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1.  HIV-1 Nef Induces CCL5 production in astrocytes through p38-MAPK and PI3K/Akt pathway and utilizes NF-kB, CEBP and AP-1 transcription factors 
Scientific Reports  2014;4:4450.
The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients infected with HIV-1. The production of pro-inflammatory cytokines by astrocytes/microglia exposed to viral proteins is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. In the present study we examined the effects of Nef on CCL5 induction in astrocytes. The results demonstrate that CCL5 is significantly induced in Nef-transfected SVGA astrocytes. To determine the mechanisms responsible for the increased CCL5 caused by Nef, we employed siRNA and chemical antagonists. Antagonists of NF-κB, PI3K, and p38 significantly reduced the expression levels of CCL5 induced by Nef transfection. Furthermore, specific siRNAs demonstrated that the Akt, p38MAPK, NF-κB, CEBP, and AP-1 pathways play a role in Nef-mediated CCL5 expression. The results demonstrated that the PI3K/Akt and p38 MAPK pathways, along with the transcription factors NF-κB, CEBP, and AP-1, are involved in Nef-induced CCL5 production in astrocytes.
doi:10.1038/srep04450
PMCID: PMC3963078  PMID: 24658403
2.  Chemokine CXCL8 Promotes HIV-1 Replication in Human Monocyte-Derived Macrophages and Primary Microglia via Nuclear Factor-κB Pathway 
PLoS ONE  2014;9(3):e92145.
Background
Chemokine CXCL8 is an important neutrophil chemoattractant implicated in various neurodegenerative disorders. Cytokine/chemokine imbalance, with an increase in proinflammatory cytokines like interleukin-1β and tumor necrosis factor-α within the central nervous system, is a hallmark of human immunodeficiency virus (HIV)-1 infection. We previously reported that HIV-1 infection is linked to upregulation of CXCL8 in brain tissues and human astrocytes. Chemokines play crucial roles in trafficking of leukocytes and trafficking of HIV-1-infected across the blood-brain barrier play an important role in HIV-1 central nervous system disease. In the post-antiretroviral therapy era, low level of productive replication of HIV-1 in brain is a critical component of neuropathogenesis regulation. The present study investigated the effect of CXCL8 on productive infection of HIV-1 in human monocytes-derived macrophages (MDM) and primary human microglia.
Results
Human MDM and microglia were infected with the blood or brain derived HIV-1 isolates, HIV-1ADA or HIV-1JRFL. Treatment with CXCL8 significantly upregulated HIV-1p24 levels in supernatants of both HIV-1-infected MDM as well as microglia. In addition, the formation of 2-long terminal repeat (LTR) circles, a measure of viral genome integration, was significantly higher in CXCL8-treated, HIV-1-infected MDM and microglia. Transient transfection of U937 cells with HIV-1 LTR luciferase reporter construct resulted in increased promoter activity when treated with CXCL8. Moreover, increased nuclear translocation of nuclear factor-κB was seen in HIV-1-infected MDM following CXCL8 treatment. Blocking CXCL8 receptors CXCR1 and CXCR2 abrogated the CXCL8-mediated enhanced HIV-1 replication.
Conclusion
Our results show that CXCL8 mediates productive infection of HIV-1 in MDM and microglia via receptors CXCR1 and CXCR2. These results demonstrate that CXCL8 exerts its downstream effects by increasing translocation of nuclear factor-κB into the nucleus, thereby promoting HIV-1 LTR activity.
doi:10.1371/journal.pone.0092145
PMCID: PMC3963875  PMID: 24662979
3.  Clinical Evaluation of a Low Cost, In-House Developed Real-Time RT-PCR Human Immunodeficiency Virus Type 1 (HIV-1) Quantitation Assay for HIV-1 Infected Patients 
PLoS ONE  2014;9(3):e89826.
Objectives
HIV-1 viral quantitation is essential for treatment monitoring. An in-house assay would decrease financial barriers to access.
Materials and Methods
A real-time competitive RT-PCR in house assay (Sing-IH) was developed in Singapore. Using HXB2 as reference, the assay's primers and probes were designed to generate a 183-bp product that overlaps a portion of the LTR region and gag region. A competitive internal control (IC) was included in each assay to monitor false negative results due to inhibition or human error. Clinical evaluation was performed on 249 HIV-1 positive patient samples in comparison with the commercially available Generic HIV Viral Load assay. Correlation and agreement of results were assessed for plasma HIV-1 quantification with both assays.
Results
The assay has a lower limit of detection equivalent to 126 copies/mL of HIV-1 RNA and a linear range of detection from 100–1000000 copies/mL. Comparative analysis with reference to the Generic assay demonstrated good agreement between both assays with a mean difference of 0.22 log10 copies/mL and 98.8% of values within 1 log10 copies/mL range. Furthermore, the Sing-IH assay can quantify HIV-1 group M subtypes A–H and group N isolates adequately, making it highly suitable for our region, where subtype B and CRF01_AE predominate.
Conclusions
With a significantly lower running cost compared to commercially available assays, the broadly sensitive Sing-IH assay could help to overcome the cost barriers and serve as a useful addition to the currently limited HIV viral load assay options for resource-limited settings.
doi:10.1371/journal.pone.0089826
PMCID: PMC3945479  PMID: 24603460
4.  Solar Disinfection of Pseudomonas aeruginosa in Harvested Rainwater: A Step towards Potability of Rainwater 
PLoS ONE  2014;9(3):e90743.
