Further Evidence of Neuroprotective Effects of Recombinant Human Erythropoietin and Growth Hormone in Hypoxic Brain Injury in Neonatal Mice.

Experimental in vivo data have recently shown complementary neuroprotective actions of rhEPO and growth hormone (rhGH) in a neonatal murine model of hypoxic brain injury. Here, we hypothesized that rhGH and rhEPO mediate stabilization of the blood−brain barrier (BBB) and regenerative vascular effects in hypoxic injury to the developing brain. Using an established model of neonatal hypoxia, neonatal mice (P7) were treated i.p. with rhGH (4000 µg/kg) or rhEPO (5000 IU/kg) 0/12/24 h after hypoxic exposure. After a regeneration period of 48 h or 7 d, cerebral mRNA expression of Vegf-A, its receptors and co-receptors, and selected tight junction proteins were determined using qRT-PCR and ELISA. Vessel structures were assessed by Pecam-1 and occludin (Ocln) IHC. While Vegf-A expression increased significantly with rhGH treatment (p < 0.01), expression of the Vegfr and TEK receptor tyrosine kinase (Tie-2) system remained unchanged. RhEPO increased Vegf-A (p < 0.05) and Angpt-2 (p < 0.05) expression. While hypoxia reduced the mean vessel area in the parietal cortex compared to controls (p < 0.05), rhGH and rhEPO prevented this reduction after 48 h of regeneration. Hypoxia significantly reduced the Ocln+ fraction of cortical vascular endothelial cells. Ocln signal intensity increased in the cortex in response to rhGH (p < 0.05) and in the cortex and hippocampus in response to rhEPO (p < 0.05). Our data indicate that rhGH and rhEPO have protective effects on hypoxia-induced BBB disruption and regenerative vascular effects during the post-hypoxic period in the developing brain.

Recombinant Human Growth Hormone Activates Neuroprotective Growth Factors in Hypoxic Brain Injury in Neonatal Mice.

Perinatal hypoxia severely disrupts cerebral metabolic and maturational programs beyond apoptotic cell death. Antiapoptotic treatments such as erythropoietin are suggested to improve outcomes in hypoxic brain injury; however, the results are controversial. We analyzed the neuroprotective effects of recombinant human growth hormone (rhGH) on regenerative mechanisms in the hypoxic developing mouse brain in comparison to controls. Using an established model of neonatal acute hypoxia (8% O2, 6 hours), P7 mice were treated intraperitoneally with rhGH (4000 µg/kg) 0, 12, and 24 hours after hypoxic exposure. After a regeneration period of 48 hours, expression of hypoxia-inducible neurotrophic factors (erythropoietin [EPO], vascular endothelial growth factor A [VEGF-A], insulin-like growth factors 1 and 2 [IGF-1/-2], IGF binding proteins) and proinflammatory markers was analyzed. In vitro experiments were performed using primary mouse cortical neurons (E14, DIV6). rhGH increased neuronal gene expression of EPO, IGF-1, and VEGF (P < .05) in vitro and diminished apoptosis of hypoxic neurons in a dose-dependent manner. In the developing brain, rhGH treatment led to a notable reduction of apoptosis in the subventricular zone and hippocampus (P < .05), abolished hypoxia-induced downregulation of IGF-1/IGF-2 expression (P < .05), and led to a significant accumulation of endogenous EPO protein and anti-inflammatory effects through modulation of interleukin-1ß and tumor necrosis factor α signaling as well as upregulation of cerebral phosphorylated extracellularly regulated kinase 1/2 levels (ERK1/2). Indicating stabilizing effects on the blood-brain barrier (BBB), rhGH significantly modified cerebrovascular occludin expression. Thus, we conclude that rhGH mediates neuroprotective effects by the activation of endogenous neurotrophic growth factors and BBB stabilization. In addition, the modification of ERK1/2 pathways is involved in neuroprotective actions of rhGH. The present study adds further evidence that pharmacologic activation of neurotrophic growth factors may be a promising target for neonatal neuroprotection.

DMBT1 is upregulated in lung epithelial cells after hypoxia and changes surfactant ultrastructure.

