Feili Wei

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.

Accumulation of nuclear and mitochondrial DNA damage in the frontal cortex cells of patients with HIV-associated neurocognitive disorders.

Oxidative stress has been suggested to play a key role in the neuropathogenesis of HIV infection. HIV proteins (gp120, Tat) and proinflammatory cytokines can trigger the production of reactive oxygen species (ROS), resulting in DNA and RNA lesions. Among all the lesions induced by ROS, one of the most abundant lesions in DNA and RNA is 8-hydroxydeoxyguanosine (8-oxoG). Here, we studied accumulated DNA oxidative damage induced by ROS in the central nervous system (CNS) in tissue from neuro-AIDS patients. The frontal cortex of autopsy tissue from HIV-1 infected patients was adopted for analysis for HIV-1 subtype, nuclear and mitochondrial DNA lesions by immunofluorescence staining, qPCR and sequencing of PCR cloning. This study provides evidence that HIV infection in the CNS leads to nuclear and mitochondrial genomic DNA damage in the brain. High level of nuclear and mtDNA 8-oxoG damage were identified in the cortex autopsy tissue of HAND patients. Increased accumulation of mtDNA mutations and depletion occurs in brain tissue in a subset of HAND cases, and is significantly different from that observed in control cases. These findings suggest that higher level of ROS in the CNS of HAND patients would contribute to the HIV induced neuro-inflammation and apoptosis of neuronal and glial cells.

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.

Mutations in the major hydrophilic region (MHR) of hepatitis B virus genotype C in North China.

Hepatitis B virus (HBV) can evolve by mutations in the major hydrophilic region (MHR) of the HBV surface antigen (HBsAg) to permit its escape from neutralization by antibodies such as HBV surface antibody (anti‐HBs) and from host immune responses. This study investigated the prevalence and pattern of MHR mutations in North China and the clinical correlations of these mutations. The MHRs of 161 HBsAg‐positive patients were amplified using nested PCR, and directly sequenced to identify MHR mutations. It was showed that among the 161 patients infected with HBV genotype C in North China, the overall frequency of MHR mutation was 46.6%. Furthermore, MHR mutations were associated with high white blood cell counts (P = 0.025), high bilirubin levels (P = 0.048), and cirrhosis (P = 0.010). The most frequent mutations in patients with both HBsAg‐positive and anti‐HBs‐positive were located in subregion 1 and 3 of MHR, specifically, residue Q101 (29.9%) and I126 (70.6%), which was different from the mutation pattern found in Western Europe and the United States. Taken together, these data indicated important virological and clinical aspects of HBV evolution in terms of the surface gene of genotype C, which might be important for its response to the currently available HBV vaccine. J. Med. Virol. 84:1901–1906, 2012.

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.

A novel mutant 10Ala/Arg together with mutant 144Ser/Arg of hepatitis B virus X protein involved in hepatitis B virus-related hepatocarcinogenesis in HepG2 cell lines

Hepatitis B virus (HBV) infection-related hepatocellular carcinoma (HCC) represents a major health problem worldwide. HBV X (HBx) protein is the most common open reading frame that may undergo mutations, resulting in the development of HCC. This study aimed to determine specific HBx mutations that differentiate the central- and para-tumor tissues, and identify their association with HCC development. HBx gene from HCC tumor and para-tumor tissues of 47 HCC patients was amplified, sequenced and statistically analyzed. A novel combination of 2 mutations at residues 10 and 144 was identified which might play a significant role in HCC development. Expression vectors carrying HBx with the specific mutations were constructed and transfected into HepG2 and p53-null HepG2 cells. Compared to wild type (WT) and single mutation of HBx at residue 10 or 144, the 10/144 double mutations strongly up-regulated p21 expression and prolonged G1/S transition in WT- and p53-null HepG2 cells. Apoptosis was also inhibited by HBx harboring 10/44 double-mutation. Binding of 10/144 double-mutant HBx to p53 was lower than WT HBx. Conclusively, the 10/144 double mutation of HBx might play a crucial role in HCC formation.

