Mechanisms of Platelet-Derived Growth Factor-BB in Restoring HIV Tat-Cocaine-Mediated Impairment of Neuronal Differentiation.

Diminished adult neurogenesis is known to play a key role in the pathogenesis of diverse neurodegenerative disorders such as HIV-associated neurological disorders (HAND). Cocaine, often abused by HIV-infected patients, has been suggested to worsen HIV-associated CNS disease. Mounting evidence also indicates that HIV infection can lead not only to neuronal dysfunction or loss, but can also negatively impact neurogenesis, resulting in generation of fewer adult neural progenitor cells (NPCs) in the dentate gyrus of the hippocampus, brain area critical for memory and learning. The crucial role of platelet-derived growth factor-BB (PDGF-BB) in providing tropic support for the neurons as well as in promoting NPC proliferation has been demonstrated by us previously. However, whether PDGF-BB regulates neuronal differentiation especially in the context of HAND and drug abuse remains poorly understood. In this study, we demonstrate that pretreatment of rat hippocampal NPCs with PDGF-BB restored neuronal differentiation that had been impaired by HIV Tat and cocaine. To further study the intracellular mechanism(s) involved in this process, we examined the role of transient receptor potential canonical (TRPC) channels in mediating neuronal differentiation in the presence of PDGF-BB. TRPC channels are Ca2+-permeable, nonselective cationic channels that elicit a variety of physiological functions. Parallel but distinct ERK, Akt signaling pathways with downstream activation of CREB were found to be critical for neuronal differentiation. Pharmacological blocking of TRPC channels resulted in suppression of PDGF-mediated differentiation and PDGF-BB-induced activation of ERK and Akt, culminating also to inhibition of PDGF-induced activation of CREB. Taken together, these findings underpin the role of TRPC channel as a novel target regulating cell differentiation mediated by PDGF-BB. This finding could have implications for development of therapeutic interventions aimed at restoration of Tat and cocaine-mediated impairment of neurogenesis in drug abusing HAND patients.

Involvement of miR-9/MCPIP1 axis in PDGF-BB-mediated neurogenesis in neuronal progenitor cells.

Highly conserved microRNA-9 (miR-9) has a critical role in various cellular processes including neurogenesis. However, its regulation by neurotropins that are known to mediate neurogenesis remains poorly defined. In this study, we identify platelet-derived growth factor-BB (PDGF-BB)-mediated upregulation of miR-9, which in turn downregulates its target gene monocyte chemotactic protein-induced protein 1 (MCPIP1), as a key player in modulating proliferation, neuronal differentiation as well as migration of neuronal progenitor cells (NPCs). Results indicate that miR-9-mediated NPC proliferation and neuronal differentiation involves signaling via the nuclear factor-kappa B (NF-κB) and cAMP response element-binding protein (CREB) pathways, and that NPC migration involves CREB but not the NF-κB signaling. These findings thus suggest that miR-9-mediated downregulation of MCPIP1 acts as a molecular switch regulation of neurogenesis.

Improving susceptibility mapping using a threshold-based K-space/image domain iterative reconstruction approach.

To improve susceptibility quantification, a threshold-based k-space/image domain iterative approach that uses geometric information from the susceptibility map itself as a constraint to overcome the ill-posed nature of the inverse filter is introduced. Simulations were used to study the accuracy of the method and its robustness in the presence of noise. In vivo data were processed and analyzed using this method. Both simulations and in vivo results show that most streaking artifacts inside the susceptibility map caused by the ill-defined inverse filter were suppressed by the iterative approach. In simulated data, the bias toward lower mean susceptibility values inside vessels has been shown to decrease from around 10% to 2% when choosing an appropriate threshold value for the proposed iterative method. Typically, three iterations are sufficient for this approach to converge and this process takes less than 30 s to process a 512×512×256 dataset. This iterative method improves quantification of susceptibility inside vessels and reduces streaking artifacts throughout the brain for data collected from a single-orientation acquisition. This approach has been applied to vessels alone as well as to vessels and other structures with lower susceptibility to generate whole brain susceptibility maps with significantly reduced streaking artifacts.

