Luxin Qiao

Cytokines in CSF correlate with HIV-associated neurocognitive disorders in the post-HAART era in China

In the current era of highly active antiretroviral therapy (HAART), the incidence of HIV dementia has declined, but the prevalence of HIV-associated neurocognitive disorder (HAND) remains high. HIV-induced systemic and localized inflammation is considered to be one of the mechanisms of HAND. Changes in cytokine levels in the cerebrospinal fluid (CSF) during HIV infection might help to identify HAND. To investigate whether the cytokine profile of the CSF during HIV infection could be used as a biomarker of HAND, we compared cytokine levels in the CSF of HIV-infected cases with and without neurocognitive impairment. Cytokine concentrations in the CSF were measured by quantification bioassays (Luminex xMAP). HIV-infected cases with neurocognitive impairment demonstrated higher levels of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, induced protein (IP)-10, and granulocyte colony-stimulating factor (G-CSF) in the CSF than those without neurocognitive impairment (G-CSF (p = 0.0003), IL-8 (p = 0.0046), IP-10 (p < 0.0001), and MCP-1 (p < 0.0001)). There was no significant impact of HAART on cytokine levels in the CSF, except for IP-10, which was higher in HAART-treated patients with impaired cognition (p = 0.0182). Findings from this preliminary study suggest that elevated levels of the cytokines IL-8, MCP-1, G-CSF, and IP-10 in the CSF are associated with neurocognitive impairment in HIV infection, and these cytokines likely represent a biomarker profile for HAND.

Sigma-1 receptor agonists provide neuroprotection against gp120 via a change in bcl-2 expression in mouse neuronal cultures.

Although combined antiretroviral therapy has significantly improved the prognosis of HIV-1 infected patients and decreased the incidence of HIV-1 associated dementia, the cumulative prevalence of this disease, in particular, mild or asymptomatic neurocognitive impairment, has not decreased. Thus, in addition to active antiretroviral therapy, the search for an effective neuroprotective approach is very important. Sigma-1 receptors are widely distributed in the central nervous system. Sigma-1 receptor agonists are robustly neuroprotective in many neuropathy and neurotoxicity in vivo and in vitro studies. This study aims to investigate possible neuroprotective effects of sigma-1 receptor agonist, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) against HIV-1 protein gp120. Primary cortical neuronal cultures were exposed to gp120 in different concentrations; to investigate neuroprotective effects of sigma-1 receptor agonist, cells were pre-treated with PPBP (10μM) in the presence or absence of pre-incubated sigma-1 receptor antagonist rimcazole (5μM). Cell apoptosis was confirmed with calcein/PI uptake test, lactate dehydrogenase (LDH) leakage assay or TUNEL assay and neurite degeneration was evaluated with morphometry via MAP-2 stained immunofluorescence. The mRNA and protein levels of apoptosis associated bax and bcl-2 were determined with real-time qPCR and Western blot. The results showed that gp120 could induce neuronal apoptosis and neurite degeneration in a concentration dependent manner and PPBP could attenuate the neurotoxicity of gp120. Simultaneously, gp120 could induce low expression of bcl-2 and bax, but only low expression of bcl-2 could be reversed by PPBP. The present data suggest that PPBP, at least, in part protects the neuron against gp120 by regulating bcl-2 expression.

Long-Term Exposure of Mice to Nucleoside Analogues Disrupts Mitochondrial DNA Maintenance in Cortical Neurons

