Clinical trial evidence shows minimal survival gains and higher complication rates from radical prostatectomy (RP) versus watchful waiting (WW) for elderly men with localized prostate cancer (PCa). It is believed that these patients are overtreated. The current analyses aim to explore patient-level heterogeneity in survival effects, examine matching of patients to treatments in practice, and identify patient characteristics driving heterogenous effects, in order to present more comprehensive evidence about the concerns of overtreatment.
The interaction of amaranth with two homologous serum albumins from human and bovine (HSA and BSA) was studied by microcalorimetry. The binding stoichiometry for the complexation of amaranth to both BSA and HSA was around 1, and the equilibrium constants were (5.79 ± 0.07) × 10(5) and (1.76 ± 0.05) × 10(5) M(-1), respectively. The binding reaction to HSA at 298.15 K was driven by a large negative enthalpic contribution and a small but positive entropic contribution, while to BSA, it was entirely enthalpy-driven and the entropic contribution was unfavorable. Parsing of the standard molar Gibbs energy revealed that the complexation was dominated by non-polyelectrolytic forces. Temperature-dependent isothermal titration calorimetry studies revealed that the enthalpic contribution increased and the entropic contribution decreased with the rise in the temperature but the Gibbs energy change remained almost unaltered. Differential scanning calorimetry results revealed that the binding reaction stabilized the serum albumins significantly against thermal unfolding.
Thermodynamics of the interaction of the chemotherapeutic and chemopreventive dietary pigment, curcumin, with hemoglobin was studied by isothermal titration calorimetry. The binding was characterized to be exothermic. At 293.15 K, the equilibrium constant for curcumin-Hb complexation was found to be (4.88 ± 0.06) × 10(5) M(-1). The binding stoichiometry was calculated to be 1.08 ± 0.05, confirming a 1:1 complexation. The binding was driven by a large negative standard molar enthalpy change (?H(0) = -118.45 ± 0.05 kJ mol(-1)) and an unfavorable standard molar entropy change (T?S(0) = -86.53 ± 0.01 kJ mol(-1)) at 293.15 K. Increasing the temperature favoured the binding, and the magnitude of the negative standard molar heat capacity change suggested the involvement of significant hydrophobic forces in the binding process. With increasing salt concentration, the magnitude of the equilibrium constant decreased slightly; and the complexation mostly involved non-polyelectrolytic forces contributing about 92-94% of the standard molar Gibbs energy change. DSC studies revealed that curcumin binding caused a partial unfolding of the protein.
Care coordination services that link pregnant women to health-promoting resources, avoid duplication of effort, and improve communication between families and providers have been endorsed as a strategy for reducing disparities in adverse pregnancy outcomes, however empirical evidence regarding the effects of these services is contradictory and incomplete. This study investigates the effects of maternity care coordination (MCC) on pregnancy outcomes in North Carolina. Birth certificate and Medicaid claims data were analyzed for 7,124 women delivering live infants in North Carolina from October 2008 through September 2010, of whom 2,255 received MCC services. Propensity-weighted analyses were conducted to reduce the influence of selection bias in evaluating program participation. Sensitivity analyses compared these results to conventional ordinary least squares analyses. The unadjusted preterm birth rate was lower among women who received MCC services (7.0 % compared to 8.3 % among controls). Propensity-weighted analyses demonstrated that women receiving services had a 1.8 % point reduction in preterm birth risk; p < 0.05). MCC services were also associated with lower pregnancy weight gain (p = 0.10). No effects of MCC were seen for birthweight. These findings suggest that coordination of care in pregnancy can significantly reduce the risk of preterm delivery among Medicaid-enrolled women. Further research evaluating specific components of care coordination services and their effects on preterm birth risk among racial/ethnic and geographic subgroups of Medicaid enrolled mothers could inform efforts to reduce disparities in pregnancy outcome.
The present study was aimed to evaluate the anti-inflammatory potentials of Vespa tropica (VT) venom and its isolated peptides. Effects of whole venom and its two peptides (Vt1512 and Vt1386) on lipopolysaccharide (LPS) challenged BV-2 murine microglial cells was evaluated.
Retinoic acid inducible gene I (RIG-I) is a well established pattern recognition receptor (PRR) in neurons infected with Japanese encephalitis virus (JEV) as reported previously from our laboratory. Japanese encephalitis (JE) virus infection in brain has been shown to decrease the proliferation of neural stem/progenitor cells (NSPCs) which has its implications in neurological sequelae in JE survivors. We have found that ablation of RIG-I both in vivo and in vitro models results in significant decrease in NSPC proliferation post JEV infection. We hypothesize that knockdown of RIG-I diminishes the expression of antiviral molecules resulting in an increase in viral replication, which in turn results in enhancement of the expression of cell cycle inhibitors, hence affecting the proliferation of NSPCs.
Toll-like receptor 7 (TLR7) known to recognize guanidine-rich ssRNA has been shown to mount vital host defense mechanism against many viruses including flaviviruses. Signal transduction through TLR7 has been shown to produce type-1 interferon and proinflammatory mediators, thereby initiating essential innate immune response against ssRNA viruses in hosts. Systemic and brain specific TLR7 knock-down mice (TLR7(KD)) were generated using vivo-morpholinos. These mice were then subcutaneously challenged with lethal dose of JEV (GP78 strain) and were subsequently analyzed for survival. Significant difference in susceptibility to JEV between wild-type and systemic TLR7(KD) mice was observed whereas, no difference in susceptibility to JEV infection was seen in brain-specific TLR7(KD) mice. Significant decreases in IFN-? and antiviral proteins were also observed in both TLR7(KD) mice along with increased viral loads in their brain. Owing to increased viral load, increases in levels of various proinflammatory cyto/chemokines, increased microglial activation and infiltration of peripheral immune cells in brain of TLR7(KD) mice were also observed. Immunocytochemistry and RNA co-immunoprecipitation performed with JEV-infected N2a or HT22 cells indicated endosomal localization and confirmed interaction between JEV ssRNA with TLR7. Treatment of mice with imiquimod, a TLR7 agonist, prior to JEV infection resulted in their increased survival. Overall, our results suggest that the TLR7 response following JEV infection promotes type-1 interferon production and generation of antiviral state which might contribute to protective effect in systemic infection.
An immune role of neural stem/progenitor cells (NSPCs) has been proposed in many recent studies; however much still remains to be elucidated. In the current investigation, we report that NSPCs possess the ability to convert encephalitogenic T cells into CD4(+)-CD25(+)-FOXP3(+) regulatory T cells (T(reg)). Encephalitogenic and nonencephalitogenic T cells isolated from sham and Japanese encephalitis virus (JEV) infected animals were co-cultured with mouse NSPCs. Post co-culture, significant increase in the number of T(regs) was observed from encephalitogenic T cells co-cultured with NSPCs. This increased conversion was found to be dependent on direct contact between T cells and NSPCs. Neutralization of TGF-? and IFN-? in NSPC cultures abrogated this increased conversion of encephalitogenic T cells into T(regs). Flow cytometric, quantitative RT-PCR, and immunoblot analysis of both T cells and NSPCs revealed surface and intracellular changes post co-culture. Co-stimulatory molecules (B7) and ICAM-1 were increased on NSPCs post co-culture; levels of TGF?, IFN?, and TGF?R1 were also increased in NSPCs. This study provides a basic insight into the interaction between CNS-infiltrating encephalitogenic T cells and NSPCs during viral encephalitis. Conversion of encephalitogenic T cells into CD4(+)-CD25(+)-FOXP3(+) T(regs) through interaction with NSPCs indicates an attempt in regulation of excessive inflammation in the CNS.