Exogenous p53 and ASPP2 expression enhances rAdV-TK/ GCV-induced death in hepatocellular carcinoma cells lacking functional p53

Suicide gene therapy using herpes simplex virus-1 thymidine kinase (HSV-TK) in combination with ganciclovir (GCV) has emerged as a potential new method for treating cancer. We hypothesize that the efficacy of HSV-TK/GCV therapy is at least partially dependent on p53 status in hepatocellular carcinoma (HCC) patients. Using recombinant adenoviral vectors (rAdV), TK, p53, and ASPP2 were overexpressed individually and in combination in Hep3B (p53 null) and HepG2 (p53 wild-type) cell lines and in primary HCC tumor cells. p53 overexpression induced death in Hep3B cells, but not HepG2 cells. ASPP2 overexpression increased rAdV-TK/GCV-induced HepG2 cell death by interacting with endogenous p53. Similarly, ASPP2 reduced survival in rAdV-TK/GCV-treated primary HCC cells expressing p53 wild-type but not a p53 R249S mutant. Mutated p53 was unable to bind to ASPP2, suggesting that the increase in rAdV-TK/GCV-induced cell death resulting from ASPP2 overexpression was dependent on its interaction with p53. Additionally, γ-H2AX foci, ATM phosphorylation, Bax, and p21 expression increased in rAdV-TK/GCV-treated HepG2 cells as compared to Hep3B cells. This suggests that the combined use of HSV-TK, GCV, rAdV-p53 and rAdV-ASPP2 may improve therapeutic efficacy in HCC patients lacking functional p53.

Discordant genotypic resistance and HIV-1 genetic diversity from paired plasma and cerebrospinal fluid samples in Chinese settings

In the highly active antiretroviral therapy era, the incidence of human immunodeficiency virus (HIV)-associated neurocognitive disorder remains high with the improved survival. The prevalence of resistance differs across geographical areas and HIV subtypes. Currently, little information on the resistance patterns in the central nervous system (CNS) is available in Chinese settings. In this study, we sequenced and analyzed the pol gene from paired cerebrospinal fluid and plasma samples of 34 Chinese HIV-infected patients. We found that eight subjects harbored mutations that confer drug resistance, and of these, six subjects exhibited discordant resistance patterns between the CNS and the blood. The levels of viral diversity in the CNS were significantly higher than those in the blood (p < 0.0001). Our results contribute to improving our understanding of HIV neuropathogenesis and help to optimize neuro-acquired immunodeficiency syndrome treatment.

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.

The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T

The mitochondrial toxicity of nucleoside reverse transcriptase inhibitors (NRTIs) is due to the inhibition of mitochondrial DNA (mtDNA) polymerase γ (pol γ). Previous studies have shown that wild type p53 (wtp53) can interact with pol γ and mtDNA to enhance mitochondrial DNA base excision repair (mtBER) activity and increase the accuracy of DNA synthesis. The N-terminal transactivation domain and central specific DNA-binding domain of p53 play critical roles in the stimulation of BER. In this study, we identified the possible roles of wtp53, Δ40p53 and Δ133p53 in regulating mtDNA pol γ activity in cells with d4T treatment. The results show that Δ40p53 and Δ133p53 can exist in mitochondrial fragments and form polymers with themselves or wtp53. Unlike wtP53, Δ133p53 alone cannot increase DNA pol γ activity. More importantly, we found that Δ133p53 played a negative role in p53 stimulation of DNA pol γ activity when studied in d4T-treated and d4T-untreated mitochondrial extracts. Gel shift data also indicate that Δ40p53 and Δ133p53 cannot interact with APE. Wtp53 and Δ40p53 can act antagonize the effect of d4T inhibition of DNA pol γ activity. However, when wtp53 interacted with Δ133p53, DNA pol γ activity was significantly decreased. Conclusion: Δ133p53 negatively regulates p53’s stimulation of pol γ in the presence and absence of d4T.