Nucleoside analogue reverse transcriptase inhibitor (NRTI), an integral component of highly active antiretroviral therapy (HAART), was widely used to inhibit HIV replication. Long-term exposure to NRTIs can result in mitochondrial toxicity which manifests as lipoatrophy, lactic acidosis, cardiomyopathy and myopathy, as well as polyneuropathy. But the cerebral neurotoxicity of NRTIs is still not well known partly due to the restriction of blood-brain barrier (BBB) and the complex microenvironment of the central nervous system (CNS). In this study, the Balb/c mice were administered 50 mg/kg stavudine (D4T), 100 mg/kg zidovudine (AZT), 50 mg/kg lamivudine (3TC) or 50 mg/kg didanosine (DDI) per day by intraperitoneal injection, five days per week for one or four months, and primary cortical neurons were cultured and exposed to 25 µM D4T, 50 µM AZT, 25 µM 3TC or 25 µM DDI for seven days. Then, single neuron was captured from mouse cerebral cortical tissues by laser capture microdissection. Mitochondrial DNA (mtDNA) levels of the primary cultured cortical neurons, and captured neurons or glial cells, and the tissues of brains and livers and muscles were analyzed by relative quantitative real-time PCR. The data showed that mtDNA did not lose in both NRTIs exposed cultured neurons and one month NRTIs treated mouse brains. In four months NRTIs treated mice, brain mtDNA levels remained unchanged even if the mtDNA levels of liver (except for 3TC) and muscle significantly decreased. However, mtDNA deletion was significantly higher in the captured neurons from mtDNA unchanged brains. These results suggest that long-term exposure to NRTIs can result in mtDNA deletion in mouse cortical neurons.

An initial screening for HIV-associated neurocognitive disorders of HIV-1 infected patients in China

HIV-associated neurocognitive disorders (HAND), characterized by cognitive, motor, and behavioral abnormalities, are common among people living with HIV and AIDS. In combined antiretroviral therapy era in Western countries, nearly 40% of HIV-infected patients continue to suffer from HAND, mainly with mild or asymptomatic cognitive impairment. However, the prevalence and the clinical features of HAND in China are still not well known. In this study, a multi-center cross-sectional study was performed to determine the prevalence and clinical features of HAND in 134 HIV-1 infected patients in China. The International HIV Dementia Scale and a neuropsychological test battery were administered for screening and diagnosis HAND. Subjective complaints, CD4 count and viral loads in both blood plasma and cerebrospinal fluid were correlated with diagnosis of HAND. The results showed that the prevalence of HAND was approximately 37% in these patients. CD4 counts at time of sampling were significant lower in the HAND group than in the non-HAND group. But the distribution of the HAND severity did not differ by CD4 count or viral load. The presence of HAND was associated with cognitive and behavior disorder complaints (4.9- and 4.1-fold higher than those without HAND, respectively). The present data suggest that CD4 count and viral load cannot predict the severity of HAND, although the prevalence of HAND is similar to previous report in these patients. Cognitive and behavioral disorder is major complaint rather than cognitive and motor impairment. A larger prospective study is needed to obtain better estimates of HAND in China.

The Relationship of CSF and Plasma Cytokine Levels in HIV Infected Patients with Neurocognitive Impairment

Although HAD is now rare due to HAART, the milder forms of HAND persist in HIV-infected patients. HIV-induced systemic and localized inflammation is considered to be one of the mechanisms of HAND. The levels of cytokines in CSF were associated with neurocognitive impairment in HIV infection. However, the changes of cytokines involved in cognition impairment in plasma have not been shown, and their relationships between CSF and plasma require to be addressed. We compared cytokine levels in paired CSF and plasma samples from HIV-infected individuals with or without neurocognitive impairment. Cytokine concentrations were measured by Luminex xMAP. In comparing the expression levels of cytokines in plasma and CSF, IFN-α2, IL-8, IP-10, and MCP-1 were significantly higher in CSF. Eotaxin was significantly higher in plasma, whereas G-CSF showed no difference between plasma and CSF. G-CSF (P = 0.0079), IL-8 (P = 0.0223), IP-10 (P = 0.0109), and MCP-1 (P = 0.0497) in CSF showed significant difference between HIV-CI and HIV-NC group, which may indicate their relationship to HIV associated neurocognitive impairment. In addition, G-CSF (P = 0.0191) and IP-10 (P = 0.0377) in plasma were significantly higher in HIV-CI than HIV-NC. The consistent changes of G-CSF and IP-10 in paired plasma and CSF samples might enhance their potential for predicting HAND.