The interaction of the synthetic azo dye and food colorant carmoisine with human and bovine serum albumins was studied by microcalorimetric techniques. A complete thermodynamic profile of the interaction was obtained from isothermal titration calorimetry studies. The equilibrium constant of the complexation process was of the order of 10(6)M(-1) and the binding stoichiometry was found to be 1:1 with both the serum albumins. The binding was driven by negative standard molar enthalpy and positive standard molar entropy contributions. The binding affinity was lower at higher salt concentrations in both cases but the same was dominated by mostly non-electrostatic forces at all salt concentrations. The polyelectrolytic forces contributed only 5-8% of the total standard molar Gibbs energy change. The standard molar enthalpy change enhanced whereas the standard molar entropic contribution decreased with rise in temperature but they compensated each other to keep the standard molar Gibbs energy change almost invariant. The negative standard molar heat capacity values suggested the involvement of a significant hydrophobic contribution in the complexation process. Besides, enthalpy-entropy compensation phenomenon was also observed in both the systems. The thermal stability of the serum proteins was found to be remarkably enhanced on binding to carmoisine.
Emergence of multidrug resistance among Streptococcus pneumoniae (SP), has limited the available options used to treat infections caused by this organism. The objective of this study was to compare the role of monotherapy and combination therapy with ampicillin (AMP) and azithromycin (AZM) in eradicating bacterial burden and down regulating lung inflammation in a murine experimental pneumococcal infection model.
MicroRNAs (miRNAs) are single-stranded small RNA molecules that regulate various cellular processes. miRNA 155 (miR-155) regulates various aspects of innate and adaptive immune responses and plays a key role in various viral infections and the resulting neuroinflammation. The present study evaluated the involvement of miR-155 in modulating Japanese encephalitis virus (JEV)-induced neuroinflammation. We observed that miR-155 expression was upregulated during JEV infection of mouse primary microglia, the BV-2 microglia cell line, and in both mouse and human brains. In vitro and in vivo knockdown of miR-155 minimized JEV-induced inflammatory responses. In the present study, we confirmed targeting of the Src homology 2-containing inositol phosphatase 1 (SHIP1) 3' untranslated region (UTR) by miR-155 in the context of JEV infection. Inhibition of SHIP1 by miR-155 resulted in higher beta interferon (IFN-?) and proinflammatory cytokine production through activation of TANK-binding kinase 1 (TBK-1). Based on these observations, we conclude that miR-155 modulates the neuroinflammatory response during JEV infection via negative regulation of SHIP1 expression. Thus, modulation of miR-155 could be a novel strategy to regulate JEV-induced neuroinflammation.
Tumor necrosis factor ? (TNF-?) is an essential player in infection with Leishmania, controlling inflammatory lesion and parasite killing. We recently have shown the leishmanicidal activity of transmembrane form of TNF (mTNF) derived from allogeneic natural killer (NK) cells in experimental visceral leishmaniasis. Allogeneic macrophages and human monocytes derived mTNF has significantly higher antileishmanial activity compared to allogeneic NK cells. Unlike NK cells, syngeneic macrophages also possess antileishmanial activity, although degree of activity is significantly less compared to allogeneic macrophages. Cellular therapy by intravenous transfer of allogeneic macrophages enhances leishmanicidal effect against the established infection in susceptible animal by reducing the splenic parasite burden to 28.3 ± 4.71 × 10(5) compared to 256.00 ± 17.36 × 10(5) in control group. In vivo treatment with anti-mouse TNF-? reduces the therapeutic efficacy of the allogeneic macrophages by increasing the parasite load in spleen of infected mice. These results demonstrated that allogeneic and xenogeneic macrophages induce cytokine mediated protective mechanism against infected macrophages via TNF-? in vitro and, possibly in vivo. The macrophage mediated protective role in absence of T cell help demonstrate an unique property of the mononuclear phagocytes in controlling infection and inflammation in visceral leishmaniasis, despite being acts as a host cell for the same parasite.
Studies on the molecular aspects of alkaloid-RNA complexation are of prime importance for the development of rational RNA targeted drug design strategies. Towards this goal, the binding aspects of three novel 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs to four single stranded ribonucleotides, poly(G), poly(I), poly(C) and poly(U), were studied for the first time employing multifaceted biophysical tools. Absorbance and fluorescence studies revealed that these analogs bound non-cooperatively to poly(G) and poly(I) with binding affinities remarkably higher than berberine. The binding of these analogs to poly(U) and poly(C) was weaker in comparison to poly(G) and poly(I) but were one order higher in comparison to berberine. Quantum efficiency values revealed that energy transfer occurred from the RNA bases to the analogs upon complexation. The binding was dominated by large positive entropic contributions and small but favorable enthalpic contributions. Salt dependent studies established that the binding was dominated by hydrophobic forces that contributed around 90% of the total standard molar Gibbs energy. The chain length of the substitution at the 9-position was found to be critical in modulating the binding affinities. These results provide new insights into the binding efficacy of these novel berberine analogs to single stranded RNA sequences.
The flaviviral encephalitis has now become a major health concern in global scale. The efficient detection of viral infection and induction of the innate antiviral response by host's innate immune system are crucial to determine the outcome of infection. The intracellular pattern recognition receptors TLRs, RLRs, NLRs and CLRs play a central role in detection and initiation of robust antiviral response against flaviviral infection. Both cytoplasmic RLRs, RIG-I and MDA5 have been shown to be implicated in sensing flaviviral genomic RNA. Similarly among TLRs mainly TLR3 and TLR7 are known to respond in flaviviral infections as they are known to sense dsRNA and ssRNA moiety as their natural cognate ligand. Several studies have also shown the roles of NLRs and CLRs in mounting an innate antiviral response against flavivirus but, it is yet to be completely understood. Until now only few reports have implicated NLRs and CLRs in induction of antiviral and proinflammatory state following flaviviral infection. The current review therefore aims to comprehensively analyze past as well as current understanding on the role of PRRs in flaviviral infections.
Lead is one of the major pollutants of environment and is highly toxic to the functioning of central nervous system (CNS). The chronic exposure of this heavy metal is debilitating to the functional behavior of an organism. Studies have shown that acute exposure to Pb can lead to glial activation and secretion of cyto-chemokines in both in vitro and in vivo models. However, the cellular source of secretion of these cyto-chemokines remains to be identified. Microglia are monocytes of the brain, and are primary source of cytokine secretion in the CNS. We hypothesized that microglia exposed to Pb can secrete cyto-chemokines, thereby resulting in subsequent neuronal death. Our studies show that stimulation of BV-2 mouse microglia with 10?? dose of Pb resulted in up-regulation of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) pathways, along with activation of an important transcription factor, nuclear factor-?B (NF-?B). Further, we found that the levels of tumor necrosis factor-? (TNF-?), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), and cyclooxygenase-2 (COX-2) pro-inflammatory enzyme were increased in response to Pb exposure. Furthermore, treatment with conditioned media from Pb treated BV-2 cells lead to neuronal death in neuroblastoma cells, which potentially involved the activation of caspase-3 enzyme. In all, the current study brings forth critical involvement of microglial activation in mediating the neurotoxicity associated with lead exposure.
