Hepatitis C Virus Core Protein Induces Neuroimmune Activation and Potentiates Human Immunodeficiency Virus-1 Neurotoxicity

Background

Hepatitis C virus (HCV) genomes and proteins are present in human brain tissues although the impact of HIV/HCV co-infection on neuropathogenesis remains unclear. Herein, we investigate HCV infectivity and effects on neuronal survival and neuroinflammation in conjunction with HIV infection.

Methodology

Human microglia, astrocyte and neuron cultures were infected with cell culture-derived HCV or exposed to HCV core protein with or without HIV-1 infection or HIV-1 Viral Protein R (Vpr) exposure. Host immune gene expression and cell viability were measured. Patch-clamp studies of human neurons were performed in the presence or absence of HCV core protein. Neurobehavioral performance and neuropathology were examined in HIV-1 Vpr-transgenic mice in which stereotaxic intrastriatal implants of HCV core protein were performed.

Principal Findings

HCV-encoded RNA as well as HCV core and non-structural 3 (NS3) proteins were detectable in human microglia and astrocytes infected with HCV. HCV core protein exposure induced expression of pro-inflammatory cytokines including interleukin-1β, interleukin-6 and tumor necrosis factor-α in microglia (p<0.05) but not in astrocytes while increased chemokine (e.g. CXCL10 and interleukin-8) expression was observed in both microglia and astrocytes (p<0.05). HCV core protein modulated neuronal membrane currents and reduced both β-III-tubulin and lipidated LC3-II expression (p<0.05). Neurons exposed to supernatants from HCV core-activated microglia exhibited reduced β-III-tubulin expression (p<0.05). HCV core protein neurotoxicity and interleukin-6 induction were potentiated by HIV-1 Vpr protein (p<0.05). HIV-1 Vpr transgenic mice implanted with HCV core protein showed gliosis, reduced neuronal counts together with diminished LC3 immunoreactivity. HCV core-implanted animals displayed neurobehavioral deficits at days 7 and 14 post-implantation (p<0.05).

Conclusions

HCV core protein exposure caused neuronal injury through suppression of neuronal autophagy in addition to neuroimmune activation. The additive neurotoxic effects of HCV- and HIV-encoded proteins highlight extrahepatic mechanisms by which HCV infection worsens the disease course of HIV infection.     

Regulation of lipopolysaccharide‐induced inflammatory response and endotoxemia by β‐arrestins

β‐Arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that β‐arrestin‐1 (β‐arr‐1) and ‐2 knockout (KO) mice are protected from TLR4‐mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild‐type (WT) and β‐arr‐1 and ‐2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS‐induced inflammatory cytokine levels in the plasma were markedly decreased in both β‐arr‐1 and ‐2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11^b+ and CD11^b? populations) from WT, β‐arr‐1, and ‐2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS‐induced inflammatory cytokines were significantly blocked in both splenocyte populations from the β‐arr‐2 KO compared to the WT mice. This effect in the β‐arr‐1 KO mice, however, was restricted to the CD11^b? splenocytes. Our studies further indicate that regulation of cytokine production by β‐arrestins is likely independent of MAPK and IκBα‐NFκB pathways. Our results, however, suggest that LPS‐induced chromatin modification is dependent on β‐arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by β‐arrestins in vivo. Taken together, these results indicate that β‐arr‐1 and ‐2 mediate LPS‐induced cytokine secretion in a cell‐type specific manner and that both β‐arrestins have overlapping but non‐redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice. J. Cell. Physiol. 225: 406–416, 2010. © 2010 Wiley‐Liss, Inc.     

In vitro degradation of fluoranthene by bacteria isolated from petroleum sludge

An investigation was carried out for in vitro degradation of fluoranthene by four bacterial strains (PSM6, PSM7, PSM10 and PSM11) isolated from the petroleum sludge. Although all the strains registered their growth in MSM with 100 ppm fluoranthene, PSM11 growth was better than other strains. Growth of bacterial strains invariably corresponded to their degradation potential of fluoranthene. After 168 h of incubation, 61% fluoranthene was degraded by PSM11, followed by PSM10 (48%) and PSM6 (42%) and the least was recorded in PSM7 (41%). Besides, 11% loss in fluoranthene was attributed to abiotic factors. Thirty-eight times more activity of catechol 2,3-dioxygenase than catechol 1,2-dioxygenase showed that it played a significant role in fluoranthene degradation. Molecular weight of catechol 2,3-dioxygenase isolated from PSM11 was determined as ∼136 kDa by size exclusion chromatography and 34 kDa on denaturing SDS-PAGE, indicating tetrameric nature of the enzyme.     

Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry

MAb1, a human IgG1 monoclonal antibody produced in a NS0 cell line, exhibits charge heterogeneity because of the presence of variants formed by processes such as N-terminal glutamate cyclization, C-terminal lysine truncation, deamidation, aspartate isomerization and sialylation in the carbohydrate moiety. Four major charge variants of MAb1 were isolated and the conformations of these charge variants were studied using hydrogen/deuterium exchange mass spectrometry, including the H/D exchange time course (HX-MS) and the stability of unpurified proteins from rates of H/D exchange (SUPREX) techniques. HX-MS was used to evaluate the conformation and solution dynamics of MAb1 charge variants by measuring their deuterium buildup over time at the peptide level. The SUPREX technique evaluated the unfolding profile and relative stability of the charge variants by measuring the exchange properties of globally protected amide protons in the presence of a chemical denaturant. The H/D exchange profiles from both techniques were compared among the four charge variants of MAb1. The two techniques together offered extensive understanding about the local and subglobal/global unfolding of the charge variants of MAb1. Our results demonstrated that all four charge variants of MAb1 were not significantly different in conformation, solution dynamics and chemical denaturant-induced unfolding profile and stability, which aids in understanding the biofunctions of the molecules. The analytical strategy used for conformational characterization may also be applicable to comparability studies done for antibody therapeutics.     

Recent advances in novel heterocyclic scaffolds for the treatment of drug-resistant malaria

Malaria is a major public health problem all over the world, particularly in tropical and subtropical countries due to the development of resistance and most deadly infection is caused by Plasmodium falciparum. There is a direct need for the discovery of new drugs with unique structures and mechanism of action to treat sensitive and drug-resistant strains of various plasmodia for radical cure of this disease. Traditional compounds such as quinine and related derivatives represent a major source for the development of new drugs. This review presents recent modifications of 4-aminoquinoline and 8-aminoquinolone rings as leads to novel active molecules which are under clinical trials. The review also encompasses the other heterocyclic compounds emerged as potential antimalarial agents with promising results such as acridinediones and acridinone analogues, pyridines and quinolones as antimalarials. Miscellaneous heterocyclics such as tetroxane derivatives, indole derivatives, imidazolopiperazine derivatives, biscationic choline-based compounds and polymer-linked combined antimalarial drugs are also discussed. At last brief introduction to heterocyclics in natural products is also reviewed. Most of them have been under clinical trials and found to be promising in the treatment of drug-resistant strains of Plasmodium and others can be explored for the same purpose.     

Adenovirus vector-induced innate inflammatory mediators, MAPK signaling, as well as adaptive immune responses are dependent upon both TLR2 and TLR9 in vivo

Adenovirus (Ad) vectors are promising candidates for both gene transfer and vaccine applications. In this study, we investigated the role of TLR2 in innate and adaptive immune responses to Ad and/or the transgene it expresses following systemic injection. We found that Ad directly activates ERK1/2 in vivo, but that initiation of ERK1/2 activation is primarily a MyD88/TLR2-independent, but Kupffer cell-dependent, event. The complexity of Ad-induced innate immune responses was confirmed when we also found that both TLR2 and MyD88 functions are required for the sustained activation of ERK1/2. Although we found that the initial activation of NF-kappaB by Ads is dependent upon MyD88, but independent of TLR2 in (non-Kupffer cells) the liver, TLR2 significantly influenced the Ad-induced late phase NF-kappaB activation. These very rapid responses were positively correlated with subsequent innate immune responses to the Ad vector, as our results confirmed that the induction of several cytokines and chemokines, and the expression of innate immune response genes following Ad injection were TLR2 dependent in vivo. The requirement of TLR2 in Ad-induced innate responses also correlated with significantly altered adaptive immune responses. For example, our results demonstrate that the generation of Ad-neutralizing Abs, and anti-transgene-specific Abs elicited subsequent to Ad vector treatments, are both dependent upon TLR2 functionality. Finally, we found that several Ad-induced innate immune responses are dependent on both TLR2 and TLR9. Therefore, this study confirms that several (but not all) Ad-induced innate and adaptive immune responses are TLR dependent.     