BACKGROUND: Hypoxia and asphyxia are known to induce surfactant inactivation in newborns. Deleted in Malignant Brain Tumors 1 (DMBT1) is an innate immunity protein with functions in epithelial differentiation and angiogenesis. It was detected in hyaline membranes of infants with respiratory distress syndrome. Human recombinant DMBT1 is able to increase the surface tension of exogenous surfactant preparations in a dose-dependent manner. METHODS: Immunohistochemistry was performed on lung sections of infants who died due to pre-, peri- or postnatal hypoxia. The lung epithelial cell line A549 was stably transfected with a DMBT1 (DMBT1+ cells) expression plasmid or with an empty plasmid (DMBT1- cells). The cells were cultured in normoxic or hypoxic conditions, and then DMBT1 as well as HIF-1α RNA expression were analyzed by using real-time-polymerase chain reaction. Human recombinant DMBT1 was added to the modified porcine natural surfactant Curosurf to examine the effect of DMBT1 on surfactant ultrastructure with electron microscopy. RESULTS: DMBT1 expression was upregulated in human lung tissue after fetal/peri-/postnatal hypoxia. In addition, in vitro experiments showed increased DMBT1 RNA expression in A549 cells after hypoxia. HIF-1α was upregulated in both DMBT1+ and DMBT1- cells in response to hypoxia. The addition of human recombinant DMBT1 to Curosurf caused an impaired surfactant ultrastructure. CONCLUSIONS: DMBT1 is upregulated in response to hypoxia and there seems to be a link between hypoxia and surfactant inactivation.

Seizure-induced neuronal apoptosis is related to dysregulation of the RNA-edited GluR2 subunit in the developing mouse brain.

Ca2+-permeable AMPA receptors (AMPAR) which crucially modify maturational programs of the developing brain are involved in seizure-induced glutamate excitotoxicity and apoptosis. Regulatory effects on AMPAR subunit composition and RNA-editing in the developing brain and their significance as therapeutic targets are not well understood. Here, we analyzed acute effects of recurrent pilocarpine-induced neonatal seizures on age- and region-specific expression of AMPAR subunits and adenosine deaminases (ADAR) in the developing mouse brain (P10). After recurrent seizure activity and regeneration periods of 6-72 h cerebral mRNA levels of GluR (glutamate receptor subunit) 1, GluR2, GluR3, and GluR4 were unaffected compared to controls. However, ratio of GluR2 and GluR4 to pooled GluR1-4 mRNA concentration significantly decreased in seizure-exposed brains in comparison to controls. After a regeneration period of 24-72 h ADAR1 and ADAR2 mRNA expression was significantly lower in seizure-exposed brains than in those of controls. This was confirmed at the protein level in the hippocampal CA3 region. We observed a regionally increased apoptosis (TUNEL+ and CC3+ cells) in the hippocampus, parietal cortex and subventricular zone of seizure-exposed brains in comparison to controls. Together, present in vivo data demonstrate the maturational age-specific, functional role of RNA-edited GluR2 in seizure-induced excitotoxicity in the developing mouse brain. In response to recurrent seizure activity, we observed reduced expression of GluR2 and the GluR2 mRNA-editing enzymes ADAR1 and ADAR2 accompanied by increased apoptosis in a region-specific manner. Thus, AMPA receptor subtype-specific mRNA editing is assessed as a promising target of novel neuroprotective treatment strategies in consideration of age-related developmental mechanisms.

C1 Esterase Inhibitor Reduces BBB Leakage and Apoptosis in the Hypoxic Developing Mouse Brain.

Inflammatory pathways involved in blood-brain barrier (BBB) vulnerability and hypoxic brain oedema in models of perinatal brain injury seem to provide putative therapeutic targets. To investigate impacts of C1-esterase inhibitor (C1-INH; 7.5-30 IU/kg, i.p.) on functional BBB properties in the hypoxic developing mouse brain (P7; 8% O2 for 6 h), expression of pro-apoptotic genes (BNIP3, DUSP1), inflammatory markers (IL-1ß, TNF-alpha, IL-6, MMP), and tight junction proteins (ZO-1, occludin, claudin-1, -5), and S100b protein concentrations were analysed after a regeneration period of 24 h. Apoptotic cell death was quantified by CC3 immunohistochemistry and TUNEL staining. In addition to increased apoptosis in the parietal cortex, hippocampus, and subventricular zone, hypoxia significantly enhanced the brain-to-plasma albumin ratio, the cerebral S100b protein levels, BNIP3 and DUSP1 mRNA concentrations as well as mRNA expression of pro-inflammatory cytokines (IL-1ß, TNF-alpha). In response to C1-INH, albumin ratio and S100b concentrations were similar to those of controls. However, the mRNA expression of BNIP3 and DUSP1 and pro-inflammatory cytokines as well as the degree of apoptosis were significantly decreased compared to non-treated controls. In addition, occludin mRNA levels were elevated in response to C1-INH (p < 0.01). Here, we demonstrate for the first time that C1-INH significantly decreased hypoxia-induced BBB leakage and apoptosis in the developing mouse brain, indicating its significance as a promising target for neuroprotective therapy.