Safety and efficacy of decitabine in combination with temozolomide in metastatic melanoma: a phase I/II study and pharmacokinetic analysis.

BACKGROUND: Temozolomide (TMZ) is widely used for chemotherapy of metastatic melanoma. We hypothesized that epigenetic modulators will reverse chemotherapy resistance, and in this article, we report studies that sought to determine the recommended phase 2 dose (RP2D), safety, and efficacy of decitabine (DAC) combined with TMZ. PATIENTS AND METHODS: In phase I, DAC was given at two dose levels: 0.075 and 0.15 mg/kg intravenously daily × 5 days/week for 2 weeks, TMZ orally 75 mg/m(2) qd for weeks 2-5 of a 6-week cycle. The phase II portion used a two-stage Simon design with a primary end point of objective response rate (ORR). RESULTS: The RP2D is DAC 0.15 mg/kg and TMZ 75 mg/m(2). The phase II portion enrolled 35 patients, 88% had M1c disease; 42% had history of brain metastases. The best responses were 2 complete response (CR), 4 partial response (PR), 14 stable disease (SD), and 13 progressive disease (PD); 18% ORR and 61% clinical benefit rate (CR + PR + SD). The median overall survival (OS) was 12.4 months; the 1-year OS rate was 56%. Grade 3/4 neutropenia was common but lasted >7 days in six patients. CONCLUSIONS: The combination of DAC and TMZ is safe, leads to 18% ORR and 12.4-month median OS, suggesting possible superiority over the historical 1-year OS rate, and warrants further evaluation in a randomized setting.

Exosome-mediated shuttling of microRNA-29 regulates HIV Tat and morphine-mediated neuronal dysfunction.

Neuronal damage is a hallmark feature of HIV-associated neurological disorders (HANDs). Opiate drug abuse accelerates the incidence and progression of HAND; however, the mechanisms underlying the potentiation of neuropathogenesis by these drugs remain elusive. Opiates such as morphine have been shown to enhance HIV transactivation protein Tat-mediated toxicity in both human neurons and neuroblastoma cells. In the present study, we demonstrate reduced expression of the tropic factor platelet-derived growth factor (PDGF)-B with a concomitant increase in miR-29b in the basal ganglia region of the brains of morphine-dependent simian immunodeficiency virus (SIV)-infected macaques compared with the SIV-infected controls. In vitro relevance of these findings was corroborated in cultures of astrocytes exposed to morphine and HIV Tat that led to increased release of miR-29b in exosomes. Subsequent treatment of neuronal SH-SY5Y cell line with exosomes from treated astrocytes resulted in decreased expression of PDGF-B, with a concomitant decrease in viability of neurons. Furthermore, it was shown that PDGF-B was a target for miR-29b as evidenced by the fact that binding of miR-29 to the 3'-untranslated region of PDGF-B mRNA resulted in its translational repression in SH-SY5Y cells. Understanding the regulation of PDGF-B expression may provide insights into the development of potential therapeutic targets for neuronal loss in HIV-1-infected opiate abusers.

Association of genetic polymorphisms in ESR2, HSD17B1, ABCB1, and SHBG genes with colorectal cancer risk.