Efficiency of solar based disinfection of Pseudomonas aeruginosa (P. aeruginosa) in rooftop harvested rainwater was evaluated aiming the potability of rainwater. The rainwater samples were exposed to direct sunlight for about 8–9 hours and the effects of water temperature (°C), sunlight irradiance (W/m2), different rear surfaces of polyethylene terephthalate bottles, variable microbial concentrations, pH and turbidity were observed on P. aeruginosa inactivation at different weathers. In simple solar disinfection (SODIS), the complete inactivation of P. aeruginosa was obtained only under sunny weather conditions (>50°C and >700 W/m2) with absorptive rear surface. Solar collector disinfection (SOCODIS) system, used to improve the efficiency of simple SODIS under mild and weak weather, completely inactivated the P. aeruginosa by enhancing the disinfection efficiency of about 20% only at mild weather. Both SODIS and SOCODIS systems, however, were found inefficient at weak weather. Different initial concentrations of P. aeruginosa and/or Escherichia coli had little effects on the disinfection efficiency except for the SODIS with highest initial concentrations. The inactivation of P. aeruginosa increased by about 10–15% by lowering the initial pH values from 10 to 3. A high initial turbidity, adjusted by adding kaolin, adversely affected the efficiency of both systems and a decrease, about 15–25%; in inactivation of P. aeruginosa was observed. The kinetics of this study was investigated by Geeraerd Model for highlighting the best disinfection system based on reaction rate constant. The unique detailed investigation of P. aeruginosa disinfection with sunlight based disinfection systems under different weather conditions and variable parameters will help researchers to understand and further improve the newly invented SOCODIS system.
doi:10.1371/journal.pone.0090743
PMCID: PMC3940928  PMID: 24595188
5.  Epigenetic repression of LEDGF during UVB exposure by recruitment of SUV39H1 and HDAC1 to the Sp1-responsive elements within LEDGF promoter CpG island 
Epigenetics  2013;8(3):268-280.
Expression level of lens epithelial derived growth factor (LEDGF) is vital for LEDGF-mediated cell survival and cytoprotection against proapoptotic stimuli. We previously demonstrated that LEDGF is transcriptionally regulated by Sp1-responsive elements within a CpG island in the LEDGF promoter. Herein, we report on the existence of epigenetic signaling involved in the repression of LEDGF transcription in lens epithelial cells (LECs) facing UVB. UVB exposure led to histone H3 dimethylation and deacetylation at its CpG island, where a histone deacetylase/histone methylase (HDAC1/SUV39H1) complex was recruited. Exposure of LECs to UVB stress altered LEDGF protein and mRNA expression as well as promoter activity, while failing to methylate the CpG island. These events were correlated with increased reactive oxygen species (ROS) and increased cell death. LEDGF promoter activity and expression remained unaltered after 5-Aza treatment, but were relieved with tricostatin A, an inhibitor of HDACs. Expression analysis disclosed that UVB radiation altered the global expression levels of acetylated histone proteins, diminished total histone acetyltransferase (HAT) activity and increased HDAC activity and HDAC1 expression. In silico analysis of LEDGF proximal promoter and ChIP analyses disclosed HDAC1/SUV39H1 complex anchored to the -170/-10 nt promoter regions at Sp1-responsive elements and also attenuated Sp1 binding, resulting in HDAC1- and SUV39H1-dependent deacetylation and dimethylation of H3 at K9. Acetylation of H3K9 was essential for LEDGF active transcription, while enrichment of H3K9me2 at Sp1-responsive elements within CpGs (-170/-10) by UVB radiation repressed LEDGF transcription. Our study may contribute to understanding diseases associated with LEDGF aberrant expression due to specific epigenetic modifications, including blinding disorders.
doi:10.4161/epi.23861
PMCID: PMC3669119  PMID: 23386123
LEDGF; Sp1; epigenetics; transcription; histone acetyltransferases; DNA methyltransferase; histone deacetylase
6.  Hydroxyl ion addition to one-electron oxidized thymine: Unimolecular interconversion of C5 to C6 OH-adducts 
In this work, addition of OH− to one-electron oxidized thymidine (dThd) and thymine nucleotides in basic aqueous glasses is investigated. At pHs ca. 9–10 where the thymine base is largely deprotonated at N3, one-electron oxidation of the thymine base by Cl2•− at ca. 155 K results in formation of a neutral thyminyl radical, T(−H)•. Assignment to T(−H)• is confirmed by employing 15N substituted 5'-TMP. At pH ≥ ca. 11.5, formation of the 5-hydroxythymin-6-yl radical, T(5OH)•, is identified as a metastable intermediate produced by OH− addition to T(−H)• at C5 at ca. 155 K. Upon further annealing to ca. 170 K, T(5OH)• readily converts to the 6-hydroxythymin-5-yl radical, T(6OH)•. One-electron oxidation of N3-methyl-thymidine (N3-Me-dThd) by Cl2•− at ca. 155 K produces the cation radical (N3-Me-dThd•+) for which we find a pH dependent competition between deprotonation from the methyl group at C5 and addition of OH− to C5. At pH 7 the 5-methyl deprotonated species is found; however, at pH ca. 9, N3-Me-dThd•+ produces T(5OH)• that on annealing up to 180 K forms T(6OH)•. Through use of deuterium substitution at C5' and on the thymine base, i.e., specifically employing [5',5”-D,D]-5'-dThd, [5',5”-D,D]-5'-TMP, [CD3]-dThd and [CD3,6D]-dThd, we find unequivocal evidence for T(5OH)• formation and its conversion to T(6OH)•. The addition of OH− to the C5 position in T(−H)• and N3-Me-dThd•+ is governed by spin and charge localization. DFT calculations predict that the conversion of the “reducing” T(5OH)• to the “oxidizing” T(6OH)• occurs by a unimolecular OH group transfer from C5 to C6 in the thymine base. The T(5OH)• to T(6OH)• conversion is found to occur more readily for deprotonated dThd and its nucleotides than for N3-Me-dThd. In agreement, calculations predict that the deprotonated thymine base has a lower energy barrier (ca. 6 kcal/mol) for OH transfer than its corresponding N3-protonated thymine base (14 kcal/mol).
doi:10.1021/ja310650n
PMCID: PMC3616772  PMID: 23362972
7.  Innovative pharmaceutical development based on unique properties of nanoscale delivery formulation 
Nanoscale  2013;5(18):8307-8325.