HIV-1 integrase strand-transfer inhibitor resistance in southern Taiwan.

The use of antiretroviral therapy has reduced rates of mortality and morbidity in patients with human immunodeficiency virus/acquired immune deficiency syndrome(HIV/AIDS). However, transmission of drug-resistant strains poses a challenge to control the spread of HIV-1. Primary resistance to integrase strand-transfer inhibitors (INSTIs) is rare despite their increased use. The prevalence of transmitted drug resistance (TDR) to INSTIs was 0.9% in northern Taiwan. This study was to analyse the prevalence and risk factors of TDR to INSTIs in southern Taiwan. In this study, we enrolled antiretroviral treatment-naïve HIV-1-infected subjects who underwent voluntary counselling and testing from 2013 to 2016 in southern Taiwan. Genotypic drug resistance, coreceptor tropism (CRT) and INSTI resistance were determined. Logistic regression was used to analyse the risk factors for INSTI polymorphic substitution. Sequences were obtained from 184 consecutive individuals, of whom 96.7% were men who have sex with men and 3.3% were heterosexual. Of the patients, 10% (19/183) had hepatitis B and 33.3% (61/183) had syphilis infection. Subtype B HIV-1 strains were found in 96.1% of the patients. Fifteen patients (8.4%, 15/178) harboured nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors or protease inhibitors resistance. CCR-5 coreceptors were used by 71.4% (130/184) of the patients. None of the patients had INSTI resistance-associated mutations, however 16 patients had INSTI polymorphic substitutions, and they were associated with a higher HIV viral load (p = 0.03, OR 2.4, CI 1.1-5.3) and syphilis infection (p = 0.03, OR 3.7, CI 1.1-12.0). In conclusion, no signature INSTI resistance-associated mutations were detected in our cohort. Continued monitoring of TDR to INSTI is needed due to the increased use of INSTIs.

Differential regulation of angiogenesis in the developing mouse brain in response to exogenous activation of the hypoxia-inducible transcription factor system.

Angiogenesis due to hypoxic-ischemic (HI) injury represents a crucial compensatory mechanism of the developing brain that is mainly regulated by hypoxia-inducible transcription factors (HIF). Pharmacological stimulation of HIF is suggested as a neuroprotective option, however, studies of its effects on vascular development are limited. We analyzed the influence of the prolyl-4-hydroxylase inhibitor (PHI), FG-4497, and erythropoietin (rhEPO) on post-hypoxic angiogenesis (angiogenic growth factors, vessel structures) in the developing mouse brain (P7) assessed after a regeneration period of 72 h. Exposure to systemic hypoxia (8% O2, 6 h) was followed by treatment (i.p.) with rhEPO (2500/5000 IU/kg) at 0, 24 and 48 h or FG-4497 (60/100 mg/kg) compared to controls. In response to FG-4497 treatment cortical and hippocampal vessel area and branching were significantly increased compared to controls. This was associated with elevated ANGPT-2 as well as decreased ANGPT-1 and TIE-2 mRNA levels. In response to rhEPO, mildly increased angiogenesis was associated with elevated ANGPT-2 but also TIE-2 mRNA levels in comparison to controls. In conclusion, present data demonstrate a differential regulation of the angiopoietin/TIE-2 system in response to PHI and rhEPO in the post-hypoxic developing brain pointing to potential functional consequences for vascular regeneration and vessel development.

Oxygen-sensitive regulation and neuroprotective effects of growth hormone-dependent growth factors during early postnatal development.

Perinatal hypoxia severely disrupts metabolic and somatotrophic development, as well as cerebral maturational programs. Hypoxia-inducible transcription factors (HIFs) represent the most important endogenous adaptive mechanisms to hypoxia, activating a broad spectrum of growth factors that contribute to cell survival and energy homeostasis. To analyze effects of systemic hypoxia and growth hormone (GH) therapy (rhGH) on HIF-dependent growth factors during early postnatal development, we compared protein (using ELISA) and mRNA (using quantitative RT PCR) levels of growth factors in plasma and brain between normoxic and hypoxic mice (8% O2, 6 h; postnatal day 7, P7) at P14. Exposure to hypoxia led to reduced body weight (P < 0.001) and length (P < 0.04) compared with controls and was associated with significantly reduced plasma levels of mouse GH (P < 0.01) and IGF-1 (P < 0.01). RhGH abrogated these hypoxia-induced changes of the GH/IGF-1 axis associated with normalization of weight and length gain until P14 compared with controls. In addition, rhGH treatment increased cerebral IGF-1, IGF-2, IGFBP-2, and erythropoietin mRNA levels, resulting in significantly reduced apoptotic cell death in the hypoxic, developing mouse brain. These data indicate that rhGH may functionally restore hypoxia-induced systemic dysregulation of the GH/IGF-1 axis and induce upregulation of neuroprotective, HIF-dependent growth factors in the hypoxic developing brain.

