Morphine Enhances HIV-1SF162-Mediated Neuron Death and Delays Recovery of Injured Neurites

HIV-1 enters the CNS soon after initial systemic infection; within the CNS parenchyma infected and/or activated perivascular macrophages, microglia and astrocytes release viral and cellular toxins that drive secondary toxicity in neurons and other cell types. Our previous work has largely modeled HIV-neuropathology using the individual viral proteins Tat or gp120, with murine striatal neurons as targets. To model disease processes more closely, the current study uses supernatant from HIV-1-infected cells. Supernatant from HIV-1SF162-infected differentiated-U937 cells (HIV+sup) was collected and p24 level was measured by ELISA to assess the infection. Injection drug abuse is a significant risk factor for HIV-infection, and opiate drug abusers show increased HIV-neuropathology, even with anti-retroviral treatments. We therefore assessed HIV+sup effects on neuronal survival and neurite growth/pruning with or without concurrent exposure to morphine, an opiate that preferentially acts through µ-opioid receptors. Effects of HIV+sup ± morphine were assessed on neuronal populations, and also by time-lapse imaging of individual cells. HIV+sup caused dose-dependent toxicity over a range of p24 levels (10–500 pg/ml). Significant interactions occurred with morphine at lower p24 levels (10 and 25 pg/ml), and GSK3β was implicated as a point of convergence. In the presence of glia, selective neurotoxic measures were significantly enhanced and interactions with morphine were also augmented, perhaps related to a decreased level of BDNF. Importantly, the arrest of neurite growth that occurred with exposure to HIV+sup was reversible unless neurons were continuously exposed to morphine. Thus, while reducing HIV-infection levels may be protective, ongoing exposure to opiates may limit recovery. Opiate interactions observed in this HIV-infective environment were similar, though not entirely concordant, with Tat/gp120 interactions reported previously, suggesting unique interactions with virions or other viral or cellular proteins released by infected and/or activated cells

Exploration of bivalent ligands targeting putative mu opioid receptor and chemokine receptor CCR5 dimerization

Modern antiretroviral therapies have provided HIV-1 infected patients longer lifespans and better quality of life. However, several neurological complications are now being seen in these patients due to HIV-1 associated injury of neurons by infected microglia and astrocytes. In addition, these effects can be further exacerbated with opiate use and abuse. One possible mechanism for such potentiation effects of opiates is the interaction of the mu opioid receptor (MOR) with the chemokine receptor CCR5 (CCR5), a known HIV-1 co-receptor, to form MOR-CCR5 heterodimer. In an attempt to understand this putative interaction and its relevance to neuroAIDS, we designed and synthesized a series of bivalent ligands targeting the putative CCR5-MOR heterodimer. To understand how these bivalent ligands may interact with the heterodimer, biological studies including calcium mobilization inhibition, binding affinity, HIV-1 invasion, and cell fusion assays were applied. In particular, HIV-1 infection assays using human peripheral blood mononuclear cells, macrophages, and astrocytes revealed a notable synergy in activity for one particular bivalent ligand. Further, a molecular model of the putative CCR5-MOR heterodimer was constructed, docked with the bivalent ligand, and molecular dynamics simulations of the complex was performed in a membrane-water system to help understand the biological observation

HIV⁺_sup ± morphine-mediated effects on secretion of growth factors and cytokines by glia.

<p>After specified times and treatments, conditioned medium from mixed glial cultures was collected and assessed for levels of BDNF, GDNF, IL-6 and TNFα by ELISA; Growth factor/cytokine levels were determined based on a standard curve. The findings were reported as average concentrations (pg/ml) ± SEM. Significance was analyzed using a one-way ANOVA and Duncan's post hoc test, from <i>n</i> = 3 separate experiments. <b>BDNF:</b> HIV⁺_sup ± morphine treatments significantly reduced levels of BDNF (*<i>p</i><0.05 vs. ‘72 h (C)’); after removal of HIV⁺_sup, BDNF returned to control levels (^{p<0.05). GDNF: HIV⁺_sup ± morphine treatments did not have significant effects on GDNF levels. IL-6: HIV⁺_sup treatment significantly enhanced levels of IL-6; morphine treatment alone also significantly elevated IL-6 levels (*p<0.05 vs. ‘72 h (C)’), and morphine co-treatment significantly augmented HIV⁺_sup-mediated effects (^#p<0.05). After removal of HIV⁺_sup, IL-6 returned to control levels (}<i>p</i><0.05); in the continuous presence of morphine, IL-6 remained at a significantly higher level than control (*<i>p</i><0.05) and [24 h (H) then 48 h (C)]-treatment group (^#<i>p</i><0.05). <b>TNFα:</b> HIV⁺_sup treatment significantly enhanced levels of TNFα (*<i>p</i><0.05 vs. ‘72 h (C)’); morphine co-treatment significantly enhanced the HIV⁺_sup-mediated effect (^#<i>p</i><0.05). After removal of HIV⁺_sup, TNFα levels returned to control values (^$<i>p</i><0.05). C =  Control_sup; H =  HIV⁺_sup (p24 = 25 pg/ml); M =  morphine sulfate (500 nM).</p

HIV⁺_sup ± morphine-mediated neuronal death.