The advent of nanotechnology has reignited interest in the field of pharmaceutical science for the development of nanomedicine. Nanomedicinal formulations are nanometer-sized carrier materials designed for increasing the drug tissue bioavailability, thereby improving the treatment of systemically applied chemotherapeutic drugs. Nanomedicine is a new approach to deliver the pharmaceuticals through different routes of administration with safer and more effective therapies compared to conventional methods. To date, various kinds of nanomaterials have been developed over the years to make delivery systems more effective for the treatment of various diseases. Even though nanomaterials have significant advantages due to their unique nanoscale properties, there are still significant challenges in the improvement and development of nanoformulations with composites and other materials. Here in this review, we highlight the nanomedicinal formulations aiming to improve the balance between the efficacy and the toxicity of therapeutic interventions through different routes of administration and how to design nanomedicine for safer and more effective ways to improve the treatment quality. We also emphasize the environmental and health prospects of nanomaterials for human health care.
doi:10.1039/c3nr01525d
PMCID: PMC3934102  PMID: 23860639
8.  Prostaglandin E2 Reduces the Release and Infectivity of New Cell-Free Virions and Cell-To-Cell HIV-1 Transfer 
PLoS ONE  2014;9(2):e85230.
Background
The course of human immunodeficiency virus type-1 (HIV-1) infection is influenced by a complex interplay between viral and host factors. HIV infection stimulates several proinflammatory genes, such as cyclooxigense-2 (COX-2), which leads to an increase in prostaglandin (PG) levels in the plasma of HIV-1-infected patients. These genes play an indeterminate role in HIV replication and pathogenesis. The effect of prostaglandin E2 (PGE2) on HIV infection is quite controversial and even contradictory, so we sought to determine the role of PGE2 and the signal transduction pathways involved in HIV infection to elucidate possible new targets for antiretrovirals.
Results
Our results suggest that PGE2 post-infection treatment acts in the late stages of the viral cycle to reduce HIV replication. Interestingly, viral protein synthesis was not affected, but a loss of progeny virus production was observed. No modulation of CD4 CXCR4 and CCR5 receptor expression, cell proliferation, or activation after PGE2 treatment was detected. Moreover, PGE2 induced an increase in intracellular cAMP (cyclic AMP) levels through the EP2/EP4 receptors. PGE2 effects were mimicked by dbcAMP and by a specific Epac (exchange protein directly activated by cyclic AMP) agonist, 8-Cpt-cAMP. Treatment with PGE2 increased Rap1 activity, decreased RhoA activity and subsequently reduced the polymerization of actin by approximately 30% compared with untreated cells. In connection with this finding, polarized viral assembly platforms enriched in Gag were disrupted, altering HIV cell-to-cell transfer and the infectivity of new virions.
Conclusions
Our results demonstrate that PGE2, through Epac and Rap activation, alters the transport of newly synthesized HIV-1 components to the assembly site, reducing the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer.
doi:10.1371/journal.pone.0085230
PMCID: PMC3934822  PMID: 24586238
9.  Neurofibromatosis type I with breast cancer: not only for women! 
The association of neurofibromatosis type I with invasive male breast cancer is a rare clinical entity with only one case in literature reported in 1953. Women with NF1 are at risk of developing breast cancer and men also may be at risk but there is scarce data on the risk and association of NF1 with male breast cancer due to its rarity. Established clinical trials in male breast cancer patients are lacking and the results are extrapolated from female breast cancer patients. The treatment of male breast cancer is followed as per the guidelines of premenopausal female breast cancer and tamoxifen is the hormone treatment in them. Mendes et al suggests that silencing of NF1 gene confers resistance to tamoxifen. Our conclusions are that since NF1 is mutated or deleted in one third of sporadic breast cancers, its role as a molecular driver for treatment has to be further explored.
doi:10.1186/1897-4287-12-5
PMCID: PMC3974064  PMID: 24565603
10.  HSV-2 Co-Infection as a Driver of HIV Transmission among Heterosexual Non-Injecting Drug Users in New York City 
PLoS ONE  2014;9(1):e87993.
Objective
To examine herpes simplex virus 2 (HSV-2)/HIV co-infection as a contributing factor in the increase in HIV infection among non-injecting heroin and cocaine users in New York City.
Methods
Subjects were recruited from the Beth Israel Medical Center drug detoxification and methadone maintenance programs in New York City in 1995–1999 and 2005–2011. All reported current heroin and/or cocaine use and no injection drug use. A structured questionnaire was administered and serum samples collected for HIV and HSV-2 testing. Population-attributable risk percentages (PAR%s) were estimated for associations between HSV-2 and increased susceptibility to and increased transmissibility of HIV among female NIDUs.
Results
785 subjects were recruited from 1995–1999, and 1764 subjects from 2005–2011. HIV prevalence increased from 7% to 13%, with nearly uniform increases among all demographic subgroups. HSV-2/HIV co-infection was common in both time periods, with an average (over the two time periods) of 80% of HIV negative females infected with HSV-2, an average of 43% of HIV negative males infected with HSV-2; an average of 97% of HIV positive females also infected with HSV-2 and an average of 67% of HIV positive males also infected with HSV-2. The increase in HIV prevalence was predominantly an increase in HSV-2/HIV co-infection, with relatively little HIV mono-infection in either time period. The estimated PAR%s indicate that approximately half of HIV acquisition among females was caused by HSV-2 infection and approximately 60% of HIV transmission from females was due to HSV-2 co-infection.
