial gene expression was higher in exercised NamptTg mice than in sedentary WT mice. CONCLUSIONS: Our studies have unveiled a fascinating interaction between elevated NAMPT activity in skeletal muscle and voluntary exercise that was manifest as a striking improvement in exercise endurance.

Hepatocyte ABCA1 Deletion Impairs Liver Insulin Signaling and Lipogenesis.

Plasma membrane (PM) free cholesterol (FC) is emerging as an important modulator of signal transduction. Here, we show that hepatocyte-specific knockout (HSKO) of the cellular FC exporter, ATP-binding cassette transporter A1 (ABCA1), leads to decreased PM FC content and defective trafficking of lysosomal FC to the PM. Compared with controls, chow-fed HSKO mice had reduced hepatic (1) insulin-stimulated Akt phosphorylation, (2) activation of the lipogenic transcription factor Sterol Regulatory Element Binding Protein (SREBP)-1c, and (3) lipogenic gene expression. Consequently, Western-type diet-fed HSKO mice were protected from steatosis. Surprisingly, HSKO mice had intact glucose metabolism; they showed normal gluconeogenic gene suppression in response to re-feeding and normal glucose and insulin tolerance. We conclude that: (1) ABCA1 maintains optimal hepatocyte PM FC, through intracellular FC trafficking, for efficient insulin signaling; and (2) hepatocyte ABCA1 deletion produces a form of selective insulin resistance so that lipogenesis is suppressed but glucose metabolism remains normal.

SETDB2 Links Glucocorticoid to Lipid Metabolism through Insig2a Regulation.

Transcriptional and chromatin regulations mediate the liver response to nutrient availability. The role of chromatin factors involved in hormonal regulation in response to fasting is not fully understood. We have identified SETDB2, a glucocorticoid-induced putative epigenetic modifier, as a positive regulator of GR-mediated gene activation in liver. Insig2a increases during fasting to limit lipid synthesis, but the mechanism of induction is unknown. We show Insig2a induction is GR-SETDB2 dependent. SETDB2 facilitates GR chromatin enrichment and is key to glucocorticoid-dependent enhancer-promoter interactions. INSIG2 is a negative regulator of SREBP, and acute glucocorticoid treatment decreased active SREBP during refeeding or in livers of Ob/Ob mice, both systems of elevated SREBP-1c-driven lipogenesis. Knockdown of SETDB2 or INSIG2 reversed the inhibition of SREBP processing. Overall, these studies identify a GR-SETDB2 regulatory axis of hepatic transcriptional reprogramming and identify SETDB2 as a potential target for metabolic disorders with aberrant glucocorticoid actions.

An SREBP-responsive microRNA operon contributes to a regulatory loop for intracellular lipid homeostasis.

Sterol regulatory element-binding proteins (SREBPs) have evolved as a focal point for linking lipid synthesis with other pathways that regulate cell growth and survival. Here, we have uncovered a polycistrionic microRNA (miRNA) locus that is activated directly by SREBP-2. Two of the encoded miRNAs, miR-182 and miR-96, negatively regulate the expression of Fbxw7 and Insig-2, respectively, and both are known to negatively affect nuclear SREBP accumulation. Direct manipulation of this miRNA pathway alters nuclear SREBP levels and endogenous lipid synthesis. Thus, we have uncovered a mechanism for the regulation of intracellular lipid metabolism mediated by the concerted action of a pair of miRNAs that are expressed from the same SREBP-2-regulated miRNA locus, and each targets a different protein of the multistep pathway that regulates SREBP function. These studies reveal an miRNA "operon" analogous to the classic model for genetic control in bacterial regulatory systems.

Host defense against viral infection involves interferon mediated down-regulation of sterol biosynthesis.