The incidence rates and relative risks for colorectal cancer (CRC) are higher in men than in women. Sex steroids may play a role in this gender-associated difference in CRC risk. This study was conducted to explore the relationship of single nucleotide polymorphisms (SNPs) in steroid hormone signaling (ESR1, ESR2, PGR, NR1I2, and SHBG), phase I- and II-metabolizing enzyme (COMT, HSD17B1, CYP1A1, CYP17A1, CYP1A2, CYP1B1, CYP2C9, CYP3A4, CYP2C19, and GSTP1), and hormone transporter (ABCB1) genes with the risk of CRC in German women and men, separately. From the population-based DACHS study (South Germany), 47 putatively functional SNPs were genotyped in 1798 CRC cases (746 women and 1052 men) and 1810 controls (732 women and 1078 men). Significant allele dose-response associations were observed with ESR2_rs1255998, ESR2_rs928554, HSD17B1_rs605059, and ABCB1_rs2229109 in women (P trend=0.004, 0.05, 0.03, and 0.05 respectively) and with ABCB1_rs1045642, ABCB1_rs9282564, and SHBG_rs6259 in men (P trend=0.01, 0.03, and 0.02 respectively). The ESR2_rs1255998_G allele showed the most significant association with risk for CRC in women, with a per-allele odds ratio (OR) of 0.68 (95% confidence interval (CI) 0.52-0.88). This finding was replicated in an independent study from North Germany including 1076 female CRC cases and 1151 controls (OR=0.84, 95% CI 0.71-1.04), yielding a per-allele OR of 0.80 (95% CI 0.69-0.93, P trend=0.003) in the pooled sample. These findings implicate a role of ESR2 in the risk for developing CRC in women and suggest that HSD17B1, ABCB1, and SHBG genes may contribute to sex steroid-mediated effects on CRC development.

TLR-3 polymorphism is an independent prognostic marker for stage II colorectal cancer.

BACKGROUND: Clinicopathologic stage is still the main parameter to evaluate the prognosis of newly diagnosed colorectal cancer (CRC) patients. Although molecular markers have been suggested for follow up of treated CRC patients, their complete clinical application is still under evaluation. MATERIALS AND METHODS: To evaluate the association of immune-related genes with CRC prognosis and survival, a total of 19 single nucleotide polymorphisms (SNPs) were genotyped in 614 German patients within the Kiel cohort (POPGEN). RESULTS: A promoter variant (rs1800872) in the Interleukin-10 (IL-10) gene was associated with an increased lymph node metastasis involvement [odds ratio (OR) = 2.1, 95% confidence interval (CI) = 1.03-4.2, for carriers of the TT genotype]. More importantly, among 582 followed up patients the SNP rs3775291 in the toll-like receptor 3 (TLR-3) gene was associated with CRC specific survival (150 events). Patients carrying the TT genotype had a 93% increased risk of death compared with the CC carriers [hazard ratio (HR) = 1.93, 95% CI 1.14-3.28]. The observed effect of the TLR-3 variant was restricted to stage II patients (HR = 4.14, 95% CI 1.24-13.84) and to patients who did not receive adjuvant therapy (HR = 3.2, 95% CI 1.4-7.7). CONCLUSIONS: Our results may provide additional candidates for risk assessment in stage II CRC patients for treatment decision. Further validation of the presented findings is warranted.

COGENT (COlorectal cancer GENeTics): an international consortium to study the role of polymorphic variation on the risk of colorectal cancer.

It is now recognised that a part of the inherited risk of colorectal cancer (CRC) can be explained by the co-inheritance of low-penetrance genetic variants. The accumulated experience to date in identifying these variants has served to highlight difficulties in conducting statistically and methodologically rigorous studies and follow-up analyses. The COGENT (COlorectal cancer GENeTics) consortium includes 20 research groups in Europe, Australia, the Americas, China and Japan. The overarching goal of COGENT is to identify and characterise low-penetrance susceptibility variants for CRC through association-based analyses. In this study, we review the rationale for identifying low-penetrance variants for CRC and our proposed strategy for establishing COGENT.

TRPC channel-mediated neuroprotection by PDGF involves Pyk2/ERK/CREB pathway.

