Neuroprotective effects of Withania somnifera in the SH-SY5Y Parkinson cell model.

Parkinson's disease is the most frequent neurodegenerative motor disorder. The clinical syndrome and pathology involve motor disturbance and the degeneration of dopaminergic neurons in the substantia nigra. Root extracts of Withania. somnifera, commonly called Ashwagandha, contain several major chemical constituents known as withanolides. Studies have shown that W. somnifera extracts exhibit numerous therapeutic effects including inflammation and oxidative stress reduction, memory and cognitive function improvement. This study aimed to evaluate the protective effects of KSM-66, W. somnifera root extract, on 6-hydroxydopamine (6-OHDA)-induced toxicity in the human neuroblastoma SH-SY5Y cell line, as well as the associated oxidative response protein expression and redox regulation activity focused on S-glutathionylation. SH-SY5Y cells were treated with 6-OHDA preceded or followed by treatment with the KSM-66 extract. Using KSM-66 concentrations ranging from 0.25 to 1 mg/ml before and after treatment of the cells with 6-OHDA has resulted in an increased viability of SH-SY5Y cells. Interestingly, the extract significantly increased glutathione peroxidase activity and thioltransferase activity upon pre- or post- 6-OHDA treatment. KSM-66 also modulated oxidative response proteins: peroxiredoxin-I, VGF and vimentin proteins upon 6-OHDA pre/post treatments. In addition, the extract controlled redox regulation via S-glutathionylation. Pre-treatment of SH-SY5Y cells with KSM-66 decreased protein-glutathionylation levels in the cells treated with 6-OHDA. The rescue of mitochondria with 0.5 mg/ml KSM-66 extract showed an increase in ATP levels. These findings suggest that W. somnifera root extract acts as a neuroprotectant, thereby introducing a potential agent for the treatment or prevention of neurodegenerative diseases.

Phosphoproteomic analysis of dengue virus infected U937 cells and identification of pyruvate kinase M2 as a differentially phosphorylated phosphoprotein.

Dengue virus (DENV) is an arthropod-borne Flavivirus that can cause a range of symptomatic disease in humans. There are four dengue viruses (DENV 1 to 4) and infection with one DENV only provides transient protection against a heterotypic virus. Second infections are often more severe as the disease is potentiated by antibodies from the first infection through a process known as antibody dependent enhancement (ADE) of infection. Phosphorylation is a major post-translational modification that can have marked effects on a number of processes. To date there has been little information on the phosphorylation changes induced by DENV infection. This study aimed to determine global phosphoproteome changes induced by DENV 2 in U937 cells infected under an ADE protocol. A 2-dimensional electrophoretic approach coupled with a phosphoprotein-specific dye and mass spectroscopic analysis identified 15 statistically significant differentially phosphorylated proteins upon DENV 2 infection. One protein identified as significantly differentially phosphorylated, pyruvate kinase M2 (PKM2) was validated. Treatment with a PKM2 inhibitor modestly reduced levels of infection and viral output, but no change was seen in cellular viral protein levels, suggesting that PKM2 acts on exocytic virus release. While the effect of inhibition of PKM2 was relatively modest, the results highlight the need for a greater understanding of the role of phosphoproteins in DENV infection.

Upregulation of Pro-inflammatory Cytokine Expression Following Chronic Paracetamol Treatment in Astrocyte.

The present study aimed to investigate the effect of APAP treatment on the expression of pro-inflammatory cytokines in the astrocytes. The mouse astrocyte cells (C8-D1A) were treated with APAP at the concentration of 100 µM for 24 h, 16 and 28 days. The expressions of pro-inflammatory cytokines and NF-kB were determined using western blot analysis. Furthermore, the expression and localization of phosphorylation of NF-kB were detected by immunohistochemical and immunofluorescent analysis. The ultrastructure of C8-D1A cells was as well monitored. The results revealed that acute APAP treatment (24 h) had no effect on the expression of pro-inflammatory cytokines and pNF-kB. This treatment did not alter the ultrastructure of C8-D1A cells when compared with those in the control cells. However, the results obtained from the study on chronic APAP-treated cells (16 and 28 days) showed the different effect of APAP treatment. The results obtained from western blot analysis showed the increment of pro-inflammatory cytokine (IL-1ß and TNF-α) expressions and the activation of NF-kB signaling pathway. Nuclear translocation of pNF-kB and alteration of several cell structures were well observed in the C8-D1A cells with chronic APAP treatment. The results obtained from this study suggest that chronic APAP treatment can induce an upregulation of pro-inflammatory cytokines (IL-1ß and TNFα) in astrocytes. This alteration implies the involvement of the activation of NF-kB signaling pathway.

Identification of novel biomarkers for adult-onset-immunodeficiency (AOID) syndrome using serum proteomics.