Japanese encephalitis virus (JEV) infection leads to Japanese encephalitis (JE) in humans. JEV is transmitted through mosquitoes and maintained in a zoonotic cycle. This cycle involves pigs as the major reservoir, water birds as carriers and mosquitoes as vectors. JEV invasion into the central nervous system (CNS) may occur via antipodal transport of virions or through the vascular endothelial cells. Microglial cells get activated in response to pathogenic insults. JEV infection induces the innate immune response and triggers the production of type I interferons. The signaling pathway of type I interferon production is regulated by a number of molecules. TRIM proteins are known to regulate the expression of interferons; however, the involvement of TRIM genes and their underlying mechanism during JEV infection are not known.
Interaction of the 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted analogs of the anticancer isoquinoline alkaloid berberine with RNA triplex, poly(U)-poly(A) · poly(U) has been studied in comparison to the duplex poly(A)-poly(U), using multiple biophysical techniques. Spectrophotometric and spectrofluorimetric studies established the non-cooperative binding mode of all the analogs with both the duplex and the triplex. However, berberine exhibited cooperative binding with poly(A)-poly(U) and non-cooperative binding with poly(U)-poly(A) · poly(U). Analog BER1 showed the highest affinity to both the duplex and the triplex followed by BER2 and BER3. The overall binding affinity varied as BER1 > BER2 > BER3 > BER. The magnitude of the quantum efficiency values (Q > 1) revealed that energy was transferred from the bases of the triplex and the duplex to the analogs. Comparative ferrocyanide quenching and viscosity studies unambiguously established a stronger intercalative geometry of the analogs to both the triplex and the duplex in comparison to berberine. Circular dichroism studies revealed that the alkaloids perturbed the conformation of both RNA helices. The binding of all the alkaloids was found to be exothermic from isothermal titration studies. Binding of the analogs was highly entropy driven while that of berberine was enthalpy dominated. The results presented here reveal strong and specific binding of these new berberine analogs to the RNA triplex and duplex and highlight the remarkable influence of the 9-substitution on the interaction profile.
In this study we have reported the in vivo proteomic changes during Japanese Encephalitis Virus (JEV) infection in combination with in vitro studies which will help in the comprehensive characterization of the modifications in the host metabolism in response to JEV infection. We performed a 2-DE based quantitative proteomic study of JEV-infected mouse brain as well as mouse neuroblastoma (Neuro2a) cells to analyze the host response to this lethal virus. 56 host proteins were found to be differentially expressed post JEV infection (defined as exhibiting ? 1.5-fold change in protein abundance upon JEV infection). Bioinformatics analyses were used to generate JEV-regulated host response networks which reported that the identified proteins were found to be associated with various cellular processes ranging from intracellular protein transport, cellular metabolism and ER stress associated unfolded protein response. JEV was found to invade the host protein folding machinery to sustain its survival and replication inside the host thereby generating a vigorous unfolded protein response, subsequently triggering a number of pathways responsible for the JEV associated pathologies. The results were also validated using a human cell line to correlate them to the human response to JEV. The present investigation is the first report on JEV-host interactome in in vivo model and will be of potential interest for future antiviral research in this field.
The interaction of the food additive carmoisine with herring testes DNA was studied by multifaceted biophysical techniques. Carmoisine exhibited hypochromic effects in absorbance, whereas in fluorescence the intensity enhanced upon complexation with DNA. Energy transfer from the DNA base pairs to carmoisine molecules occurred upon complexation. A groove binding model of interaction was envisaged for carmoisine-DNA complexation from 4,6-diamidino-2-phenylindole (DAPI) and Hoechst displacement studies. The binding of carmoisine stabilized the DNA structure against thermal denaturation. The binding induced moderate conformational perturbations in the B-form structure of DNA. The binding affinity (10(4) M(-1)) values, calculated from absorbance and fluorescence data, and calorimetry titrations were in close agreement with each other. The binding was characterized to be exothermic and favored by small negative enthalpic and large positive entropic contributions. Salt-dependent calorimetric studies revealed that the binding reaction was dominated by nonpolyelectrolytic forces. The negative heat capacity value suggested the role of hydrophobic effect in the interaction.
Japanese encephalitis virus (JEV), a single-stranded RNA (ssRNA) virus, is the leading cause of encephalitis in Asia. Microglial activation is one of the key events in JEV-induced neuroinflammation. Although the various microRNAs (miRNAs) has been shown to regulate microglia activation during pathological conditions including neuroviral infections, till date, the involvement of miRNAs in JEV infection has not been evaluated. Hence, we sought to evaluate the possible role of miRNAs in mediating JEV-induced microglia activation. Initial screening revealed significant up-regulation of miR-29b in JEV-infected mouse microglial cell line (BV-2) and primary microglial cells. Furthermore, using bioinformatics tools, we identified tumor necrosis factor alpha-induced protein 3, a negative regulator of nuclear factor-kappa B signaling as a potential target of miR-29b. Interestingly, in vitro knockdown of miR-29b resulted in significant over-expression of tumor necrosis factor alpha-induced protein 3, and subsequent decrease in nuclear translocation of pNF-?B. JEV infection in BV-2 cell line elevated inducible nitric oxide synthase, cyclooxygenase-2, and pro-inflammatory cytokine expression levels, which diminished after miR-29b knockdown. Collectively, our study demonstrates involvement of miR-29b in regulating JEV- induced microglial activation. miR-29b regulates Japanese Encephalitis Virus (JEV)-induced microglia activation via inhibition of the anti-inflammatory proteinTNFAIP3, which results in sustained activation of NF-kB. Sustained NF-?Bactivation further results in augmented secretion of pro-inflammatory cytokines and induction of inflammatory mediators (iNOS and Cox-2).
Inflammation plays a critical role in the progression of neurodegenerative diseases. Microglia are the resident macrophages of the central nervous system (CNS) which actively take part in the neuronal development of CNS and are involved in clearance of pathogens as well as cellular debris from the system upon insult to this organization. Chronic activation of microglia in neurodegenerative disorders such as Alzheimers disease (AD), Parkinsons disease (PD), Huntingtons disease (HD), amyotrophic lateral sclerosis (ALS) as well as inflammatory conditions of CNS such as multiple sclerosis (MS) results in overall upregulation of pro-inflammatory cytokines and chemokines in the brain parenchyma. This compromises the neuronal health which further activates microglia by releasing death associated molecules such as neuromelanin, A? peptides and cellular debris at the lesion site thereby forming a vicious cycle of disease advancement. Targeting microglial activation has proven to be a viable option in the treatment of inflammation related neurodegenerative diseases. This review will discuss the central position of inflammation and therapeutic strategies aiming to alleviate disease progression in some of the important inflammatory conditions of CNS.
Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) is an emerging tropical pathogen with a case fatality rate of 55 to 75% that predominantly affects children in the age group of 2 to 16 years. Although it has been established as a neurotropic virus causing encephalitis, the molecular pathology leading to neuronal death is unknown. The present study elucidates for the first time the mechanism of cell death in neurons after CHPV infection that answers the basic cause of CHPV-mediated neurodegeneration. Through various cell death assays in vitro and in vivo, a relationship between viral replication within neuron and neuronal apoptosis has been established. We report that expression of CHPV phosphoprotein increases up to 6 h postinfection and diminishes thereafter in neuronal cell lines, signifying the replicative phase of CHPV. Various analyses conducted during the investigation established that CHPV-infected neurons are undergoing apoptosis through an extrinsic pathway mediated through the Fas-associated death domain (FADD) following activation of caspase-8 and -3 and prominent cleavage of poly(ADP-ribose) polymerase (PARP). Knocking down the expression of caspase-3, the final executioner of apoptosis, in a neuronal cell line by endoribonuclease-prepared small interfering RNA (siRNA) validated its pivotal role in CHPV-mediated neurodegeneration by showing reduction in apoptosis after CHPV infection.