Platelet-derived growth factor-BB (PDGF) has been reported to provide tropic support for neurons in the central nervous system. The protective role of PDGF on dopaminergic neurons, especially in the context of HIV-associated dementia (HAD), however, remains largely unknown. Here, we show that exogenous PDGF was neuroprotective against toxicity induced by HIV-1 Tat in primary midbrain neurons. Furthermore, we report the involvement of transient receptor potential canonical (TRPC) channels in PDGF-mediated neuroprotection. TRPC channels are Ca(2+)-permeable, nonselective cation channels with a variety of physiological functions. Blocking TRPC channels with either a blocker or short-interfering RNAs (specific for TRPC 5 and 6) in primary neurons resulted in suppression of both PDGF-mediated neuroprotection as well as elevations in intracellular Ca(2+). PDGF-mediated neuroprotection involved parallel but distinct ERK/CREB and PI3K/Akt pathways. TRPC channel blocking also resulted in suppression of PDGF-induced Pyk2/ERK/CREB activation, but not Akt activation. Relevance of these findings in vivo was further corroborated by intrastriatal injections of PDGF and HIV-1 Tat in mice. Administration of PDGF was able to rescue the dopaminergic neurons in the substantia nigra from Tat-induced neurotoxicity. This effect was attenuated by pre-treatment of mice with the TRP blocker, thus underscoring the novel role of TRPC channels in the neuroprotection mediated by PDGF.

Glial-restricted precursors: patterns of expression of opioid receptors and relationship to human immunodeficiency virus-1 Tat and morphine susceptibility in vitro.

Recent evidence suggests that human immunodeficiency virus (HIV)-induced pathogenesis is exacerbated by opioid abuse and that the synergistic toxicity may result from direct actions of opioids in immature glia or glial precursors. To assess whether opioids and HIV proteins are directly toxic to glial-restricted precursors (GRPs), we isolated neural stem cells from the incipient spinal cord of embryonic day 10.5 ICR mice. GRPs were characterized immunocytochemically and by reverse transcriptase-polymerase chain reaction (RT-PCR). At 1 day in vitro (DIV), GRPs failed to express mu opioid receptors (MOR or MOP) or kappa-opioid receptors (KOR or KOP); however, at 5 DIV, most GRPs expressed MOR and KOR. The effects of morphine (500 nM) and/or Tat (100 nM) on GRP viability were assessed in GRPs at 5 DIV by examining the apoptotic effector caspase-3 and cell viability (ethidium monoazide exclusion) at 96 h following continuous exposure. Tat or morphine alone or in combination caused significant increases in GRP cell death at 96 h, but not at 24 h, following exposure. Although morphine or Tat caused increases in caspase-3 activity at 4 h, this was not accompanied with increased cleaved caspase-3 immunoreactive or ethidium monoazide-positive dying cells at 24 h. The results indicate that prolonged morphine or Tat exposure is intrinsically toxic to isolated GRPs and/or their progeny in vitro. Moreover, MOR and KOR are widely expressed by Sox2 and/or Nkx2.2-positive GRPs in vitro and the pattern of receptor expression appears to be developmentally regulated. The temporal requirement for prolonged morphine and HIV-1 Tat exposure to evoke toxicity in glia may coincide with the attainment of a particular stage of maturation and/or the development of particular apoptotic effector pathways and may be unique to spinal cord GRPs. Should similar patterns occur in vivo then we predict that immature astroglia and oligodendroglia may be preferentially vulnerable to HIV-1 infection or chronic opiate exposure.

Retention rate of levetiracetam in children with intractable epilepsy at 1 year.