<p>Neurons were repeatedly imaged for 72-treatment neuron count ± SEM. Significance was analyzed by repeated measures ANOVA and Duncan's post hoc test, from <i>n</i> = 6 separate experiments. Over the period of 72 h, in both culture systems, all groups exposed to HIV⁺_sup showed significantly reduced neuronal survival (*<i>p</i><0.05 vs. C). Morphine significantly enhanced HIV⁺_sup-mediated neuronal death (^#<i>p</i><0.05 vs. H), and the interactive effects of morphine were blocked by naloxone. In the presence of glia, HIV⁺_sup ± morphine-mediated neuronal death was significantly enhanced (^$<i>p</i><0.05 vs. corresponding treatment in neuronal cultures; compare panels). C =  Control_sup; H =  HIV⁺_sup (p24 = 25 pg/ml); M =  morphine sulfate (500 nM); N =  naloxone (1.5 µM).</p

Reversibility of HIV⁺_sup ± morphine-mediated neurite damage.

<p>Images of pre-selected neurons were captured for 24 h after initial treatments, and for an additional 48 h after treatments were changed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100196#pone-0100196-t001" target="_blank">Table 1</a>. (<b>A</b>) Digital images of neuronal cultures after specified time and treatments (white arrowheads indicate area of neurite outgrowth since previous image); scale bar  = 40 µm. (<b>B</b>) Neurons that remained alive until the experiment end (72 h) were assessed for their arborization in images taken at 0, 24 and 72 h, using Sholl analysis. The findings were reported as average Sholl scores at each time, normalized to pre-treatment (0 h) scores ± SEM. Significance was analyzed by repeated measures ANOVA and Duncan's post hoc test, from <i>n</i> = 45–60 neurons per treatment group (sampled from 3 separate experiments; at least 15 neurons per group per experiment). Over the period of 24 h, and in both culture systems, HIV⁺_sup ± morphine treatments induced neurite growth arrest; in neuron-glia co-cultures, HIV⁺_sup + morphine treatment appeared to cause neurite pruning (*<i>p</i><0.05 vs. 0 h, for corresponding treatment). After removing HIV⁺_sup at 24 h, neurite growth arrest was reversible (^$<i>p</i><0.05 vs. 24 h, for corresponding treatment); however, if HIV⁺_sup ± morphine treatments were continued for 72 h, then neurite growth arrest was persisted. If morphine treatment continued after the removal of HIV⁺_sup, neurite outgrowth was significantly reduced/delayed compared to neurons returned to Control_sup (^#<i>p</i><0.05 vs. ‘24 h (H) then 48 h (C)’). This effect of morphine was blocked by naloxone. In the presence of glia, neurite outgrowth after removal of HIV⁺_sup was significantly enhanced, even in the continued presence of morphine (^§<i>p</i><0.05 vs. corresponding treatment and time point in neuronal cultures; compare panels). C =  Control_sup; H =  HIV⁺_sup (p24 = 25 pg/ml); M =  morphine sulfate (500 nM); N =  naloxone (1.5 µM).</p

Concentration-dependent change in MTT reduction.

<p>Cell toxicity/proliferation was analyzed in neuron cultures at 48 h after treatment using an MTT assay. The findings were reported as percent of control absorbance at 540 nm (A₅₄₀) ± SEM. Significance was analyzed using a one-way ANOVA and Duncan's post hoc test, from <i>n</i>  = 3 separate experiments. All treatment groups, except morphine alone [Control + Mor] and p24 = 10 pg/ml of HIV⁺_sup [HIV(10)], showed significantly decreased absorbance at 540 nm (*<i>p</i><0.05 vs. Control), likely reflecting neurotoxicity. HIV⁺_sup caused a concentration-dependent reduction in A₅₄₀ (^$<i>p</i><0.05). Morphine did not show a significant interaction with HIV⁺_sup at any p24 level. Control  =  Control_sup; HIV  =  HIV⁺_sup (concentration of p24 in pg/ml is specified in parentheses); Mor  =  morphine sulfate (500 nM).</p

HIV⁺_sup ± morphine-mediated GSK3β activation.

<p>Cells were lysed and immunoblotted for p-GSKβ-S9 (an inactive form of GSKβ), t- GSKβ (total GSKβ) and GAPDH in neuronal cultures at 24 h after treatment. Findings were reported as a percent of control values of p-GSKβ-S9 levels normalized with t-GSKβ (p-GSKβ-S9/t-GSKβ) ± SEM. Significance was analyzed using a one-way ANOVA and Duncan's post hoc test, from <i>n</i> = 3 separate experiments. HIV⁺_sup caused significant loss of p-GSKβ-S9 (*<i>p</i><0.05 vs. C). Treatment with morphine alone also caused significant loss of p-GSKβ-S9, and morphine co-treatment significantly augmented the HIV⁺_sup-mediated effect (^#<i>p</i><0.05). The effects of morphine were blocked by naloxone. C =  Control_sup; H =  HIV⁺_sup (p24 = 25 pg/ml); M =  morphine sulfate (500 nM); N =  naloxone (1.5 µM).</p

Neuronal apoptosis induced by HIV⁺_sup ± morphine.