Japanese encephalitis (JE) is a mosquitoborne viral disease that is the primary cause of acute encephalitis syndrome in India. This virus mainly infects the central nervous system and causes massive inflammation which, if left unchecked, may prove fatal. Survivors often suffer from mild to severe neuropsychiatric sequelae. JE is a major cause of death in many parts of India and there is a possibility of it spreading into the National Capital Region from highly endemic neighbouring states. Fourteen cases of JE were reported in Delhi in 2011 compared with none in the previous 4 years from 2007 to 2010. Unless immediate preventive measures are taken this trend could continue and the disease could spread with increasing prevalence.
Viruses have evolved various mechanisms to subvert the hosts immune system and one of them is preventing the infected cells from sending out chemotactic signals to activate the adaptive immune response. Japanese encephalitis virus (JEV) is a neuropathologic flavivirus that is responsible for significant number of child mortalities in various parts of South-East Asia. In this study we show that JEV modulates suppressors of cytokine signaling (SOCS)1 and 3 expression in macrophages to bring about changes in the JAK-STAT signaling cascade, so as to inhibit proinflammatory cyto/chemokine release. Using real time PCR, immunoblotting and immunofluorescent staining, we show that the expression of type 1 interferons and intracellular expression of viral genes are also affected over time. Also, following the initial activation of SOCS1 and 3, there is production of interferon-inducible anti-viral proteins in the cells which may be responsible for inhibiting viral replication. However, even at later time points, viral genes were still detected from the macrophages, albeit at lesser quantities, than earlier time points, indicative of intracellular persistence of the virus in a latent form. On knocking down SOCS1 and SOCS3 we found a significant decrease in viral gene expression at an early time point, indicating the dysregulation of the signaling cascade leading to increased production of interferon-inducible anti-viral proteins. Taken together, our study provides an insight into the role of JEV infection in modulating the JAK-STAT pathway with the help of SOCS leading to the generation of an antiviral innate immune response.
Cytokine measurement is a prerequisite to understand the inflammatory state of the body. Quantitative analysis of cytokines by Western blotting and ELISA is a daunting task as these are time-consuming and error-prone protocols. With the advent of flow cytometry, the estimation of cytokines using the classical antigen-antibody reaction has become a popular choice with researchers/clinicians. Here, we describe a protocol for multiple cytokine analysis using flow cytometry.
Microglia are the resident macrophages of the CNS, which secrete several pro- and anti-inflammatory cyto-chemokines including interleukin-1? (IL-1?), in response to pathogenic stimuli. Once secreted, IL-1? binds to IL-1 receptor present on microglia and initiates the production of inflammatory cytokines in microglia. However, the detailed information regarding the molecular mechanisms of IL-1? triggered inflammatory pathways in microglia is lacking. Our studies focused on the role of Krüppel-like factor 4 (Klf4) in mediating the regulation of pro-inflammatory gene expression upon IL-1? stimulation in microglia. Our studies show that stimulation of microglia with IL-1? robustly induces Klf4 via PI3K/Akt pathway which positively regulates the production of endogenous IL-1? as well as other pro-inflammatory markers, cyclooxygenase-2, monocyte chemoattractant protein-1 and interleukin-6 (IL-6). In addition, we report that Klf4 negatively regulates the expression of inducible nitric oxide synthase, thereby playing a key role in regulating the immunomodulatory activities of microglia.
The interaction of the dietary pigment curcumin with herring testes deoxyribonucleic acid was studied by biophysical and microcalorimetric techniques. Curcumin bound to DNA exhibiting hypochromic effect in absorbance and enhanced intensity of its fluorescence. The binding a affinity value evaluated from spectroscopy data was of the order 10(4)M(-1). The quantum efficiency value testified the occurrence of energy transfer from the DNA base pairs to the curcumin molecules. Displacement studies of DNA bound DAPI, Hoechst and ethidium bromide suggested binding of curcumin to be in the minor groove of the DNA. Moderate conformational perturbations of the B-form structure of DNA occurred on binding. The binding affinity weakened as the DNA GC content enhanced. The binding was characterized by negative enthalpy and positive entropy changes; the binding affinity from calorimetry was in good agreement with that evaluated from the spectral data. The binding was dominated by hydrophobic and other non-polyelectrolytic forces; the polyelectrolytic forces contributing only a quarter to the total Gibbs energy at 50mM [Na(+)].
Japanese encephalitis virus (JEV) is a common cause of encephalitis in humans who are dead-end hosts producing negligible viremia. The virus reaches the brain and causes massive inflammation. Our study seeks to understand the virus-host interaction using the murine monocyte/macrophage cell line RAW264.7, an antigen presenting cell involved in eliciting an innate immune response. We have discovered several interesting phenomena occurring in JEV-infected RAW264.7 cells which diverge from established observations. JEV remains inside RAW264.7 and appears to have little negative effect on cell viability. Expression studies of major histocompatibility complexes (MHC) and co-stimulatory molecules show inhibition of antigen presentation. There is enhanced immune suppression creating an anti-viral milieu. Expression of pro-inflammatory cytokines and chemokines is suppressed along with increased expression of anti-inflammatory molecules. Histone deacetylases (HDACs) have known inflammatory properties. In our study, through modulation of HDACs JEV seems to induce a crucial anti-inflammatory and anti-viral role in host macrophages.
A comprehensive pharmacokinetic profiling of novel drugs and therapeutics is a definite prerequisite of drug discovery and development. The present study expedites the in vivo and in vitro pharmacokinetic properties of colloidal sulfur nanoparticles (SNPs). In vitro dissolution properties of SNPs have been demonstrated and compared with the in vivo pharmacokinetic parameters of rabbit (Oryctolagus cuniculus) serum sample. The present study was also aimed at developing levels of correlation between in vitro and in vivo pharmacokinetic parameters. Cumulative results of the proposed study also suggest good in vitro-in vivo correlation of these novel nanocolloids and suggest their immediate profiling as an antimicrobial drug.
This interaction of four novel 9-O-substituted analogs of the plant alkaloid berberine with double stranded poly(A) was studied using a variety of biophysical techniques. Remarkably higher binding of two 9-O-?-amino alkyl ether analogs compared to the two 9-O-N-aryl/arylalkyl amino carbonyl methyl berberine analogs was observed. Quantum efficiency values suggested that energy was transferred from the adenine base pairs to the analogs on binding. Ferrocyanide quenching and viscosity studies revealed the binding mode to be intercalative for these analogs. Circular dichroism studies showed that these analogs induced significant conformational changes in the secondary structure of ds poly(A). Energetics of the binding suggested that 9-O-N-aryl/arylalkyl amino carbonyl methyl berberines bound very weakly to ds poly(A). The binding of 9-O-?-amino alkyl ether analogs was entropy dominated with a smaller but favorable enthalpic contribution to the Gibbs energy. Increasing the temperature resulted in weaker binding; the enthalpic contribution increased and the entropic contribution decreased. A small negative heat capacity change with significant enthalpy-entropy compensation established the involvement of multiple weak noncovalent interactions in the binding process.