DNA methylation and transcription in HERV (K, W, E) and LINE sequences remain unchanged upon foreign DNA insertions.

AIM: DNA methylation and transcriptional profiles were determined in the regulatory sequences of the human endogenous retroviral (HERV-K, -W, -E) and LINE-1.2 elements and were compared between non-transgenomic and plasmid-transgenomic cells. METHODS: DNA methylation profiles in the HERV (K, W, E) and LINE sequences were determined by bisulfite genomic sequencing. The transcription of these genome segments was assessed by quantitative real-time PCR. RESULTS: In HERV-K, HERV-W and LINE-1.2 the levels of DNA methylation ranged between 75 and 98%, while in HERV-E they were around 60%. Nevertheless, the HERV and LINE-1.2 sequences were actively transcribed. No differences were found in comparisons of HERV and LINE-1.2 CpG methylation and transcription patterns between non-transgenomic and plasmid-transgenomic HCT116 cells. CONCLUSION: The insertion of a 5.6 kbp plasmid into the HCT116 genome had no effect on the HERV and LINE-1.2 methylation and transcription profiles, although other parts of the HCT116 genome had shown marked changes. These repetitive sequences are transcribed, probably because the large number of HERV and LINE-1.2 elements harbor copies with non- or hypo-methylated long terminal repeat sequences.

Hypoxia Potentiates LPS-Mediated Cytotoxicity of BV2 Microglial Cells In Vitro by Synergistic Effects on Glial Cytokine and Nitric Oxide System.

BACKGROUND: Microglial activation due to a variety of stimuli induces secretion of neurotoxic substances including inflammatory cytokines and nitric oxide (NO). Clinical studies indicate a cross-link between inflammatory and hypoxia-regulated pathways suggesting that bacterial infections markedly sensitize the immature brain to hypoxic injury. METHODS: The impact of inflammation and hypoxia on interleukin (IL)-1ß, IL-6, tumor necrosis factor α (TNF-α), and NO secretion and microglia-induced cytotoxicity was investigated exposing BV2 cells to lipopolysaccharides (LPS) and hypoxia (1% O2). Cytotoxicity, NO, and cytokine release was quantified by MTS and Griess assays and by enzyme-linked immunosorbent assays, respectively. RESULTS: LPS exposure of BV2 cells induced a significant, persistent production of NO, IL-1ß, IL-6, and TNF-α. Even after LPS removal, ongoing NO and cytokine secretion was observed. Hypoxia mediated exclusively a significant, short-term IL-1ß increase, but enhanced LPS-induced cytokine and NO secretion significantly. In addition, LPS-induced supernatants exhibited a stronger cytotoxic effect in glial and neuronal cells than LPS exposition (p < 0.001). Hypoxia potentiated LPS-induced cytotoxicity. CONCLUSION: Present data prove that LPS-induced soluble factors rather than LPS exposure mediate microglial toxicity under conditions of hypoxia in vitro. Apart from potential protective effects of the hypoxia-inducible transcription factor (HIF)-1α system, activation of proinflammatory pathways may markedly sensitize microglial cells to promote hypoxia-induced injuries of the developing brain.

Activin A regulation under global hypoxia in developing mouse brain.

Activin A is a multifunctional growth and differentiation factor with pronounced neuroprotective properties that is strongly up-regulated in various forms of acute brain disorders and injuries including epilepsy, stroke and trauma. In a pediatric context, activin A has been advanced as a potential marker for the severity of perinatal hypoxic-ischemic brain injury. Here we investigated the regulation of activin A under global hypoxia without ischemia in primary cultures of cortical neurons and in neonatal and adult mice of two strains (C57BL/6 and CD-1). From birth to adulthood, activin ßA subunit, activin receptors, and functional activin antagonists were all expressed at roughly similar mRNA levels in the brain of C57BL/6 mice. Independent of mouse line and age, we found both moderate (11% O2, 2h) and severe hypoxia (8%, 6h) to be consistently associated with normal or even reduced levels of activin ßA (Inhba) mRNA. The surprising unresponsiveness of Inhba expression to hypoxia was confirmed at the protein level. In situ hybridization did not indicate regional, hypoxia-related differences in Inhba expression. Pharmacologic stabilization of hypoxia inducible factors with the prolyl hydroxylase inhibitor FG-4497 did not influence Inhba mRNA levels in neonatal mice. Our data indicate that pure hypoxia differs from other, more complex types of brain damage in that it appears not to recruit activin A as an endogenous neuroprotective agent.