Conclusions
The increase in HIV infection among these non-injecting drug users is better considered as an increase in HSV-2/HIV co-infection rather than simply an increase in HIV prevalence. Additional interventions (such as treatment as prevention and suppressing the effects of HSV-2 on HIV transmission) are needed to reduce further HIV transmission from HSV-2/HIV co-infected non-injecting drug users.
doi:10.1371/journal.pone.0087993
PMCID: PMC3909306  PMID: 24498235
11.  Thermally Triggered Mucoadhesive In Situ Gel of Loratadine: β-Cyclodextrin Complex for Nasal Delivery 
AAPS PharmSciTech  2013;14(1):412-424.
The aim of the present study was to increase the solubility of an anti-allergic drug loratadine by making its inclusion complex with β-cyclodextrin and to develop it’s thermally triggered mucoadhesive in situ nasal gel so as to overcome first-pass effect and consequently enhance its bioavailability. A total of eight formulations were prepared by cold method and optimized by 23 full factorial design. Independent variables (concentration of poloxamer 407, concentration of carbopol 934 P, and pure drug or its inclusion complex) were optimized in order to achieve desired gelling temperature with sufficient mucoadhesive strength and maximum permeation across experimental nasal membrane. The design was validated by extra design checkpoint formulation (F9) and Pareto charts were used to help eliminate terms that did not have a statistically significant effect. The response surface plots and possible interactions between independent variables were analyzed using Design Expert Software 8.0.2 (Stat Ease, Inc., USA). Faster drug permeation with zero-order kinetics and target flux was achieved with formulation containing drug: β-cyclodextrin complex rather than those made with free drug. The optimized formulation (F8) with a gelling temperature of 28.6 ± 0.47°C and highest mucoadhesive strength of 7,676.0 ± 0.97 dyn/cm2 displayed 97.74 ± 0.87% cumulative drug permeation at 6 h. It was stable for over 3 months and histological examination revealed no remarkable damage to the nasal tissue.
doi:10.1208/s12249-013-9921-9
PMCID: PMC3581637  PMID: 23358934
23 factorial design; in situ nasal gel; loratadine; mucoadhesion; temperature-induced gelation
12.  Mobile HIV Screening in Cape Town, South Africa: Clinical Impact, Cost and Cost-Effectiveness 
PLoS ONE  2014;9(1):e85197.
Background
Mobile HIV screening may facilitate early HIV diagnosis. Our objective was to examine the cost-effectiveness of adding a mobile screening unit to current medical facility-based HIV testing in Cape Town, South Africa.
Methods and Findings
We used the Cost Effectiveness of Preventing AIDS Complications International (CEPAC-I) computer simulation model to evaluate two HIV screening strategies in Cape Town: 1) medical facility-based testing (the current standard of care) and 2) addition of a mobile HIV-testing unit intervention in the same community. Baseline input parameters were derived from a Cape Town-based mobile unit that tested 18,870 individuals over 2 years: prevalence of previously undiagnosed HIV (6.6%), mean CD4 count at diagnosis (males 423/µL, females 516/µL), CD4 count-dependent linkage to care rates (males 31%–58%, females 49%–58%), mobile unit intervention cost (includes acquisition, operation and HIV test costs, $29.30 per negative result and $31.30 per positive result). We conducted extensive sensitivity analyses to evaluate input uncertainty. Model outcomes included site of HIV diagnosis, life expectancy, medical costs, and the incremental cost-effectiveness ratio (ICER) of the intervention compared to medical facility-based testing. We considered the intervention to be “very cost-effective” when the ICER was less than South Africa's annual per capita Gross Domestic Product (GDP) ($8,200 in 2012). We projected that, with medical facility-based testing, the discounted (undiscounted) HIV-infected population life expectancy was 132.2 (197.7) months; this increased to 140.7 (211.7) months with the addition of the mobile unit. The ICER for the mobile unit was $2,400/year of life saved (YLS). Results were most sensitive to the previously undiagnosed HIV prevalence, linkage to care rates, and frequency of HIV testing at medical facilities.
Conclusion
The addition of mobile HIV screening to current testing programs can improve survival and be very cost-effective in South Africa and other resource-limited settings, and should be a priority.
doi:10.1371/journal.pone.0085197
PMCID: PMC3898963  PMID: 24465503
13.  Porphyromonas Gingivalis and E-coli Induce Different Cytokine Production Patterns in Pregnant Women 
PLoS ONE  2014;9(1):e86355.
Objective
Pregnant individuals of many species, including humans, are more sensitive to various bacteria or their products as compared with non-pregnant individuals. Pregnant individuals also respond differently to different bacteria or their products. Therefore, in the present study, we evaluated whether the increased sensitivity of pregnant women to bacterial products and their heterogeneous response to different bacteria was associated with differences in whole blood cytokine production upon stimulation with bacteria or their products.
Methods
Blood samples were taken from healthy pregnant and age-matched non-pregnant women and ex vivo stimulated with bacteria or LPS from Porphyromonas Gingivalis (Pg) or E-coli for 24 hrs. TNFα, IL-1ß, IL-6, IL-12 and IL-10 were measured using a multiplex Luminex system.
Results
We observed a generally lower cytokine production after stimulation with Pg bacteria or it’s LPS as compared with E-coli bacteria. However, there was also an effect of pregnancy upon cytokine production: in pregnant women the production of IL-6 upon Pg stimulation was decreased as compared with non-pregnant women. After stimulation with E-coli, the production of IL-12 and TNFα was decreased in pregnant women as compared with non-pregnant women.