Japanese encephalitis virus (JEV) predominantly infects neurons and causes damage to the central nervous system (CNS). Neural stem/progenitor cells (NSPCs) constitute multi-potent stem cell population in postnatal/adult brain, with capacity to differentiate into neurons, astrocytes or oligodendrocytes. NSPCs are known to play a pivotal role in CNS repair mechanisms during various neurological disorders. Previous studies from our laboratory have shown that JEV infection of NSPCs depletes the stem-cell pool, which may result in impaired repair functions leading to motor and cognitive deficits in survivors. In the present study, we evaluated the effect of JEV infection on differentiation potential of NSPCs isolated from BALB/c mouse pups (Post natal day 7). Results clearly indicated that, JEV infection was more robust in undifferentiated NSPCs as compared to differentiated ones. Further, JEV infected NSPCs showed hampered differentiation and arrested migration in adherent neurosphere cultures. Interestingly, the neuronal differentiation appeared to be more severely affected by JEV as compared to astrocyte differentiation. The transcription factors involved in both neuronal and astrocyte differentiations were significantly decreased upon JEV infection. Overall, results presented in this study comprehensively provide first evidence for JEV induced alteration of neuronal and astrocyte differentiation.
Three new analogs of berberine with aryl/ arylalkyl amino carbonyl methyl substituent at the 9-position of the isoquinoline chromophore along with berberrubine were studied for their binding to tRNA(phe) by wide variety of biophysical techniques like spectrophotometry, spectrofluorimetry, circular dichroism, thermal melting, viscosity and isothermal titration calorimetry.
The quantile regression (QR) framework provides a pragmatic approach in understanding the differential impacts of covariates along the distribution of an outcome. However, the QR framework that has pervaded the applied economics literature is based on the conditional quantile regression method. It is used to assess the impact of a covariate on a quantile of the outcome conditional on specific values of other covariates. In most cases, conditional quantile regression may generate results that are often not generalizable or interpretable in a policy or population context. In contrast, the unconditional quantile regression method provides more interpretable results as it marginalizes the effect over the distributions of other covariates in the model. In this paper, the differences between these two regression frameworks are highlighted, both conceptually and econometrically. Additionally, using real-world claims data from a large US health insurer, alternative QR frameworks are implemented to assess the differential impacts of covariates along the distribution of medication adherence among elderly patients with Alzheimers disease.
Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in South and South-East Asia. Lack of antivirals and non-availability of affordable vaccines in these endemic areas are a major setback in combating JEV and other closely related viruses such as West Nile virus and dengue virus. Protein secondary structure mimetics are excellent candidates for inhibiting the protein-protein interactions and therefore serve as an attractive tool in drug development. We synthesized derivatives containing the backbone of naturally occurring lupin alkaloid, sparteine, which act as protein secondary structure mimetics and show that these compounds exhibit antiviral properties.
MicroRNAs (miRNAs) are single-stranded noncoding regions of approximately 21 nucleotides that regulate protein synthesis by targeting mRNAs for translational repression or degradation at the post-transcriptional level. These classes of RNAs are highly conserved across species and are known to regulate several protein-coding genes in humans. Therefore, their dysregulation is synonymous with inflammation, autoimmunity, neurodegeneration, viral infections, heart diseases, and cancer, among other conditions. Recent years have witnessed considerable amount of research interest in studies on miRNA-mediated modulation of gene function during neuroinflammation. This review is a meticulous compilation of information on biogenesis of miRNAs and their role in neuroinflammatory diseases. Further, their potential as markers of inflammatory diseases or novel therapeutic agents against neuroinflammation has also been discussed in detail.
Methods for estimating average treatment effects, under the assumption of no unmeasured confounders, include regression models; propensity score adjustments using stratification, weighting, or matching; and doubly robust estimators (a combination of both). Researchers continue to debate about the best estimator for outcomes such as health care cost data, as they are usually characterized by an asymmetric distribution and heterogeneous treatment effects,. Challenges in finding the right specifications for regression models are well documented in the literature. Propensity score estimators are proposed as alternatives to overcoming these challenges. Using simulations, we find that in moderate size samples (n= 5000), balancing on propensity scores that are estimated from saturated specifications can balance the covariate means across treatment arms but fails to balance higher-order moments and covariances amongst covariates. Therefore, unlike regression model, even if a formal model for outcomes is not required, propensity score estimators can be inefficient at best and biased at worst for health care cost data. Our simulation study, designed to take a proof by contradiction approach, proves that no one estimator can be considered the best under all data generating processes for outcomes such as costs. The inverse-propensity weighted estimator is most likely to be unbiased under alternate data generating processes but is prone to bias under misspecification of the propensity score model and is inefficient compared to an unbiased regression estimator. Our results show that there are no magic bullets when it comes to estimating treatment effects in health care costs. Care should be taken before naively applying any one estimator to estimate average treatment effects in these data. We illustrate the performance of alternative methods in a cost dataset on breast cancer treatment.
Instrumental variables methods (IV) are widely used in the health economics literature to adjust for hidden selection biases in observational studies when estimating treatment effects. Less attention has been paid in the applied literature to the proper use of IVs if treatment effects are heterogeneous across subjects. Such a heterogeneity in effects becomes an issue for IV estimators when individuals self-selected choices of treatments are correlated with expected idiosyncratic gains or losses from treatments. We present an overview of the challenges that arise with IV estimators in the presence of effect heterogeneity and self-selection and compare conventional IV analysis with alternative approaches that use IVs to directly address these challenges. Using a Medicare sample of clinically localized breast cancer patients, we study the impact of breast-conserving surgery and radiation with mastectomy on 3-year survival rates. Our results reveal the traditional IV results may have masked important heterogeneity in treatment effects. In the context of these results, we discuss the advantages and limitations of conventional and alternative IV methods in estimating mean treatment-effect parameters, the role of heterogeneity in comparative effectiveness research and the implications for diffusion of technology.
To develop regression models for outcomes with truncated supports, such as health-related quality of life (HRQoL) data, and account for features typical of such data such as a skewed distribution, spikes at 1 or 0, and heteroskedasticity.
Hypoxic preconditioning reprogrammes the brains response to subsequent H/I (hypoxia-ischaemia) injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O2). Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic protein)-positive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase), EAAT-1 (excitatory amino acid transporter-1; also known as GLAST), MCT-1 (monocarboxylate transporter-1) and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP), which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.
Clinical research needs to be more useful to patients, clinicians, and other decision makers. To meet this need, more research should focus on patient-centered outcomes, compare viable alternatives, and be responsive to individual patients preferences, needs, pathobiology, settings, and values. These features, which make comparative effectiveness research (CER) fundamentally patient-centered, challenge researchers to adopt or develop methods that improve the timeliness, relevance, and practical application of clinical studies. In this paper, we describe 10 priority areas that address 3 critical needs for research on patient-centered outcomes (PCOR): (1) developing and testing trustworthy methods to identify and prioritize important questions for research; (2) improving the design, conduct, and analysis of clinical research studies; and (3) linking the process and outcomes of actual practice to priorities for research on patient-centered outcomes. We argue that the National Institutes of Health, through its clinical and translational research program, should accelerate the development and refinement of methods for CER by linking a program of methods research to the broader portfolio of large, prospective clinical and health system studies it supports. Insights generated by this work should be of enormous value to PCORI and to the broad range of organizations that will be funding and implementing CER.