Little is known about the protective role of inflammatory processes in modulating lipid metabolism in infection. Here we report an intimate link between the innate immune response to infection and regulation of the sterol metabolic network characterized by down-regulation of sterol biosynthesis by an interferon regulatory loop mechanism. In time-series experiments profiling genome-wide lipid-associated gene expression of macrophages, we show a selective and coordinated negative regulation of the complete sterol pathway upon viral infection or cytokine treatment with IFNγ or ß but not TNF, IL1ß, or IL6. Quantitative analysis at the protein level of selected sterol metabolic enzymes upon infection shows a similar level of suppression. Experimental testing of sterol metabolite levels using lipidomic-based measurements shows a reduction in metabolic output. On the basis of pharmacologic and RNAi inhibition of the sterol pathway we show augmented protection against viral infection, and in combination with metabolite rescue experiments, we identify the requirement of the mevalonate-isoprenoid branch of the sterol metabolic network in the protective response upon statin or IFNß treatment. Conditioned media experiments from infected cells support an involvement of secreted type 1 interferon(s) to be sufficient for reducing the sterol pathway upon infection. Moreover, we show that infection of primary macrophages containing a genetic knockout of the major type I interferon, IFNß, leads to only a partial suppression of the sterol pathway, while genetic knockout of the receptor for all type I interferon family members, ifnar1, or associated signaling component, tyk2, completely abolishes the reduction of the sterol biosynthetic activity upon infection. Levels of the proteolytically cleaved nuclear forms of SREBP2, a key transcriptional regulator of sterol biosynthesis, are reduced upon infection and IFNß treatment at both the protein and de novo transcription level. The reduction in srebf2 gene transcription upon infection and IFN treatment is also found to be strictly dependent on ifnar1. Altogether these results show that type 1 IFN signaling is both necessary and sufficient for reducing the sterol metabolic network activity upon infection, thereby linking the regulation of the sterol pathway with interferon anti-viral defense responses. These findings bring a new link between sterol metabolism and interferon antiviral response and support the idea of using host metabolic modifiers of innate immunity as a potential antiviral strategy.

Viscogonioplasty in narrow angle glaucoma: a randomized controlled trial.

PURPOSE: To determine the effect of viscogonioplasty and cataract extraction on intraocular pressure in patients with narrow angle glaucoma. METHODS: This was a double-masked randomized controlled trial involving 50 eyes (25 cases and 25 controls) from 38 consecutive patients. All patients underwent phacoemulsification with or without viscogonioplasty. The main outcome measures were intraocular pressure post-treatment and number of glaucoma medications post-treatment. RESULTS: Cases had a greater reduction in intraocular pressure than controls, with a mean intraocular pressure (standard deviation) at 12 months of 13.7 (±2.89) mmHg compared with 16.2 (±3.55) mmHg in controls (P = 0.009). Cases had a greater reduction in mean number of antiglaucoma medications than controls at 12-month review, with 13 of 25 eyes (52%) of cases controlled without any antiglaucoma therapy versus 9 of 25 (36%) of the controls (P = 0.005). CONCLUSIONS: Viscogonioplasty combined with cataract extraction has a significantly greater effect than cataract extraction alone on lowering intraocular pressure in patients with poorly controlled narrow angle glaucoma and should therefore be considered as a treatment option for patients with this condition.

Visual and refractive outcome of one-site phacotrabeculectomy compared with temporal approach phacoemulsification.

BACKGROUND: We aimed to compare visual and refractive outcome following phacoemulsification and intraocular lens implant (IOL) and combined one-site phacotrabeculectomy. METHOD: We performed a retrospective study of case records of patients who had temporal incision phacoemulsification with IOL or one-site phacotrabeculectomy, between June 1997 and June 2001. The patients were matched for age group, operating list and IOL type. All patients were operated on under local anesthesia by the same surgeon. Each arm of the study had 90 patients, age range 60 to 75 years. We collected pre- and postoperative visual acuity, pre- and postoperative refraction within six months after surgery, and intended refraction. The intraocular pressure control was not recorded, as it was not the aim of our study. RESULTS: In the phacotrabeculectomy group, 76.6% of patients achieved aimed spherical equivalent, 15.5% of patients had against-the-rule (ATR) astigmatism induced by the surgery, and 90% of the patients had best corrected visual acuity (BCVA) more than 6/12. In the temporal incision phacoemulsification group, 81.1% of patients achieved aimed spherical equivalent, 10% of the patients had induced ATR by the surgery and 95.55% of patients achieved BCVA more than 6/12. CONCLUSION: In this study the visual outcome of the phacotrabeculectomy group did not differ significantly from the visual outcome of temporal approach phacoemulsification.

Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio).

Recently, the zebrafish, Danio rerio, has been recognized as a useful model for infectious disease and immunity. The Toll-like receptor (TLR) family is an evolutionarily conserved component of the innate immune system that responds to specific pathogen-associated molecular patterns (PAMPs) during an infection. This study reports the identification and characterization of a full-length orthologue of mammalian TLR3, and the key TLR pathway signaling molecules IRAK-4 and TRAF6 in the zebrafish. Sequence analysis of zebrafish TLR3 (zfTLR3), IRAK-4 (zfIRAK-4), and TRAF6 (zfTRAF6) revealed conserved domains shared with insect and mammalian genes. Quantitative real-time PCR showed that all three genes are expressed in a variety of adult tissues and during embryonic development. In in situ hybridization, we showed that zfTLR3, zfIRAK-4, and zfTRAF6 are present in distinct regions of the developing brain at 22hpf and that zfTRAF6 was observed in the developing medial neural tube. Overexpression of zfIRAK-4, zfTRAF6, or a mutant zfTLR3 construct was able to stimulate NF-kappaB activation in ZFL cells as measured by a cotransfected NF-kappaB-luciferase reporter plasmid. Messenger RNA expression profiles of each gene in zebrafish embryos and adults were examined by quantitative real-time PCR following infection with snakehead rhabdovirus (SHRV) or Edwardsiella tarda. Following exposure to SHRV, only zfTLR3 and zfTRAF6 mRNA transcripts were upregulated. Interestingly, exposure of fish to E. tarda resulted in an unexpected increase in mRNA expression of zfTLR3, as well as the anticipated upregulation of zfIRAK-4 and zfTRAF6 mRNA transcripts. These results demonstrate that zebrafish possess conserved TLR-signaling pathways, further emphasizing the utility of the zebrafish as a model for vertebrate immunology.

Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish.

The zebrafish (Danio rerio) is a widely used model for developmental biology, neurobiology, toxicology, and genetic disease. Recently, the zebrafish has been recognized as a valuable model for infectious disease and immunity. In this study the pathogenesis and inflammatory cytokine response of zebrafish to experimental Edwardsiella tarda infection was characterized. In challenge experiments, zebrafish embryos were susceptible to infection by immersion. Adult fish were susceptible to challenge by intraperitoneal (ip) injection but not static immersion unless the epithelial layer was perturbed by scraping prior to exposure. To determine if E. tarda infection induces a typical acute inflammatory response, mRNA expression levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha) were assessed by quantitative real-time PCR. The expression levels of IL-1beta and TNFalpha were significantly upregulated in infected zebrafish embryos and adults. The methods developed in this study will be particularly valuable for targeted gene disruption studies of host immune components and in zebrafish genetic screens.

Characterization of snakehead rhabdovirus infection in zebrafish (Danio rerio).

The zebrafish, Danio rerio, has become recognized as a valuable model for the study of development, genetics, and toxicology. Recently, the zebrafish has been recognized as a useful model for infectious disease and immunity. In this study, the pathogenesis and antiviral immune response of zebrafish to experimental snakehead rhabdovirus (SHRV) infection was characterized. Zebrafish 24 h postfertilization to 30 days postfertilization were susceptible to infection by immersion in 10(6) 50% tissue culture infective doses (TCID50) of SHRV/ml, and adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection of 10(5) TCID50 of SHRV/ml. Mortalities exceeded 40% in infected fish, and clinical presentation of infection included petechial hemorrhaging, redness of the abdomen, and erratic swim behavior. Virus reisolation and reverse transcription-PCR analysis of the viral nucleocapsid gene confirmed the presence of SHRV. Histological sections of moribund embryonic and juvenile fish revealed necrosis of the pharyngeal epithelium and liver, in addition to congestion of the swim bladder by cell debris. Histopathology in adult fish injected i.p. was confined to the site of injection. The antiviral response in zebrafish was monitored by quantitative real-time PCR analysis of zebrafish interferon (IFN) and Mx expression. IFN and Mx levels were elevated in zebrafish exposed to SHRV, although expression and intensity differed with age and route of infection. This study is the first to examine the pathogenesis of SHRV infection in zebrafish. Furthermore, this study is the first to describe experimental infection of zebrafish embryos with a viral pathogen, which will be important for future experiments involving targeted gene disruption and forward genetic screens.