Conclusion
Our results showed that cytokine production upon bacterial stimulation of whole blood differed between pregnant and non-pregnant women, showing that the increased sensitivity of pregnant women may be due to differences in cytokine production. Moreover, pregnancy also affected whole blood cytokine production upon Pg or E-coli stimulation differently. Thus, the different responses of pregnant women to different bacteria or their products may result from variations in cytokine production.
doi:10.1371/journal.pone.0086355
PMCID: PMC3899230  PMID: 24466049
14.  Immunomodulation in Human Dendritic Cells Leads to Induction of Interferon-Gamma Production by Leishmania donovani Derived KMP-11 Antigen via Activation of NF-κB in Indian Kala-Azar Patients 
BioMed Research International  2014;2014:947606.
Dendritic cells (DCs) and macrophages (MΦs) are well-known antigen presenting cells with an ability to produce IL-12 which indicates that they have potential of directing acquired immunity toward a Th1-biased response. The aim of this study was to examine the effect of Leishmania specific KMP-11 antigen through comparison of immune responses after presentation by DCs and MΦs to T cells in Indian patients with VL. Patients with DCS and MΦs were directed against a purified Leishmania donovani antigen (KMP-11) and phytohaemagglutinin (PHA). The cytokines (IL-12, IL-10, and TGF-β) producing abilities of the DCs and MΦs against these antigens were determined by flow cytometry. The transcription factor (NF-κB) and T-cell cytokine support (IFN-γ, IL-10), which could be significant in effector immune function, were also determined. Severe hindrance in the immune protection due to Leishmania parasites, as revealed by decreased expression of IL-12 and upregulation of IL-10 and TGF-β expression in the MΦs compared to DCs, occurred in VL patients. The production of IL-12 in response to L. donovani KMP-11 antigen was increased in DCs which was reduced in MΦs of VL patients. In contrast, the presentation of KMP-11 antigen by DCs to T-lymphocytes in VL patients significantly increased the IFN-γ produced by these immune cells, whereas the levels of IL-10 were significantly elevated after presentation of KMP-11antigen by MΦs. The VL patients were observed with severely dysfunctional MΦs in terms of NF-κB activity that could be recovered only after stimulation of DCs with L. donovani KMP-11 antigen. Immunologically the better competitiveness of KMP-11 antigen through a dendritic cell delivery system may be used to revert T-cell anergy, and control strategy can be designed accordingly against kala-azar.
doi:10.1155/2014/947606
PMCID: PMC3919100  PMID: 24587999
15.  Enhanced Expression of WD Repeat-Containing Protein 35 via Nuclear Factor-Kappa B Activation in Bupivacaine-Treated Neuro2a Cells 
PLoS ONE  2014;9(1):e86336.
The family of WD repeat proteins comprises a large number of proteins and is involved in a wide variety of cellular processes such as signal transduction, cell growth, proliferation, and apoptosis. Bupivacaine is a sodium channel blocker administered for local infiltration, nerve block, epidural, and intrathecal anesthesia. Recently, we reported that bupivacaine induces reactive oxygen species (ROS) generation and p38 mitogen-activated protein kinase (MAPK) activation, resulting in an increase in the expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. It has been shown that ROS activate MAPK through phosphorylation, followed by activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1). The present study was undertaken to test whether NF-κB and c-Jun/AP-1 are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Bupivacaine activated both NF-κB and c-Jun in Neuro2a cells. APDC, an NF-κB inhibitor, attenuated the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. GW9662, a selective peroxisome proliferator-activated receptor-γ antagonist, enhanced the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. In contrast, c-Jun siRNA did not inhibit the bupivacaine-induced increase in WDR35 mRNA expression. These results indicate that bupivacaine induces the activation of transcription factors NF-κB and c-Jun/AP-1 in Neuro2a cells, while activation of NF-κB is involved in bupivacaine-induced increases in WDR35 expression.
doi:10.1371/journal.pone.0086336
PMCID: PMC3897669  PMID: 24466034
16.  Detection of Fusobacterium Nucleatum and fadA Adhesin Gene in Patients with Orthodontic Gingivitis and Non-Orthodontic Periodontal Inflammation 
PLoS ONE  2014;9(1):e85280.
Fusobacterium nucleatum is one of the most abundant gram-negative bacilli colonizing the subgingival plaque and closely associated with periodontal disease. However it is unclear whether F. nucleatum is involved in gingival inflammation under orthodontic appliance. A novel adhesin, FadA, which is unique to oral Fusobacteria, is required for F. nucleatum binding and invasion to epithelial cells and thus may play an important role in colonization of Fusobacterium in the host. In this study, we evaluated the prevalence of F. nucleatum and its virulence factor FadA adhesion gene (fadA) in 169 subgingival biofilm samples from 55 cases of gingivitis patients with orthodontic appliances, 49 cases of gingivitis patients without orthodontic treatment, 35 cases of periodontitis patients and 30 cases of periodontally healthy people via PCR. The correlations between the F. nucleatum/fadA and gingivitis index(GI)was also analyzed. The detection rate of F. nucleatum/fadA in periodontitis group and non-orthodontic gingivitis group was higher than the other two groups (p<0.01) while it was higher in orthodontic gingivitis group than in health people (p<0.05). An obviously positive correlation was observed between the prevalence of F. nucleatum/fadA and GI. F. nucleatum carrying fadA may be more closely related to the development of gingivitis and periodontal disease compared with orthodontic gingivitis.
doi:10.1371/journal.pone.0085280
PMCID: PMC3887018  PMID: 24416378
17.  Comparison of Audio Computer Assisted Self-Interview and Face-To-Face Interview Methods in Eliciting HIV-Related Risks among Men Who Have Sex with Men and Men Who Inject Drugs in Nigeria 
PLoS ONE  2014;9(1):e81981.