Value of information (VOI) techniques can provide estimates of the expected benefits from clinical research studies that can inform decisions about the design and priority of those studies. Most VOI studies use decision-analytic models to characterize the uncertainty of the effects of interventions on health outcomes, but the complexity of constructing such models can pose barriers to some practical applications of VOI. However, because some clinical studies can directly characterize uncertainty in health outcomes, it may sometimes be possible to perform VOI analysis with only minimal modeling. This article 1) develops a framework to define and classify minimal modeling approaches to VOI, 2) reviews existing VOI studies that apply minimal modeling approaches, and 3) illustrates and discusses the application of the minimal modeling to 2 new clinical applications to which the approach appears well suited because clinical trials with comprehensive outcomes provide preliminary estimates of the uncertainty in outcomes. The authors conclude that minimal modeling approaches to VOI can be readily applied in some instances to estimate the expected benefits of clinical research.
Repurposing of old drugs is a useful concept as it helps to minimize costs associated with the research and development of a new drug. Minocycline, a common second generation antibiotic, has been shown to possess several other beneficial effects other than its intended uses. The antiviral role of minocycline has generated considerable interest from the last decade. It was first shown to be beneficial in preventing human immunodeficiency virus (HIV) infections and later it was reported to improve cognitive deficiencies associate with neuroAIDS. However, its antiviral efficacies are not limited to retroviruses alone. In animal models or in vitro systems of flaviviral infections (especially Japanese encephalitis virus), minocycline has been shown to be highly effective. However, not all effects are based on direct inhibition of viral replication. The general anti-inflammatory and immunomodulatory properties of minocycline are also responsible in part, in imparting the protective effects. Owing to the fact that minocycline is well tolerated by most people and that the drug has nearly 40 years history of usage, it is an exciting prospect to try out in other viral infections.
Microglia, the resident macrophages of the Central Nervous System (CNS) mediate key innate immune responses against foreign invasions within the CNS and clear the debris after any damage to the nearby tissue. Blood Brain Barrier (BBB) segregates the CNS from the rest of the lymphatic system and prevents the entry of foreign molecules into the brain. Pathogens still cross the BBB via different mechanisms and can cause severe infections of the CNS. Viral encephalitis is the most common form of brain infection and the causative agents include Japanese Encephalitis Virus (JEV), West Nile Virus (WNV), Murray Valley Encephalitis Virus (MVEV), Herpes Simplex Virus (HSV), Human Immunodeficiency Virus (HIV), Cytomegalovirus (CMV) and Hepatitis C Virus (HCV) among several others. Microglia expresses various Pattern Recognition Receptors (PRRs) to identify viral signatures called Pathogen Associated Molecular Patterns (PAMPs) to which microglia respond by releasing several pro and anti-inflammatory cytokines like MCP1, IL-1beta, Type I IFN, IFN-gamma, TNF-alpha etc. This review discusses the various viral infections of the brain and strategies employed by microglia to detect them.
Differentiation and self-renewal are two primary properties that characterize stem cells. Differentiation of neural stem/precursor cells (NSPCs) gives rise to multiple neural lineages, including neurons, astrocytes, and oligodendrocytes. Self-renewal, by definition, signifies the progressive growth of cells, while preserving an undifferentiated state. A large number of interdependent factors, including transcription factors, epigenetic control, and micro-RNA regulators, modulate these opposing processes without disrupting the regular neural network. The epigenetic modification of developmental genes, including alterations in DNA methylation, histone modifications, polycomb gene group and noncoding RNA expression, which are passed on through successive cell divisions, has proved to be one of the major mechanisms determining the fate of neural stem cells. Here, we review the diverse epigenetic pathways that decide whether NSPCs undergo proliferation or differentiation into different neuronal cell lineages.
Comparative effectiveness research (CER) is thought to identify what works and does not work in health care. We interpret CER as infusing evidence on product quality into markets, shifting the relative demand for products in CER studies. We analyze how shifts in demand affect health and health care spending and demonstrate that CER may raise or lower overall health when treatments have heterogeneous effects, but payers respond with product-specific coverage policies. Among patients with schizophrenia, we calibrate that subsidy policies based on the clinical trial CATIE may have reduced overall health by inducing some patients to switch away from schizophrenia treatments that were effective for them towards winners of the CER.
We studied health utilities in patients with type 1 diabetes to understand potential differences in health utilities as function of age, type of respondent (self report vs. proxy report), and method of assessment (direct vs. indirect).
Chemotherapy in Japanese encephalitis (JE) is at present entirely supportive and not targeted at the virus. There are no available drugs to effectively counter the viral infection, thereby making the fight against JE a daunting task. With approximately 50,000 reported cases per year, nearly 10,000 deaths and 3 billion people living in endemic regions, it is imperative that the hunt for an effective drug be expedited. Prophylactic measures are effective against JE, but the problem plaguing all is the underdevelopment and inefficiency of medical services in developing countries. Combined to that are difficulties to earn a living and illiteracy, that leaves significant proportions of the population in these countries uninformed about the magnitude of the threat and uninterested in the potential benefits of prophylactic strategies. Thus, for such countries coming under the JE endemic region, the need for developing therapeutic strategies that are cheap, easily available and with no or tolerable side effects, becomes significant. With rapid globalization and a gradual shift in global climate, JEV, like many other flaviviruses, may emerge in newer areas. This review is an effort to briefly outline the chemotherapeutic approaches adopted over the years in developing effective therapeutic countermeasures against this deadly disease and highlights the promising avenues that need to be treaded in order to win the war against JEV.
The United States aspires to use information from comparative effectiveness research (CER) to reduce waste and contain costs without instituting a formal rationing mechanism or compromising patient or physician autonomy with regard to treatment choices. With such ambitious goals, traditional combinations of research designs and analytical methods used in CER may lead to disappointing results. In this paper, I study how alternate regimes of comparative effectiveness information help shape the marginal benefits (demand) curve in the population and how such perceived demand curves impact decision-making at the individual patient level and welfare at the societal level. I highlight the need to individualize comparative effectiveness research in order to generate the true (normative) demand curve for treatments. I discuss methodological principles that guide research designs for such studies. Using an example of the comparative effect of substance abuse treatments on crime, I use novel econometric methods to salvage individualized information from an existing dataset.
Neuroinflammation associated with Japanese encephalitis (JE) is mainly due to the activation of glial cells with subsequent release of proinflammatory mediators from them. The recognition of viral RNA, in part, by the pattern recognition receptor retinoic acid-inducible gene I (RIG-I) has been indicated to have a role in such processes. Even though neurons are also known to express this receptor, its role after JE virus (JEV) infections is yet to be elucidated.
Inflammation in the central nervous system (CNS) in Japanese encephalitis (JE) is shown to be the result of microglial activation that leads to the release of various proinflammatory mediators. Peripheral macrophages have been reported to infiltrate into the CNS in JE, though their contribution to the inflammatory process is yet to be elucidated. In this study, using an in vitro macrophage model, we have shown that upon JE virus infection, these cells secrete various soluble factors which may significantly add to the existing inflammatory milieu and lead to apoptotic or necrotic death of neurons. However, it is difficult to quantify the extent of involvement of either the microglia or infiltrating macrophages in the inflammatory processes.