UNLABELLED: Levetiracetam (LEV) is a novel antiepileptic drug (AED) that has recently obtained marketing authorisation for use in children. The purpose of this study was to assess the efficacy, tolerability and retention rate of LEV in children with refractory epilepsies. It is a retrospective multicentre observational study reporting the use of LEV in 200 children, aged 0.3-19 years (median 9-years-old) over a 4-year period. All of the patients included in the study had refractory epilepsy with a median age of onset of epilepsy of 3 years (range 0-13 years). The 38% had failed and withdrawn 3 or more AEDs previously and 24% were taking at least 2 other AEDs in addition to LEV. The 47% had focal, and 58% had symptomatic epilepsies. The LEV dose ranged from 8 to 100 mg/kg/day (mean 39 mg/kg). The study comprised 215 person years of LEV exposure. RESULTS: LEV was well tolerated with a retention rate of 49% at 1 year. No serious adverse events were reported with possibly related adverse events reported in only 24% of patients (mainly emotional or behavioural changes). At more than 2, 6 and 12 months, worthwhile improvement (>50% seizure reduction) was noted in 60, 40 and 32%, including seizure freedom in 14, 14 and 5%, respectively. CONCLUSION: Our results confirm the efficacy and tolerability of LEV in children with refractory epilepsies and demonstrate good response and retention rates at 12 months. It represents the largest cohort of paediatric patients published so far on LEV with a 1-year follow-up.

Molecular targets of opiate drug abuse in neuroAIDS.

Opiate drug abuse, through selective actions at mu-opioid receptors (MOR), exacerbates the pathogenesis of human immunodeficiency virus-1 (HIV-1) in the CNS by disrupting glial homeostasis, increasing inflammation, and decreasing the threshold for pro-apoptotic events in neurons. Neurons are affected directly and indirectly by opiate-HIV interactions. Although most opiates drugs have some affinity for kappa (KOR) and/or delta (DOR) opioid receptors, their neurotoxic effects are largely mediated through MOR. Besides direct actions on the neurons themselves, opiates directly affect MOR-expressing astrocytes and microglia. Because of their broad-reaching actions in glia, opiate abuse causes widespread metabolic derangement, inflammation, and the disruption of neuron-glial relationships, which likely contribute to neuronal dysfunction, death, and HIV encephalitis. In addition to direct actions on neural cells, opioids modulate inflammation and disrupt normal intercellular interactions among immunocytes (macrophages and lymphocytes), which on balance further promote neuronal dysfunction and death. The neural pathways involved in opiate enhancement of HIV-induced inflammation and cell death, appear to involve MOR activation with downstream effects through PI3-kinase/Akt and/or MAPK signaling, which suggests possible targets for therapeutic intervention in neuroAIDS.

3-Nitrotyrosine immunoreactivity in olfactory receptor neurons of patients with Alzheimer's disease: implications for impaired odor sensitivity.

Olfactory sensory function is impaired in patients with the diagnosis of probable Alzheimer's disease (AD) compared to elderly controls, and the olfactory epithelium (OE) of AD patients exhibits several pathological changes characteristic of the AD brain. To confirm that the populations from whom our postmortem tissues are obtained exhibit similar decrements in sensory function, threshold testing was performed; probable AD patients had significantly higher olfactory thresholds than controls. To determine if oxidative stress contributes to decreased olfactory function in AD, we localized 3-nitrotyrosine (3-NT) immunoreactivity in OE obtained postmortem from patients with neuropathologically confirmed AD and age-matched controls with brains free of significant neurodegenerative pathology. In AD patients, immunoreactivity was localized in olfactory receptor neurons (ORNs), including dendritic knobs where ion channels that participate in sensory transduction are located, suggesting a direct mechanism for olfactory impairment. In controls, immunoreactivity occurred in blood vessel endothelium, suggesting age-related vascular dysfunction. Immunohistochemistry for CD68, a macrophage scavenger receptor, demonstrated activated macrophages, a source of free radicals contributing to 3-NT formation, in the OE of AD patients but not controls. These results demonstrate increased oxidative stress and modification of ORN proteins that may contribute directly to olfactory impairment in AD patients.

Oxidative stress in HIV demented patients and protection ex vivo with novel antioxidants.