OBJECTIVE: To identify the candidate protein biomarkers of adult-onset-immunodeficiency (AOID) syndrome using serum proteomics. METHODS: Screening and verification phases were performed in the study. A total of 97 serum samples were classified into three groups: AOID patients with opportunistic infections (active AOID), AOID patients without opportunistic infections (inactive AOID), and healthy control. In the screening phase, pooled sera collected from patients and healthy control in each group were separated by 2D-gel electrophoresis, analyzed for differentially expressed proteins and identified for biomarkers using LC/MS. In the verification phase, the protein candidates were selected for confirmation by western blotting. RESULTS: The analysis revealed 35 differentially expressed proteins. Three proteins including haptoglobin, gelsolin, and transthyretin, were selected for verification. The results showed that the levels of haptoglobin in both active and inactive AOID groups were significantly higher than that in the control group, while the levels of gelsolin in the active AOID group were significantly lower than that in the inactive AOID group. The level of transthyretin in the active AOID group was also significantly lower than that in the control group. CONCLUSIONS: The comparison of serum proteins between the three groups revealed three candidates which are related to chronic inflammatory diseases. Haptoglobin and transthyretin biomarkers could be applied in clinical assessment for monitor of disease outcome, including for the study of AOID pathogenesis.

Nevirapine induces apoptosis in liver (HepG2) cells.

OBJECTIVE: To generate insights into the mechanism of NVP induced hepatotoxicity. METHODS: Liver (HepG2) cells were cultured with various concentrations of NVP. This cell line was chosen because it has low expression of cytochrome P450, allowing evaluation of the effects of NVP rather than specific metabolites. Cytotoxicity was determined using a proliferation assay and cell numbers were monitored using trypan blue exclusion assay for long term culture experiments and apoptosis induction was determined by morphological and biochemical investigation. RESULTS: HepG2 cells treated with the highest concentration of NVP tested (819 µM) initially showed a rounded morphology and all cells had died by week three of exposure. Nuclear condensation and fragmentation, increased Annexin V/propidium iodide staining and caspase 9 activation all supported the induction of apoptosis in HepG2 cells in response to NVP treatment. CONCLUSIONS: There is a clear induction of apoptosis in response to NVP which suggests that NVP has significant cytotoxicity, over and above any cytotoxicity of metabolites and may contribute directly to patient hepatotoxicity.

Up-regulation of calcitonin gene-related peptide in trigeminal ganglion following chronic exposure to paracetamol in a CSD migraine animal model.

Previously, our group has demonstrated that chronic paracetamol (APAP) treatment induces alterations to the trigeminovascular nociceptive system in the cortical spreading depression (CSD) migraine animal model. The calcitonin gene related peptide (CGRP) is a key neuropeptide involved in the activation of the trigeminovascular nociceptive system. Therefore, this study examined the expression levels of CGRP in the trigeminal ganglion (TG) after chronic APAP exposure (0, 15, and 30 days) using a CSD model. Rats were divided into control, CSD only, APAP only and APAP treatment with CSD groups. A single injection (i.p.) of APAP (200 mg/kg body weight) was given to the 0-day APAP-treated groups, while the other APAP-treated groups received daily injections for 15 and 30 days. CSD was induced by the topical application of KCl to the parietal cortex. The protein expression of CGRP in the TG was evaluated by immunohistochemistry, and the CGRP mRNA level was investigated by real-time quantitative reverse transcription polymerase chain reaction. The results revealed that the induction of CSD significantly increased the level of CGRP protein but had no effect on CGRP mRNA level. Pretreatment with APAP 1 hour before CSD activation significantly reduced CGRP expression induced by CSD. In contrast, chronic treatment with APAP (15 and 30 days) significantly enhanced CGRP expression in both protein and mRNA levels when compared with the control groups. In combination with CSD, the expression of CGRP further increased in the animal with 30 day treatment. These findings indicate that chronic treatment with APAP induces an increase of CGRP expression in the TG. This alteration may be associated with the increased trigeminovascular nociception observed in our previous studies.

Proteomic analysis of serum and urine of HIV-monoinfected and HIV/HCV-coinfected patients undergoing long term treatment with nevirapine.

Nevirapine (NVP) is an effective nonnucleoside reverse transcriptase inhibitor (NNRTI) of particular interest as it is often used in resource limited countries. However, one of the main concerns with the use of NVP is hepatotoxicity and elevation of liver enzymes as a consequence of highly active antiretroviral therapy (HAART) containing NVP is more often reported in HIV patients coinfected with hepatitis C virus than in HIV-monoinfected patients. To discover possible markers of NVP induced hepatotoxicity, serum and urine samples from twenty-five HIV or HIV/HCV patients, all of whom had received NVP continuously for at least four months, and healthy controls were subjected to in-solution or in-gel proteomic analysis. A total of 83 differentially regulated proteins consisted of 34 proteins identified in serum by in-solution analysis, 2 proteins identified from serum in a 2D gel electrophoresis analysis, and 47 proteins identified in urine in an in-solution analysis. Three proteins, namely, haptoglobin, Rho-related BTB domain containing protein 3, and death-associated protein kinase 3, were selected for further validation by Western blot analysis and results showed that haptoglobin has potential for further development as an additional marker of NVP induced hepatotoxicity.