Viral infections in the prenatal (during pregnancy) and perinatal period have been a common cause of brain malformation. Besides the immediate neurological dysfunctions, virus infections may critically affect CNS development culminating in long-term cognitive deficits. Most of these neurotropic viruses are most damaging at a critical stage of the host, when the brain is in a dynamic stage of development. The neuropathology can be attributed to the massive neuronal loss induced by the virus as well as lack of CNS repair owing to a deficit in the neural stem/progenitor cell (NSPC) pool or aberrant formation of new neurons from NSPCs. Being one of the mitotically active populations in the post natal brain, the NSPCs have emerged as the potential targets of neurotropic viruses. The NSPCs are self-renewing and multipotent cells residing in the neurogenic niches of the brain, and, therefore, hampering the developmental fate of these cells may adversely affect the overall neurogenesis pattern. A number of neurotropic viruses utilize NSPCs as their cellular reservoirs and often establish latent and persistent infection in them. Both HIV and Herpes virus infect NSPCs over long periods of time and reactivation of the virus may occur later in life. The virus infected NSPCs either undergoes cell cycle arrest or impaired neuronal or glial differentiation, all of which leads to impaired neurogenesis. The disturbances in neurogenesis and CNS development following neurotropic virus infections have direct implications in the viral pathogenesis and long-term neurobehavioral outcome in infected individuals.
Japanese encephalitis virus (JEV) induces neuroinflammation with typical features of viral encephalitis, including inflammatory cell infiltration, activation of microglia, and neuronal degeneration. The detrimental effects of inflammation on neurogenesis have been reported in various models of acute and chronic inflammation. We investigated whether JEV-induced inflammation has similar adverse effects on neurogenesis and whether those effects can be reversed using an anti-inflammatory compound minocycline.
Variations in health state utilities can impact cost-effectiveness analyses. One potential source of error is when joint health state (JS) utilities are rated higher than the embedded single state (SS) utilities. Knowing when and in whom this occurs can improve cost-effectiveness analyses.
Mechanisms of interleukin-18 (IL-18) and interleukin-10 (IL-10) in lipopolysaccharide (LPS) induced endotoxemia are not clear; their protective role is being investigated so that they may effectively modulate the host cytokine levels during endotoxemia. The aim of the study was to evaluate protective effects of IL-18 and IL-10 in experimentally induced endotoxemia in mice correlating the changes in tissue anti-oxidant enzymes and circulating cytokines. Liver injury was determined by estimation of serum glutamate oxalate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT), serum nitric oxide (NOx), hepatic anti-oxidant enzyme and cytokine content in LPS (250 microg/kg) induced endotoxemic mice receiving either IL-18 (500 ng/mouse) or IL-10 (600 ng/mouse) treatment. Mice (87% of IL-10 treated and 74% of IL-18 treated) survived when administered prior to LPS challenge. Pre-treatment of mice with either IL-10 or IL-18 followed by LPS, lead to reduction in SGPT and SGOT level, serum NOx, and altered hepatic anti-oxidant enzymes activity and myeloperoxidase activity than the only LPS treated group. Marked reduction in the amounts of LPS-induced hepatic and splenic TNF-u content has been observed after IL-10 pre-treatment. Results suggested that attenuating the induction of TNF-alpha and IFN-gamma and subsequent induction of nitric oxide formation in response to LPS may in part account for efficient protection by IL-18 and IL-10 in the reduction of LPS-induced liver injury.
To understand the structure-activity relationship of isoquinoline alkaloids, absorption, fluorescence, circular dichroism, and thermodynamics were employed to study the interaction of five C-9-?-amino alkyl ether analogs from the plant alkaloid berberine with double-stranded calf thymus DNA. The C-9 derivatization resulted in dramatic enhancements in the fluorescence emission of these compounds. The most remarkable changes in the spectral and binding properties were in the BC4 and BC5 derivatives. Interactions of these analogs, which have an additional recognition motif with DNA, were evaluated through different spectroscopic and calorimetric titration experiments. The analogs remarkably enhanced the DNA binding affinity and the same was directly dependent on the alkyl chain length. The analog with six alkyl chains enhanced the DNA binding affinity by about 33 times compared with berberine. The binding became more entropically driven with increasing chain length. These results may be of potential use in the design of berberine derivatives and understanding of the structure-activity relationship for improved therapeutic applications.
Neurons and astrocytes differentially express isoenzymes of lactate dehydrogenase (LDH). The metabolic consequences for the variations in mRNA expression of LDH isoenzyme subtypes in neurons and astrocytes control cerebral vasoregulation. Moreover, cellular signalling consequences for functional neurovascular control may also be dependent on LDH isoenzyme subtype profiles. Initial computer simulations revealed glutamate-induced calcium waves in connected astrocytes, and showed concomitant changes in the expression of nitric oxide synthase (NOS) and lactic acid metabolism. To validate these findings, the nature and extent of glutamate-dependent signalling crosstalk in murine cell lines were investigated through correlated lactate levels and calcium upregulation. Neuro2A and C8D1A cells were separately treated with timed supernatant extracts from each other and their LDH1 and LDH5 isoenzyme responses were recorded. Western blot analysis showed LDH1/LDH5 isoenzyme ratio in the astrocytes to be positively correlated with Neuro2A-derived lactate levels estimated by the amplitude of 1.33-ppm spectral peak in 1H-NMR, and LDH1/LDH5 isoenzyme ratio in neurons is negatively correlated with CSD1A-derived lactate levels. Significant modulations of the calcium-responsive protein pCamKII levels were also observed in both cell lines, particularly correlations between pCamKII and lactate in C8D1A cells, thus explaining the calcium dependence of the lactate response. Together, these observations indicate that lactate is a key indicator of the metabolic state of these cell types, and may be a determinant of release of vasoregulatory factors.
Comparative effectiveness research (CER) can provide valuable information for patients, providers and payers. These stakeholders differ in their incentives to invest in CER. To maximize benefits from public investments in CER, it is important to understand the value of CER from the perspectives of these stakeholders and how that affects their incentives to invest in CER. This article provides a conceptual framework for valuing CER, and illustrates the potential benefits of such studies from a number of perspectives using several case studies. We examine cases in which CER provides value by identifying when one treatment is consistently better than others, when different treatments are preferred for different subgroups, and when differences are small enough that decisions can be made based on price. We illustrate these findings using value-of-information techniques to assess the value of research, and by examining changes in pharmaceutical prices following publication of a comparative effectiveness study. Our results suggest that CER may have high societal value but limited private return to providers or payers. This suggests the importance of public efforts to promote the production of CER. We also conclude that value-of-information tools may help inform policy decisions about how much public funds to invest in CER and how to prioritize the use of available public funds for CER, in particular targeting public CER spending to areas where private incentives are low relative to social benefits.
Viruses have evolved with their hosts, which include all living species. This has been partly responsible for the development of highly advanced immune systems in the hosts. However, viruses too have evolved ways to regulate and evade the hosts immune defence. In addition to mutational mechanisms that viruses employ to mimic the host genome and undergo latency to evade the hosts recognition of the pathogen, they have also developed epigenetic mechanisms by which they can render the hosts immune responses inactive to their antigens. The epigenetic regulation of gene expression is intrinsically active inside the host and is involved in regulating gene expression and cellular differentiation. Viral immune evasion strategies are an area of major concern in modern biomedical research. Immune evasion strategies may involve interference with the host antigen presentation machinery or host immune gene expression capabilities, and viruses, in these manners, introduce and propagate infection. The aim of this review is to elucidate the various epigenetic changes that viruses are capable of bringing about in their host in order to enhance their own survivability and pathogenesis.