Discordant patterns of tissue-specific genetic characteristics in the HIV-1 env gene from HIV-associated neurocognitive disorder (HAND) and non-HAND patients

The genetic evolution of HIV-1 in the central nervous system (CNS) is different from that in peripheral tissues. We analyzed 121 clonal sequences of the V3–V5 regions of the env gene generated from paired cerebrospinal fluid (CSF) and plasma samples from nine chronically infected patients (four with HIV-associated neurocognitive disorder (HAND) and five without HAND). The sequence analysis indicated the significant differences between CSF and plasma was only observed in the C4 region (P = 0.043) in HAND patients. Significant increases in synonymous substitutions (dS) within the V4 region (P = 0.020) and in nonsynonymous substitutions (dN) within the C4 region (P = 0.029) were observed in the CSF-derived sequences. By contrast, CSF-derived sequences from non-HAND patients showed similar levels of diversity; dS and dN as the plasma-derived sequences. Signature differences between the CSF- and plasma-derived sequences were found at 12 amino acid positions for HAND patients and nine positions for non-HAND patients. Interestingly, five sites (positions 388, 396, 397, 404, and 406) that all belong to signature patterns exhibited positive selection pressure in CSF samples, but only site 406 was positively selected in the plasma samples from the HAND patients. Conversely, in the non-HAND patients, there were four sites (positions 397, 404, 432, and 446) showed positive selection pressure in the plasma samples, but only site 446 in the CSF samples. These results suggest that discordant patterns of genetic evolution occur between the tissue-specific HIV-1 quasispecies in the HAND and non-HAND patients. Viral molecular heterogeneity between specific tissues is greater in patients with HAND compared to non-HAND patients.

Mitochondrial toxicity studied with the PBMC of children from the Chinese national pediatric highly active antiretroviral therapy cohort.

As the backbone of highly active antiretroviral therapy (HAART), nucleoside reverse transcriptase inhibitors (NRTIs) have effectively improved outcomes for HIV-infected patients. However, long-term treatment with NRTIs can cause a series of pathologies associated with mitochondrial toxicity. To date, the status and mechanism of mitochondrial toxicity induced by NRTIs are still not clear, especially in HIV-infected children. As part of the national pediatric HAART program in China, our study focused on mitochondrial toxicity and its potential mechanism in HIV-1-infected children who were divided into two groups based on their duration of treatment with NRTIs: one group received treatment for less than 36 months and one group was treated for 36 to 72 months. The control group comprised age-matched non-HIV-infected children. Blood lactic acid and ATP levels in peripheral blood mononuclear cells (PBMCs) were measured to evaluate mitochondrial function, and mtDNA copies and mutations in PBMCs were determined for detecting mtDNA lesions. Simultaneously, TK2 and P53R2 gene expression in PBMC was measured. As compared with the control group, blood lactic acid levels in both NRTI treatment groups were significantly higher, whereas ATP levels and mtDNA mutation rates in PBMCs did not differ between the control and the two NRTI treatment groups. Both NRTI treatment groups exhibited significant mtDNA loss. N Moreover, we found that P53R2 mRNA expression and protein levels were significantly reduced in both treatment groups and that TK2 mRNA expression and protein levels were induced in the long-term NRTI treatment group. These results suggest that mitochondrial toxicity occurs in long-term HAART patients and that P53R2 and TK2 levels in PBMCs are useful biomarkers for detecting mitochondrial toxicity in patients on long-term treatment with NRTIs.