<p>Cells were fixed at specific intervals after treatment and labeled for Hoechst 33342 (blue) and TUNEL (red). (<b>A</b>) Digital images of neuronal cultures at 72 h after treatment; scale bar  = 40 µm. (<b>B</b>) Apoptosis was assessed by manually counting the percentage of TUNEL(+) cells. Findings were reported as the average percentage of TUNEL(+) cells ± SEM. Significance was analyzed by one-way ANOVA and Duncan's post hoc test, from <i>n</i> = 4 separate experiments. At all assessed time points, in both culture systems, all groups exposed to HIV⁺_sup showed significantly enhanced neuronal apoptosis (*<i>p</i><0.05 vs. respective C group). In all cases, except at 12 h in cultures with neurons alone, morphine significantly augmented HIV⁺_sup-mediated neuronal apoptosis (^#<i>p</i><0.05 vs. respective H group). In all cases, except for 72 h in neuron-glia cultures, the interactive effects of morphine were significantly attenuated by naloxone. In most cases, the presence of glia significantly enhanced HIV⁺_sup ± morphine-mediated neuron apoptosis (^$<i>p</i><0.05 vs. corresponding treatment in neuron cultures; compare panels). C =  Control_sup; H =  HIV⁺_sup (p24 = 25 pg/ml); M =  morphine sulfate (500 nM); N =  naloxone (1.5 µM).</p

Prognostic performance of blood neurofilament light chain protein in hospitalized COVID-19 patients without major central nervous system manifestations: an individual participant data meta-analysis

BACKGROUND AND AIMS: To investigate the prognostic value of blood neurofilament light chain protein (NfL) levels in the acute phase of coronavirus disease 2019 (COVID-19). METHODS: We conducted an individual participant data (IPD) meta-analysis after screening on MEDLINE and Scopus to May 23rd 2022. We included studies with hospitalized adult COVID-19 patients without major COVID-19-associated central nervous system (CNS) manifestations and with a measurement of blood NfL in the acute phase as well as data regarding at least one clinical outcome including intensive care unit (ICU) admission, need of mechanical ventilation (MV) and death. We derived the age-adjusted measures NfL Z scores and conducted mixed-effects modelling to test associations between NfL Z scores and other variables, encompassing clinical outcomes. Summary receiver operating characteristic curves (SROCs) were used to calculate the area under the curve (AUC) for blood NfL. RESULTS: We identified 382 records, of which 7 studies were included with a total of 669 hospitalized COVID-19 cases (mean age 66.2 ± 15.0 years, 68.1% males). Median NfL Z score at admission was elevated compared to the age-corrected reference population (2.37, IQR: 1.13-3.06, referring to 99th percentile in healthy controls). NfL Z scores were significantly associated with disease duration and severity. Higher NfL Z scores were associated with a higher likelihood of ICU admission, need of MV, and death. SROCs revealed AUCs of 0.74, 0.80 and 0.71 for mortality, need of MV and ICU admission, respectively. CONCLUSIONS: Blood NfL levels were elevated in the acute phase of COVID-19 patients without major CNS manifestations and associated with clinical severity and poor outcome. The marker might ameliorate the performance of prognostic multivariable algorithms in COVID-19

Prognostic performance of blood neurofilament light chain protein in hospitalized COVID-19 patients without major central nervous system manifestations: an individual participant data meta-analysis

To investigate the prognostic value of blood neurofilament light chain protein (NfL) levels in the acute phase of coronavirus disease 2019 (COVID-19).; We conducted an individual participant data (IPD) meta-analysis after screening on MEDLINE and Scopus to May 23rd 2022. We included studies with hospitalized adult COVID-19 patients without major COVID-19-associated central nervous system (CNS) manifestations and with a measurement of blood NfL in the acute phase as well as data regarding at least one clinical outcome including intensive care unit (ICU) admission, need of mechanical ventilation (MV) and death. We derived the age-adjusted measures NfL Z scores and conducted mixed-effects modelling to test associations between NfL Z scores and other variables, encompassing clinical outcomes. Summary receiver operating characteristic curves (SROCs) were used to calculate the area under the curve (AUC) for blood NfL.; We identified 382 records, of which 7 studies were included with a total of 669 hospitalized COVID-19 cases (mean age 66.2 ± 15.0 years, 68.1% males). Median NfL Z score at admission was elevated compared to the age-corrected reference population (2.37, IQR: 1.13-3.06, referring to 99th percentile in healthy controls). NfL Z scores were significantly associated with disease duration and severity. Higher NfL Z scores were associated with a higher likelihood of ICU admission, need of MV, and death. SROCs revealed AUCs of 0.74, 0.80 and 0.71 for mortality, need of MV and ICU admission, respectively.; Blood NfL levels were elevated in the acute phase of COVID-19 patients without major CNS manifestations and associated with clinical severity and poor outcome. The marker might ameliorate the performance of prognostic multivariable algorithms in COVID-19