Comparative study of circadian variation in oral,tympanic, forehead,axillary and elbow pit temperatures measured in a cohort of young university students living their normal routines

The primary objective of the present research work is to study and compare the circadian variability in body temperature recorded from different locations of the body during subjects’ normal routines. Temperatures of oral cavity (sublingually), tympanum, forehead, axilla and the elbow pit were measured simultaneously at approximate 1-h intervals for five consecutive days during subjects’ waking span in their routine living condition. The observations were made in eight young, apparently healthy, university students. Data were analysed using cosinor rhythmometry for evaluation of circadian rhythms and two-way ANOVA with repeated measures to assess the effect of time of day and measuring site on body temperatures and their interaction. Significant circadian rhythms in body temperature, irrespective of site, were found. Based on autocorrelation analysis, it was observed that the day-to-day variability in body temperature was consistent. The acrophases of all the studied temperature rhythms were located in the afternoon, except axillary temperature, which occurred in the early evening. The mean daytime temperature was found to be the highest when recorded sublingually and it was the lowest on the forehead or elbow pit. On the basis of the results of this study, we recommend that the methods used could be introduced into laboratory courses in a curriculum of chronobiology courses for both UG and PG classes for the demonstration/study of circadian rhythms in body temperature under normal routines. The methods used are valuable as they are non-invasive, easily accepted and assessable in a student setting.     

8-Chloroadenosine Sensitivity in Renal Cell Carcinoma Is Associated with AMPK Activation and mTOR Pathway Inhibition

The adenosine analog 8-chloroadenosine has been shown to deplete ATP and inhibit tumor growth in hematological malignancies as well as in lung and breast cancer cell lines. We investigated effects of 8-chloroadenosine on clear cell (cc) renal cell carcinoma (RCC) cell lines. 8-chloroadenosine was effective against ccRCC cell viability in vitro, with IC₅₀ ranging from 2 μM in the most sensitive CAKI-1 to 36 μM in the most resistant RXF-393. Proteomic analysis by reverse-phase protein array revealed that 8-chloroadenosine treatment leads to inhibition of the mTOR pathway. In time-course experiments, 8-chloroadenosine treatment rapidly activated AMPK, measured by AMPK and ACC phosphorylation, and subsequently caused dephosphorylation of p70S6K and ribosomal protein RPS6 in the sensitive cell lines. However, in the resistant cell lines, AMPK activity and the mTOR pathway were unaffected by the treatment. We also noted that the resistant cell lines had elevated basal levels of phospho RPS6 and AKT. Inhibition of PI3K pathway enhanced the efficacy of 8-chloroadenosine across all cell lines. Our observations indicate that 8-chloroadenosine activity is associated with inhibition of the mTOR pathway, and that phospho RPS6 and PI3K pathway activation status may determine resistance. Among solid tumors, RCC is one of the few susceptible to mTOR inhibition. We thus infer that 8-chloroadenosine may be effective in RCC by activating AMPK and inhibiting the mTOR pathway.     

Comparison of cellular and humoral responses to recombinant protein and synthetic peptides of exon2 region of Plasmodium falciparum erythrocyte membrane protein1 (PfEMP1) among malaria patients from an endemic region

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the surface of parasitized red blood cells (PRBCs) mediate adhesion of PRBCs to host vascular endothelial receptors and is considered responsible for pathogenesis of severe P. falciparum malaria. The present study was undertaken to measure cellular immune responses and serum antibody responses against recombinant exon2 protein, the most conserved region of PfEMP1, and its synthetic peptides. T cell recognizing this domain could provide universal help to B cells in recognizing variant epitopes located in the extracellular region of PfEMP1. Human peripheral blood mononuclear cells from malaria-exposed immune adults (IA), malaria patients with varying severity, and malaria unexposed healthy donors were stimulated with recombinant exon2 protein and six synthetic peptides from its sequence to estimate the proliferative, IFN-gamma, and IL-4 responses. Antibody responses against these synthetic peptides and exon2 protein were also studied. Positive proliferative, IFN-gamma, and IL-4 responses in IA group each were 60% with recombinant exon2 protein and 27-47% with different synthetic peptides. Antibody recognition was observed in 67% with exon2 and between 40 and 53% with different peptides. In malaria patients, frequency and magnitude of proliferative response, IL-4 concentration, and antibody recognition were far less than immune adults but IFN-gamma response was almost similar. Proportion of positive responders and the magnitude of response to synthetic peptides were low. Also, there was no consistency in response of different peptides towards proliferative, cytokine, and antibody responses in IA and malaria patient groups except for peptide 1. We presume peptide 1 is a potential vaccine candidate and different cocktails containing peptide 1 are being evaluated for their T cell immunogenicity.