Introduction
Face-to-face (FTF) interviews are the most frequently used means of obtaining information on sexual and drug injecting behaviours from men who have sex with men (MSM) and men who inject drugs (MWID). However, accurate information on these behaviours may be difficult to elicit because of sociocultural hostility towards these populations and the criminalization associated with these behaviours. Audio computer assisted self-interview (ACASI) is an interviewing technique that may mitigate social desirability bias in this context.
Methods
This study evaluated differences in the reporting of HIV-related risky behaviours by MSM and MWID using ACASI and FTF interviews. Between August and September 2010, 712 MSM and 328 MWID in Nigeria were randomized to either ACASI or FTF interview for completion of a behavioural survey that included questions on sensitive sexual and injecting risk behaviours. Data were analyzed separately for MSM and MWID. Logistic regression was run for each behaviour as a dependent variable to determine differences in reporting methods.
Results
MSM interviewed via ACASI reported significantly higher risky behaviours with both women (multiple female sexual partners 51% vs. 43%, p = 0.04; had unprotected anal sex with women 72% vs. 57%, p = 0.05) and men (multiple male sex partners 70% vs. 54%, p≤0.001) than through FTF. Additionally, they were more likely to self-identify as homosexual (AOR: 3.3, 95%CI:2.4–4.6) and report drug use in the past 12 months (AOR:40.0, 95%CI: 9.6–166.0). MWID interviewed with ACASI were more likely to report needle sharing (AOR:3.3, 95%CI:1.2–8.9) and re-use (AOR:2.2, 95%CI:1.2–3.9) in the past month and prior HIV testing (AOR:1.6, 95%CI 1.02–2.5).
Conclusion
The feasibility of using ACASI in studies and clinics targeting key populations in Nigeria must be explored to increase the likelihood of obtaining more accurate data on high risk behaviours to inform improved risk reduction strategies that reduce HIV transmission.
doi:10.1371/journal.pone.0081981
PMCID: PMC3885382  PMID: 24416134
18.  TLR9 and RIG-I Signaling in Human Endocervical Epithelial Cells Modulates Inflammatory Responses of Macrophages and Dendritic Cells In Vitro 
PLoS ONE  2014;9(1):e83882.
The innate immune system has evolved to recognize invading pathogens through pattern recognition receptors (PRRs).Among PRRs, Toll like receptors (TLRs 3, 7/8,9) and RIG-I like receptors (RLRs) have been shown to recognize viral components. Mucosal immune responses to viral infections require coordinated actions from epithelial as well as immune cells. In this respect, endocervical epithelial cells (EEC's) play an important role in initiating innate immune responses via PRRs. It is unknown whether EEC's can alter immune responses of macrophages and dendritic cells (DC's) like its counterparts in intestinal and respiratory systems. In this study, we show that endocervical epithelial cells (End1/E6E7) express two key receptors, TLR9 and RIG-I involved in anti-viral immunity. Stimulation of End1/E6E7 cells lead to the activation of NF-κB and increased secretion of pro-inflammatory cytokines, IL-6 and IL-8. Polarized End1/E6E7 cells responded to apical stimulation with ligands of TLR9 and RIG-I, CpG-ODN and Poly(I:C)LL respectively, without compromising End1/E6E7 cell integrity. At steady state, spent medium from End1/E6E7 cells significantly reduced secretion of pro-inflammatory cytokines from LPS treated human primary monocyte derived macrophages (MDMs) and DC:T cell co-cultures. Spent medium from End1/E6E7 cells stimulated with ligands of TLR9/RIG-I restored secretion of pro-inflammatory cytokines as well as enhanced phagocytosis and chemotaxis of monocytic U937 cells. Spent medium from CpG-ODN and Poly(I:C)LL stimulated End1/E6E7 cells showed significant increased secretion of IL-12p70 from DC:T cell co-cultures. The anti-inflammatory effect of spent media of End1/E6E7 cell was observed to be TGF-β dependent. In summary, the results of our study indicate that EEC's play an indispensable role in modulating anti-viral immune responses at the female lower genital tract.
doi:10.1371/journal.pone.0083882
PMCID: PMC3883652  PMID: 24409285
19.  Plasma Free Fatty Acid Concentrations as a Marker for Acute Myocardial Infarction 
Background: Acute myocardial infarction carries a high mortality among cardiac patients.The discovery of the fact that certain enzymes like CPK, LDH liberated into circulation following necrosis of the myocardial cells came as boon for physicians and patients. There has been a constant search of different parameters for the diagnosis and management of CoronaryArtery Diseases (CAD).
Aim: The present study was undertaken to investigate a possible relation between the changes in plasma free fatty acid (FFA) concentration and acute myocardial infarction.
Material and Methods: Fifty cases (25 males and 25 females) of acute myocardial infarction were selected for the present study. All the patients were in the age group of 40-70 years. For the control group fifty (25 male and 25 female) subjects of same age group were selected from patient’s relatives and friends.
Plasma free fatty acid concentration was estimated by Titrametric method of Trout et al., (1960), a modified version of Dole (1956).
Statistical Analysis: The statistical analysis of the data of the present study was done by using SPSS, version 14.0.1 was used.
Results: Our study showed a significant increase in plasma FFA in the first 24 hours of acute myocardial infarction with subsequent normalisation on the 7th day.The difference between the first and the seventh day was statistically significant.