Japanese encephalitis virus (JEV), the leading cause of acute encephalitis in South-East Asia is a neurotropic virus infecting various CNS cell types. Most Flaviviruses including JEV get internalised into cells by receptor-mediated endocytosis, which involve clathrin and membrane cholesterol. The cholesterol-enriched membrane microdomains referred to as lipid rafts act as portals for virus entry in a number of enveloped viruses, including Flavivirus. However, the precise role played by membrane lipid rafts in JEV internalisation into neural stem cells is still unknown. We have established neural stem/progenitor cells and C17.2 cell line as models of productive JEV infection. Increase in membrane fluidity and clustering of viral envelope proteins in lipid rafts was observed in early time points of infection. Localisation of non-structural proteins to rafts at later infection stages was also observed. Co-localisation of JEV glycoprotein with Cholera toxin B confirmed that JEV internalisation occurs in a lipid-raft dependent manner. Though JEV entry is raft dependent, however, there is requirement of functional clathrin during endocytosis inside the cells. Besides virus entry, the lipid rafts act as signalling platforms for Src tyrosine kinases and result in activation of phosphoinositìde 3-kinase/Akt signalling during early JEV infection. Disruption of lipid raft formation by cholesterol depletion using Methyl ?-cyclodextrin, reduced JEV RNA levels and production of infectious virus particles as well as impaired phosphoinositìde 3-kinase/Akt signalling during initial infection. Overall, our results implicate the importance of host membrane lipid rafts in JEV entry and life cycle, besides maintaining survival of neural stem/progenitor cells during early infection.
Many analyses of healthcare costs involve use of data with varying periods of observation and right censoring of cases before death or at the end of the episode of illness. The prominence of observations with no expenditure for some short periods of observation and the extreme skewness typical of these data raise concerns about the robustness of estimators based on inverse probability weighting (IPW) with the survival from censoring probabilities. These estimators also cannot distinguish between the effects of covariates on survival and intensity of utilization, which jointly determine costs. In this paper, we propose a new estimator that extends the class of two-part models to deal with random right censoring and for continuous death and censoring times. Our model also addresses issues about the time to death in these analyses and separates the survival effects from the intensity effects. Using simulations, we compare our proposed estimator to the inverse probability estimator, which shows bias when censoring is large and covariates affect survival. We find our estimator to be unbiased and also more efficient for these designs. We apply our method and compare it with the IPW method using data from the Medicare-SEER files on prostate cancer.
Activation of microglia, the resident macrophages of the central nervous system (CNS), is the hallmark of neuroinflammation in neurodegenerative diseases and other pathological conditions associated with CNS infection. The activation of microglia is often associated with bystander neuronal death. Nuclear factor-?B (NF-?B) is one of the important transcription factors known to be associated with microglial activation which upregulates the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2) and other pro-inflammatory cytokines. Recent studies have focused on the role of Krüppel-like factor 4 (Klf4), one of the zinc-finger transcription factors, in mediating inflammation. However, these studies were limited to peripheral system and its role in CNS is not understood. Our studies focused on the possible role of Klf4 in mediating CNS inflammation.
Japanese encephalitis (JE) is caused by a neurotropic flavivirus that causes CNS damage that leads to death in acute cases or permanent neuropsychiatric sequel in survivors. The course of infection of this virus is not well defined though it is clear that it evades the hosts innate immune response in the periphery. The current study was designed to investigate the time-dependent changes in the spleen and lymph node, apart from the CNS that are infected by the Japanese encephalitis virus (JEV). Our previous studies have led to the identification of minocycline, a semi-synthetic antibiotic, as a protective drug in JE. In this study we have also investigated the role of minocycline on the peripheral organs that are infected by JEV. Levels of IL-12 and MCP-1 in the organs were estimated by cytometric bead array, and immunohistochemical studies were performed on cryosections of tissue to detect CD3- or CD11b-positive cells as well as JEV antigen. We found that the levels of T cell-activating cytokine IL-12 and MCP-1 levels were significantly elevated in JEV-infected tissue samples in a time-dependent manner. Corresponding to this increase was the increase in the number of CD3- and CD11b-positive cells in the tissues of infected animals. Minocycline treatment abrogated these changes. Minocycline treatment also resulted in the gradual decrease in the number of CD11b (but not CD3) positive cells in the lymph node and spleen, even though the virus persisted in these organs. We also observed structural changes in the spleen following minocycline treatment.
Japanese encephalitis (JE), caused by a mosquito-borne flavivirus, is endemic to the entire south-east Asian and adjoining regions. Currently no therapeutic interventions are available for JE, thereby making it one of the most dreaded encephalitides in the world. An effective way to counter the virus would be to inhibit viral replication by using anti-sense molecules directed against the viral genome. Octaguanidinium dendrimer-conjugated Morpholino (or Vivo-Morpholino) are uncharged anti-sense oligomers that can enter cells of living organisms by endocytosis and subsequently escape from endosomes into the cytosol/nuclear compartment of cells. We hypothesize that Vivo-Morpholinos generated against specific regions of 3 or 5 untranslated regions of JEV genome, when administered in an experimental model of JE, will have significant antiviral and neuroprotective effect.
There is paucity of studies on cytokines in tuberculous meningitis (TBM) and their relation with clinical and radiological changes; therefore this study was undertaken. 16 TBM patients diagnosed on the basis of clinical, CSF and radiological criteria were included. They were subjected to TNF-alpha, IL-6, IL-8, IL-10, IL-1beta, and IL-12p70 estimation in CSF. The cytokine levels were also estimated in 10 controls. Initial clinical examination, stage of TBM and MRI findings (infarct, hydrocephalus, tuberculoma and exudates) were recorded. The patients were treated with 4 drugs antitubercular (RHZE) therapy and after 3 months clinical examination, cytokine levels, and radiological studies were repeated. Outcome was defined by Barthel index score at 3 months into poor, partial and complete recovery. The patients age ranged between 10 and 50 years, 5 were females. At 3 months, all the patients were clinically followed up and 14 underwent repeat MRI. 10 patients improved, 1 remained stable and 5 deteriorated. There was worsening with respect to tuberculoma in 3, infarction in 2 and exudate in 1 patient. TNF-alpha was expressed in 32% patients, IL-6, IL-10, IL-1beta and IL-8 were significantly expressed in patients and declined after 3 months following treatment. The cytokine levels did not correlate with stage of meningitis, outcome and radiological deterioration or improvement.
Continuous glucose monitoring (CGM) has been found to improve glucose control in type 1 diabetic patients. We estimated the cost-effectiveness of CGM versus standard glucose monitoring in type 1 diabetes. RESEARCH DESIGN AND METHODS This societal cost-effectiveness analysis (CEA) was conducted in trial populations in which CGM has produced a significant glycemic benefit (A1C >or=7.0% in a cohort of adults aged >or=25 years and A1C <7.0% in a cohort of all ages). Trial data were integrated into a simulation model of type 1 diabetes complications. The main outcome was the cost per quality-adjusted life-year (QALY) gained.
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