OBJECTIVE: To determine the role of oxidative stress in mediating HIV dementia and to identify novel therapeutic compounds that may block this oxidative stress. METHODS: Brain tissue from patients with HIV encephalitis and macaques with simian immune deficiency virus encephalitis was immunostained for lipid peroxidation. Oxidized proteins in CSF of patients with various stages of HIV dementia were quantitated and we determined whether CSF from these patients could alter mitochondrial function. Several novel compounds with antioxidant effects were screened to determine their relative efficacy in protecting against CSF-induced neurotoxicity. RESULTS: Evidence for oxidative stress was present both in brain and in CSF. The presence of oxidized proteins in the CSF and CSF-induced progressive decrease in mitochondrial activity correlated with the severity of cognitive impairment, but only the group of patients with moderate to severe dementia reached statistical significance. L-deprenyl, didox, imidate, diosgenin, and ebselen blocked the CSF-induced toxicity. No effect of trimidox, ruthenium red, or Quercetin was seen. CONCLUSIONS: Increased oxidative stress is present in brain and CSF of HIV-infected patients. There is also an accumulation of toxic substances in the CSF that are capable of inducing oxidative stress. The authors have identified several novel compounds that are capable of blocking the CSF-induced toxicity, the therapeutic potential of which is worthy of further exploration.

Polymorphisms at CYP and GST gene loci. Prevalence in the Indian population.

OBJECTIVE: The aim of the present study was to investigate the distribution of the CYP1A1*3 and CYP2D6*4 alleles, the null genotype at the GSTM1 and G

Influence of smokeless tobacco exposure on detoxification status and chromosomal damage in male and female habitues.

In India, a large number of tobacco chewers and masheri users are chronically exposed to tobacco genotoxicants. Detoxification processes involving cellular glutathione (GSH) and glutathione S-transferases (GST) determine the outcome of exposure to environmental mutagens including those present in tobacco. Hence, in this study, GSH levels, GST activity, GSTM1 genotype and cytogenetic damage were determined using lymphocytes from 114 smokeless tobacco habitues and controls. The study groups comprised of male tobacco chewers, female masheri users, and age- and sex-matched controls. Irrespective of the tobacco habit, GSH levels and GST activity were higher in females than in males. In both the groups of habitues, GSH levels were similar to those in controls, while a significant reduction in GST activity was observed in tobacco chewers only. The frequency of cytogenetic alterations was significantly elevated in both the groups of habitues with respect to controls. However, break-type aberrations were more frequent in tobacco chewers while gaps were commonly observed in masheri users. Differences in the nature of chromosomal alterations in the two groups of habitues appeared to be related to variation in total tobacco exposure and gender-related differences in the efficacy of the GSH/GST detoxification system.

Inhibitory and enhancing effects of IFN-gamma and IL-4 on SHIV(KU) replication in rhesus macaque macrophages: correlation between Th2 cytokines and productive infection in tissue macrophages during late-stage infection.

HIV-1 is dual-tropic for CD4+ T lymphocytes and macrophages, but virus production in the macrophages becomes manifest only during late-stage infection, after CD4+ T cell functions are lost, and when opportunistic pathogens begin to flourish. In this study, the SHIV/macaque model of HIV pathogenesis was used to assess the role of cytokines in regulating virus replication in the two cell types. We injected complete Freund's adjuvant (CFA) intradermally into SHIV(KU)-infected macaques, and infused Schistosoma mansoni eggs into the liver and lungs of others. Tissues examined from these animals demonstrated that macrophages induced by CFA did not support viral replication while those induced by S. mansoni eggs had evidence of productive infection. RT-PCR analysis showed that both Th1 (IL-2 and IFN-gamma) and Th2 cytokines (IL-4 and IL-10) were present in the CFA lesions but only the Th2 cytokines were found in the S. mansoni lesions. Follow-up studies in macaque cell cultures showed that whereas IFN-gamma caused enhancement of virus replication in CD4+ T cells, it curtailed viral replication in infected macrophages. In contrast, IL-4 enhanced viral replication in infected macrophages. These studies strongly suggest that cytokines regulate the sequential phases of HIV replication in CD4 T cells and macrophages.