Chest physiotherapy in infants with cystic fibrosis: to tip or not? A five-year study.

There is controversy about the need for postural drainage physiotherapy in asymptomatic infants with cystic fibrosis (CF). We aimed to compare the effectiveness of standard postural drainage chest physiotherapy (SPT) with a modified physiotherapy regimen without head-down tilt (MPT) in young infants with CF. Twenty newly diagnosed infants with CF (mean age, 2.1 months; range, 1-4) were randomized to SPT or MPT. Parents kept a detailed symptom and treatment diary for the following 12 months. Serial chest radiographs, taken at diagnosis, 12 months, 2(1/2) years, and 5 years after diagnosis, were assessed using the Brasfield score. Pulmonary function tests were compared between groups after 5 years. Of the 20 infants, 16 (80%) completed the review at 12 months, and 14 (70%) at 2(1/2) and 5 years. Patients receiving SPT had more days with upper respiratory tract symptoms than those on MPT (70 +/- 32.8 vs. 37 +/- 24.9 days; P = 0.04) and required longer courses of antibiotics (23 +/- 28.5 vs. 14 +/- 11.2 days; P = 0.05). Chest x-ray scores were similar at diagnosis but were worse at 2(1/2) years for those receiving SPT (P = 0.03). Forced vital capacity and forced expired volume in 1 sec (FEV(1)) at 5-6 years was lower for SPT than for MPT (P < 0.05). In conclusion, MPT was associated with fewer respiratory complications than SPT in infants with CF.

Reappraising first-line treatment in glaucoma management.

Despite treatment, glaucoma patients may still suffer vision loss because of inadequate control of intraocular pressure or late presentation. This article reviews the latest evidence supporting a reappraisal of first-line treatment in the management of glaucoma, including a review of latanoprost, recently approved for first-line treatment of glaucoma and ocular hypertension.

Energy metabolism in infants with cystic fibrosis.

OBJECTIVE: To determine whether a defect in energy metabolism exists in infants with cystic fibrosis (CF). DESIGN: Unselected, newly-diagnosed subjects with CF (n = 46) and 24 healthy infants aged <20 weeks had measurements of resting energy expenditure (REE), total energy expenditure (TEE) (n = 25), and body composition. Metabolizable energy intake (MEI) was calculated. Genotype, energy intake, and pancreatic status was determined in all subjects with CF, and 24 underwent bronchial lavage. RESULTS: At diagnosis, infants with CF detected by newborn screening had significant anthropometric deficits (mean [SD] z-weight = 0.5 [1.0], z-length = 0.7 [1.3]) associated with pancreatic insufficiency. Their REE, TEE, or MEI (absolute measurements, per unit body weight or fat-free mass) were not increased. No relationship between REE, TEE, or MEI and Delta F(508) genotype, and no proportional differences in individual components of MEI between subjects with CF and controls, or between subjects with CF who were homozygotes or compound heterozygotes for Delta F(508) were observed. REE and TEE were not correlated with bronchial infection or inflammation. CONCLUSION: Growth impairment during the first weeks of life in infants with CF is associated with pancreatic insufficiency. However, there is no evidence for a defect of energy metabolism related to Delta F(508), and in infants with CF, minimal lung disease is unaccompanied by increased energy expenditure.