HIV-1 fusion is blocked through binding of GB Virus C E2-derived peptides to the HIV-1 gp41 disulfide loop [corrected].

A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors.

O-linked N-acetylglucosaminylation of Sp1 inhibits the human immunodeficiency virus type 1 promoter.

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication are regulated by the promoter/enhancer located in the U3 region of the proviral 5' long terminal repeat (LTR). The binding of cellular transcription factors to specific regulatory sites in the 5' LTR is a key event in the replication cycle of HIV-1. Since transcriptional activity is regulated by the posttranslational modification of transcription factors with the monosaccharide O-linked N-acetyl-D-glucosamine (O-GlcNAc), we evaluated whether increased O-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the O-GlcNAcylation-enhancing agent glucosamine (GlcN) repressed viral transcription in a dose-dependent manner. Overexpression of O-GlcNAc transferase (OGT), the sole known enzyme catalyzing the addition of O-GlcNAc to proteins, specifically inhibited the activity of the HIV-1 LTR promoter in different T-cell lines and in primary CD4(+) T lymphocytes. Inhibition of HIV-1 LTR activity in infected T cells was most efficient (>95%) when OGT was recombinantly overexpressed prior to infection. O-GlcNAcylation of the transcription factor Sp1 and the presence of Sp1-binding sites in the LTR were found to be crucial for this inhibitory effect. From this study, we conclude that O-GlcNAcylation of Sp1 inhibits the activity of the HIV-1 LTR promoter. Modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation and links viral replication to the glucose metabolism of the host cell. Hence, the establishment of a metabolic treatment might supplement the repertoire of antiretroviral therapies against AIDS.

HIV entry inhibition by the envelope 2 glycoprotein of GB virus C.

Epidemiological studies have revealed an association between GB virus C (GBV-C) long-term viraemia and ameliorated HIV disease progression. We have provided evidence that a single protein of GBV-C, the glycoprotein E2, interferes with early HIV replication steps of both X4- and R5-tropic HIV strains. Preincubation with anti-E2 antibody specifically abrogates the inhibitory effect. Results were confirmed by the in-vitro expression of GBV-C E1/E2 encoding RNA.

Inhibition of HIV strains by GB virus C in cell culture can be mediated by CD4 and CD8 T-lymphocyte derived soluble factors.

OBJECTIVE: A number of studies concerning the pathogenesis of GB virus C (GBV-C) in HIV-infected people suggest a beneficial effect and improved survival for dually infected individuals. However there has remained controversy regarding the clinical relevance of these findings, as some studies have not confirmed these observations. To address the possibility of direct inhibitory mechanisms, we studied the impact of GBV-C on HIV-1 replication in vitro. METHODS: Peripheral blood mononuclear cells (PBMC) were infected with sera from GBV-C positive individuals or transfected with GBV-C specific RNA and superinfected with HIV. Replication kinetics of HIV were studied by quantification of HIV-p24 release. Induction of soluble antiretroviral factors were monitored with an HIV infection assay and by quantification of chemokine secretion. Changes in chemokine receptor expression were analysed by flow cytometry. RESULTS: We demonstrate that GBV-C infection of PBMC leads to significant replication inhibition of R5- and X4-HIV isolates representing eight HIV clades. The inhibitory effect is mediated by GBV-C infection and also by expression of GBV-C structural glycoproteins and/or of non-structural proteins NS2/NS3. Upon GBV-C infection CD4 and CD8 T lymphocytes produce soluble HIV-suppression factors. Induction of stromal cell-derived factor (SDF)-1 and subsequent internalization of CXCR4 was not observed. CONCLUSIONS: CD4 and CD8 T lymphocytes are stimulated by GBV-C to secrete antiretroviral factors, inhibiting R5- and X4-HIV strains. As no induction of SDF-1 and no down-regulation of the respective receptor CXCR4 could be observed, it is likely that additional unidentified factors causing inhibition of X4-HIV strains are induced by GBV-C.