Conclusion: The FFA were found raised in cases of acute myocardial infarction.On the basis of present study, it is worth to say that estimation of serum free fatty acid should be done routinely at the earliest opportunity in all cases of acute myocardial infarction.
doi:10.7860/JCDR/2013/7682.3566
PMCID: PMC3879868  PMID: 24392364
Plasma free fatty acid; FFA; Acute myocardial infarction; Coronary artery disease; Marker
20.  Revised Ciprofloxacin Breakpoints for Salmonella: Is it Time to Write an Obituary? 
Objectives: To determine the minimum inhibitory concentration of ciprofloxacin among 50 blood stream isolates of Salmonella enterica.
Material and Methods: A total of 50 consecutive isolates of Salmonella enterica were tested for susceptibility to antimicrobials using the Kirby Bauer disk diffusion method. Minimum inhibitory concentrations were determined using Hi-Comb strips. All results were interpreted according to the CLSI guidelines.
Results: Of the 50 isolates 70%were Salmonella Typhi, 4% Salmonella paratyphi A, 2% Salmonella paratyphi B and the remaining 10% were identified only as Salmonella species. Using the CLSI 2011 breakpoints for disc diffusion, 86% (43/50) were resistant to nalidixic acid(NA), 22% (11/50) to ciprofloxacin, 12% to azithromycin, 6% to cotrimoxazole, 4% to ampicillin and 1% to chloramphenicol. The MIC50 and MIC90 of ciprofloxacin for S.Typhi were 0.181 μg/mL and 5.06 μg/mL respectively. While the same for S. paratyphi A was 0.212μg/mL and 0.228μg/mL respectively. None of the isolates were multi drug resistant and all were susceptible to ceftriaxone. Using the CLSI 2012 revised ciprofloxacin breakpoints for disc diffusion (>31mm) & MIC (<0.06 μg/mL), 90% (45/50) of these isolates were found to be resistant.
Conclusion: MIC’s of ciprofloxacin should be reported for all salmonella isolates and should be used to guide treatment. Blindly following western guidelines for a disease which is highly endemic in the subcontinent will spell the death knell of a cheap and effective drug in our armamentarium. Therefore it will be too premature to declare that “the concept of using ciprofloxacin in typhoid fever is dead!”
doi:10.7860/JCDR/2013/7312.3581
PMCID: PMC3879876  PMID: 24392374
Salmonella typhi; Salmonella paratyphi A; Ciprofloxacin; Resistance; Breakpoints
21.  Clonidine for management of chronic pain: A brief review of the current evidences 
Saudi Journal of Anaesthesia  2014;8(1):92-96.
Clonidine, an alpha-2 adrenergic receptor agonist, has well-established role in acute perioperative pain management. However, recently it has found increasing use in chronic pain conditions as well. In this review, we systematically searched and analyzed the clinical studies from “PubMed,” “PubMed central” and “Scopus” database for use of clonidine in the chronic pain. Quantitative meta-analysis was not possible as clonidine has been used in various patient populations through different routes. However, qualitative analysis of nearly thirty clinical studies provides some evidence that clonidine administered through epidural, intrathecal and local/topical route may be effective in chronic pain conditions where neuropathy is a predominant component. It may also be effective where opioids are of limited use due to inadequate pain relief or adverse effects.
doi:10.4103/1658-354X.125955
PMCID: PMC3950462  PMID: 24665248
Chronic pain; clonidine; neuropathy
22.  How Much Demand for New HIV Prevention Technologies Can We Really Expect? Results from a Discrete Choice Experiment in South Africa 
PLoS ONE  2013;8(12):e83193.
Background
For the first time in the history of HIV, new bio-medical interventions have been shown to be effective in preventing HIV transmission. For these new HIV prevention technologies (NPTs) to have an impact on the epidemic, they must be widely used. This study uses a discrete choice experiment (DCE) to: understand the relative strength of women’s preferences for product characteristics, understand the implications for substitution away from male condoms, and inform realistic modelling of their potential impact and cost-effectiveness.
Methods
A DCE was conducted among 1017 women in urban South Africa. Women were presented with choices between potential women’s NPTs (microbicides, diaphragm, female condom) and ‘what I did last time’ (use or not use a condom) with different HIV and pregnancy prevention effectiveness’ and prices. Choice probabilities are estimated using the nested logit model and used to predict uptake.
Results
In this high HIV prevalence setting, HIV prevention effectiveness is the main driver of uptake followed by pregnancy prevention effectiveness. For example a microbicide with poor effectiveness would have niche appeal at just 11% predicted uptake, while a highly effective microbicide (95% effective against HIV and pregnancy) would have far wider appeal (56% predicted uptake). Though women who reported not using condoms were more likely to choose the NPTs, at current very high rates of male condom use in South Africa (60%), about half of microbicide uptake is projected to be among those currently not using condoms.
Conclusions
Women are very interested in NPTs, especially if highly effective in preventing HIV and pregnancy. Women in greatest need were also most likely to switch to the new products. Where products are not yet available for distribution, proxy data, such as that generated by DCEs, can bring realism to overly optimistic uptake scenarios found in many current impact models.
doi:10.1371/journal.pone.0083193
PMCID: PMC3875434  PMID: 24386160
23.  Four Genetic Polymorphisms of Lymphotoxin-Alpha Gene and Cancer Risk: A Systematic Review and Meta-Analysis 
PLoS ONE  2013;8(12):e82519.