ASPP2 Plays a Dual Role in gp120-Induced Autophagy and Apoptosis of Neuroblastoma Cells

HIV invasion of the central nervous system (CNS) in the majority of patients infected with HIV-1, leads to dysfunction and injury within the CNS, showing a variety of neurological symptoms which was broadly termed HIV-associated neurocognitive disorder (HAND). But the molecular mechanisms are not completely understood. It has been suggested that apoptosis and autophagic dysfunction in neurons may play an important role in the development of HAND. Previous studies have indicated that p53 may be involved in the onset of neurological disorder in AIDS. Apoptosis-stimulating protein of p53-2 (ASPP2), a p53-binding protein with specific function of inducing p53, has been reported to modulate autophagy. In the present study, we observed that gp120 induces autophagy and apoptosis in SH-SY5Y neuroblastoma cells. Adenovirus-mediated overexpression of ASPP2 significantly inhibited autophagy and apoptosis induced by low dose of gp120 protein (50 ng/mL), but induced autophagy and apoptosis when treated by high dose of gp120 protein (200 ng/mL). Further, ASPP2 knockdown attenuated autophagy and apoptosis induced by gp120. Conclusion: ASPP2 had different effects on the autophagy and apoptosis of neurons induced by different concentration of gp120 protein. It may be a potential therapeutic agent for HAND through modulating autophagy and apoptosis in CNS.

ASPP2 involvement in p53-mediated HIV-1 envelope glycoprotein gp120 neurotoxicity in mice cerebrocortical neurons

The mechanisms behind HIV-1-associated neurocognitive disorders are still unclear. Apoptosis-stimulating protein 2 of p53 (ASPP2) is a damage-inducible p53-binding protein that stimulates p53-mediated apoptosis and transactivates proapoptotic and cell cycle regulatory genes. It has been reported that ASPP2 has a specific regulatory function in the death of retinal ganglion cells and the development of Alzheimer’s disease. In this study, we used p53 and ASPP2 knockout mice and primary cerebrocortical neuron culture to analyze the role of the interaction between ASPP2 with p53 in HIV-1 envelope glycoprotein gp120-induced neurotoxicity. The results showed that 10 ng/mL gp120 protein might stimulate p53 overexpression and translocation to the nucleus, and 30 ng/mL gp120 protein could stimulate both p53 and ASPP2 translocation to the nucleus, but only with p53 overexpression. The primary cultured neurons of p53−/−ASPP2+/− mice had a higher survival rate than p53−/− mice under gp120 protein stress. The interaction of ASPP2 with p53 induced by a high dose of gp120 stimulated Bax transcription and contributed to caspase-3 cleavage, and ASPP2-siRNA attenuated gp120 induced neuron death through inhibition of Bax expression. These results suggest that ASPP2 plays an important role in p53-mediated neuronal apoptosis under gp120 stress.

High levels of divergent HIV-1 quasispecies in patients with neurological opportunistic infections in China

Despite the fact that the survival of people infected with human immunodeficiency virus (HIV) has improved worldwide because of the increasingly powerful and highly active antiretroviral therapy, opportunistic infections (OIs) of the central nervous system (CNS) remain a serious burden. HIV-1 is capable of entering the CNS through infected peripheral monocytes, but its effect on OIs of CNS remains unclear. In this study, we investigated the characteristics of HIV-1 in acquired immunodeficiency syndrome (AIDS) patients with CNS OIs. A total of 24 patients with CNS OIs and 16 non-CNS OIs (control) cases were selected. These AIDS patients were infected with HIV-1 by paid blood donors in China. HIV-1 loads in plasma and cerebrospinal fluid (CSF) were detected using RT-PCR, and the C2-V5 region of HIV-1 envelope gene was amplified from viral quasispecies isolated from CSF using nested PCR. The CSF HIV-1 load of CNS OIs was higher than that of non-CNS OIs, but plasma HIV-1 load of CNS OIs was not higher than that of non-CNS OIs. The nucleotide sequence of C2-V5 region of the HIV-1 quasispecies isolated from the CSF of CNS OIs had a high diversity, and the HIV-1 quasispecies isolated from the CSF of CNS OIs revealed R5 tropism as 11/25 charge rule. These results suggest that high levels of divergent HIV-1 quasispecies in the CNS probably contribute to opportunistic infections.