The involvement of microglial cells in Japanese encephalitis infections.

Despite the availability of effective vaccines, Japanese encephalitis virus (JEV) infections remain a leading cause of encephalitis in many Asian countries. The virus is transmitted to humans by Culex mosquitoes, and, while the majority of human infections are asymptomatic, up to 30% of JE cases admitted to hospital die and 50% of the survivors suffer from neurological sequelae. Microglia are brain-resident macrophages that play key roles in both the innate and adaptive immune responses in the CNS and are thus of importance in determining the pathology of encephalitis as a result of JEV infection.

Characterization of putative Japanese encephalitis virus receptor molecules on microglial cells.

Japanese encephalitis virus (JEV) a mosquito-borne flavivirus is a major cause of viral encephalitis in Asia. While the principle target cells for JEV in the central nervous system are believed to be neurons, microglia are activated in response to JEV and have been proposed to act as a long lasting virus reservoir. Viral attachment to a host cell is the first step of the viral entry process and is a critical mediator of tissue tropism. This study sought to identify molecules associated with JEV entry to microglial cells. Virus overlay protein-binding assay (VOPBA) and liquid chromatography-mass spectrometry (LC/MS/MS) identified the 37/67 kDa high-affinity laminin receptor protein and nucleolin as a potential JEV-binding proteins. These proteins were subsequently investigated for a contribution to JEV entry to mouse microglial BV-2 cells together with other possible candidate receptor molecules including Hsp70, Hsp90, GRP78, CD14, and CD4. In antibody mediated inhibition of infection experiments, both anti-laminin receptor and anti-CD4 antibodies significantly reduced virus entry while anti-Hsp70 and 90 antibodies produced a slight reduction. Significant inhibition of virus entry (up to 80%) was observed in the presence of lipopolysaccharide (LPS) which resulted in a complete down-regulation of CD4 and moderate down-regulation of CD14. These results suggest that multiple receptor proteins may mediate the entry of JEV to microglial cells, with CD4 playing a major role.

Highly permissive infection of microglial cells by Japanese encephalitis virus: a possible role as a viral reservoir.

Japanese encephalitis virus (JEV), a mosquito-borne Flavivirus, is a major cause of acute encephalitis, and neurons have been proposed to be the principle JEV target cells in the central nervous system. However, clinically, infection with JEV leads to increased levels of cytokines and chemokines in the serum and cerebrospinal fluid (CSF) the levels of which correlate with the mortality rate of patients. This research aimed to study the role of microglial cells in JEV infection. Mouse microglial cells (BV-2) supported the replication of JEV with extracellular production of virus by 10h post-infection, and virus titer reached a maximum (2.55x10(10)pfu/ml) by day 3 post-infection. While apoptosis was induced in response to virus infection, no alteration in nitric oxide production was observed. Microglial cells remained productively infected with JEV for up to 16 weeks without significant morphological alterations, and the released virions were infectious to mouse neuroblastoma (NA) cells. The high virus production and long persistence of JEV in microglial cells suggests that these cells may serve as viral reservoirs for the infection of neurons in the CNS.

Apoptosis in dengue virus infected liver cell lines HepG2 and Hep3B.

While both in vivo and in vitro evidence has suggested that liver cells undergo apoptosis in response to dengue virus infection, little is known about the mechanism of induction. Given that the p53 tumour suppressor gene is a key mediator of apoptosis, we sought to define the role of this gene in response to dengue virus infection. After infection, a p53 wild type liver cell line (HepG2) showed changes consistent with apoptosis including alterations of cell morphology, cellular detachment and DNA laddering. However, p53 was neither up-regulated, nor showed any evidence of complexing with dengue virus proteins as determined by immunoprecipitation. Infection of a p53 null liver cell line (Hep3B) also produced changes consistent with the induction of apoptosis. While the profile of the cells undergoing apoptosis in each cell line was similar as determined by flow cytometry, the absolute levels were markedly different with up to 90% of Hep3B cells undergoing apoptosis compared to only 20% of HepG2 cells at day 5 post infection. By day 7, all Hep3B infected cells were dead. In contrast, it proved possible to culture dengue virus infected HepG2 cells for 3 months. Viral progeny released from the p53 null cell line were nine-fold higher per attached cell than from the p53 wild type cell line. These results suggest that, while induction of apoptosis in liver cells is mediated by a non-p53 regulated pathway, p53 may play a role in restricting the level of viral progeny to below a critical level at which apoptosis is triggered.