Lymphotoxin-alpha (LTA) is a pro-inflammatory cytokine that plays an important role in the inflammatory and immunologic response. Numerous studies have shown LTA polymorphisms as risk factors for cancers, but the results remain inconclusive. The goal of the present meta-analyses is to establish the associations between cancers and four LTA variants (rs1041981, rs2239704, rs2229094 and rs746868). A total of 30 case-control studies involving 58,649 participants were included in the current meta-analyses. Our results showed significant associations with increased cancer risk for rs1041981 (odd ratio (OR) = 1.15, 99% confidential interval (CI) = 1.07-1.25, P < 0.0001, I2 = 12.2%), rs2239704 (OR = 1.08, 99% CI = 1.01-1.16, P = 0.021, I2 = 0.0%) and rs2229094 (OR = 1.28, 99% CI = 1.09-1.50, P = 0.003, I2 = 0.0%). No evidence was found for the association between rs746868 and cancer risk (OR = 1.01, 99% CI = 0.93-1.10, P = 0.771, I2 = 0.0%). Subgroup meta-analysis suggested that rs2239704 was likely to increase the risk of hematological malignancy (OR = 1.10, 99% CI = 1.01–1.20, P = 0.023, I2 = 0.0%), and rs2229094 was specific for the increased risk of adenocarcinoma (OR = 1.33, 99% CI = 1.11-1.59, P = 0.002, I2 = 0.0%). In conclusion, our meta-analyses suggested that the LTA rs1041981, rs2239704 and rs2229094 polymorphisms contributed to the increased risk of cancers. Future functional studies were needed to clarify the mechanistic roles of the three variants in the cancer risk.
doi:10.1371/journal.pone.0082519
PMCID: PMC3861395  PMID: 24349304
24.  Combination of Cytokine Responses Indicative of Latent TB and Active TB in Malawian Adults 
PLoS ONE  2013;8(11):e79742.
Background
An IFN-γ response to M. tuberculosis-specific antigens is an effective biomarker for M. tuberculosis infection but it cannot discriminate between latent TB infection and active TB disease. Combining a number of cytokine/chemokine responses to M. tuberculosis antigens may enable differentiation of latent TB from active disease.
Methods
Asymptomatic recently-exposed individuals (spouses of TB patients) were recruited and tuberculin skin tested, bled and followed-up for two years. Culture supernatants, from a six-day culture of diluted whole blood samples stimulated with M. tuberculosis-derived PPD or ESAT-6, were measured for IFN-γ, IL-10, IL-13, IL-17, TNF-α and CXCL10 using cytokine ELISAs. In addition, 15 patients with sputum smear-positive pulmonary TB were recruited and tested.
Results
Spouses with positive IFN-γ responses to M. tuberculosis ESAT-6 (>62.5 pg/mL) and TB patients showed high production of IL-17, CXCL10 and TNF-α. Higher production of IL-10 and IL-17 in response to ESAT-6 was observed in the spouses compared with TB patients while the ratios of IFN-γ/IL-10 and IFN-γ/IL-17 in response to M. tuberculosis-derived PPD were significantly higher in TB patients compared with the spouses. Tuberculin skin test results did not correlate with cytokine responses.
Conclusions
CXCL10 and TNF-α may be used as adjunct markers alongside an IFN-γ release assay to diagnose M. tuberculosis infection, and IL-17 and IL-10 production may differentiate individuals with LTBI from active TB.
doi:10.1371/journal.pone.0079742
PMCID: PMC3832606  PMID: 24260295
25.  Human immunodeficiency virus type 1 viral protein R (Vpr) induces CCL5 expression in astrocytes via PI3K and MAPK signaling pathways 
Background
Neurocognitive impairments remain prevalent in HIV-1 infected individuals despite current antiretroviral therapies. It is increasingly becoming evident that astrocytes play a critical role in HIV-1 neuropathogenesis through the production of proinflammatory cytokines/chemokines. HIV-1 viral protein R (Vpr) plays an important role in neuronal dysfunction; however, its role in neuroinflammation is not well characterized. The major objective of this study was to determine the effect of Vpr in induction of proinflammatory chemokine CCL5 in astrocytes and to define the underlying mechanism(s).
Methods
SVGA astrocytes were either mock transfected or were transfected with a plasmid encoding HIV-1 Vpr, and the cells were harvested at different time intervals. The mRNA level of CCL5 expression was quantified using real-time RT-PCR, and cell culture supernatants were assayed for CCL5 protein concentration. Immunocytochemistry was performed on HIV-1 Vpr transfected astrocytes to check CCL5 expression. Various signaling mechanisms such as p38 MAPK, PI3K/Akt, NF-κB and AP-1 were explored using specific chemical inhibitors and siRNAs.
Results
HIV-1 Vpr transfected astrocytes exhibited time-dependent induction of CCL5 as compared to mock-transfected astrocytes at both the mRNA and protein level. Immunostained images of astrocytes transfected with HIV-1 Vpr also showed much higher accumulation of CCL5 in comparison to untransfected and mock-transfected astrocytes. Pre-treatment with NF-κB (SC514) and PI3K/Akt (LY294002) inhibitor partially abrogated CCL5 mRNA and protein expression levels as opposed to untreated controls after HIV-1 Vpr transfection. Specific siRNAs against p50 and p65 subunits of NF-κB, p38δ MAPK, Akt-2 and Akt-3, and AP-1 transcription factor substantially inhibited the production of CCL5 in HIV-1 Vpr transfected astrocytes.
Conclusion
These results demonstrate the ability of HIV-1 Vpr to induce CCL5 in astrocytes in a time-dependent manner. Furthermore, this effect was observed to be mediated by transcription factors NF-κB and AP-1 and involved the p38-MAPK and PI3K/Akt pathway.
doi:10.1186/1742-2094-10-136
PMCID: PMC3831867  PMID: 24225433
HIV-1; Vpr; CCL5; NF-kB; AP-1; MAPK; PI3K/Akt

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